Articles de revues sur le sujet « Active approximately 610-approximately 580 b »

Objectives In order to generate independent performance data regarding accuracy of COVID-19 antigen-based rapid diagnostic tests (Ag-RDTs), prospective diagnostic evaluation studies across multiple sites are required to evaluate their performance in different clinical settings. This report describes the clinical evaluation the GENEDIA W COVID-19 Ag Device (Green Cross Medical Science Corp., Chungbuk, Korea) and the ActiveXpress+ COVID-19 Complete Testing Kit (Edinburgh Genetics Ltd, UK), in two testing sites Peru and the United Kingdom. Methods Nasopharyngeal swabs collected from 456 symptomatic patients at primary points of care in Lima, Peru and 610 symptomatic participants at a COVID-19 Drive-Through testing site in Liverpool, England were analyzed by Ag-RDT and compared to RT-PCR. Analytical evaluation of both Ag-RDTs was assessed using serial dilutions of direct culture supernatant of a clinical SARS-CoV-2 isolate from the B.1.1.7 lineage. Results For GENEDIA brand, the values of overall sensitivity and specificity were 60.4% [95% CI 52.4–67.9%], and 99.2% [95% CI 97.6–99.7%] respectively; and for Active Xpress+ the overall values of sensitivity and specificity were 66.2% [95% CI 54.0–76.5%], and 99.6% [95% CI 97.9–99.9%] respectively. The analytical limit of detection was determined at 5.0 x 102 pfu/ml what equals to approximately 1.0 x 104 gcn/ml for both Ag-RDTs. The UK cohort had lower median Ct values compared to that of Peru during both evaluations. When split by Ct, both Ag-RDTs had optimum sensitivities at Ct<20 (in Peru; 95% [95% CI 76.4–99.1%] and 100.0% [95% CI 74.1–100.0%] and in the UK; 59.2% [95% CI 44.2–73.0%] and 100.0% [95% CI 15.8–100.0%], for the GENDIA and the ActiveXpress+, respectively). Conclusions Whilst the overall clinical sensitivity of the Genedia did not meet WHO minimum performance requirements for rapid immunoassays in either cohort, the ActiveXpress+ did so for the small UK cohort. This study illustrates comparative performance of Ag-RDTs across two global settings and considers the different approaches in evaluation methods.

570 patients with acute ankle joint distortion were randomized to four treatment groups: a combination spray of arnica tincture and hydroxyethyl salicylate (HES; group A, ), arnica (B, ), HES (C, ), and placebo (D, ). The medication was applied 4-5 times daily for 10 days. Efficacy was assessed on day 3-4 by evaluating pain on motion on a visual analogue scale (VAS). Pain improvement in group A was significantly superior over groups B–D (-test with unadjusted baseline values, and ANCOVA after adjustment, ) and approximately corresponded to the cumulative effect of the single constituents (12.1, 7.5, and 18.7 mm VAS for A versus B, A versus C, and A versus D; 95% CI 8.0–16.2, 4.7–10.4, and 14.8–22.5 mm). The combination is justified by the additive effects of the single active constituents.

Corrosion fatigue crack propagation tests were conducted on a high-sulfur ASTM A302-B plate steel overlaid with weld-deposited Alloy EN82H cladding. The specimens featured semi-elliptical surface cracks penetrating approximately 6.3 mm of cladding into the underlying steel. The initial crack sizes were relatively large with surface lengths of 22.8–27.3 mm, and depths of 10.5–14.1 mm. The experiments were initiated in a quasi-stagnant low-oxygen (O2 < 10 pph) aqueous environment at 243°C, under loading conditions (ΔK, R, cyclic frequency) conducive to environmentally assisted cracking (EAC) under quasi-stagnant conditions. Following fatigue testing under quasi-stagnant conditions where EAC was observed, the specimens were then fatigue tested under conditions where active water flow of either 1.7 m/s or 4.7 m/s was applied parallel to the crack. Earlier experiments on unclad surface-cracked specimens of the same steel exhibited EAC under quasi-stagnant conditions, but water flow rates at 1.7 m/s and 5.0 m/s parallel to the crack mitigated EAC. In the present experiments on clad specimens, water flow at approximately the same as the lower of these velocities did not mitigate EAC, and a free stream velocity approximately the same as the higher of these velocities resulted in sluggish mitigation of EAC. The lack of robust EAC mitigation was attributed to the greater crack surface roughness in the cladding interfering with flow induced within the crack cavity. An analysis employing the computational fluid dynamics code, FIDAP, confirmed that frictional forces associated with the cladding crack surface roughness reduced the interaction between the free stream and the crack cavity.

Nuclear organization and chromatin interactions are important for genome function, yet determining chromatin connections at high resolution remains a major challenge. To address this, we developed Accessible Region Conformation Capture (ARC-C), which profiles interactions between regulatory elements genome-wide without a capture step. Applied to Caenorhabditis elegans, ARC-C identifies approximately 15,000 significant interactions between regulatory elements at 500-bp resolution. Of 105 TFs or chromatin regulators tested, we find that the binding sites of 60 are enriched for interacting with each other, making them candidates for mediating interactions. These include cohesin and condensin II. Applying ARC-C to a mutant of transcription factor BLMP-1 detected changes in interactions between its targets. ARC-C simultaneously profiles domain-level architecture, and we observe that C. elegans chromatin domains defined by either active or repressive modifications form topologically associating domains (TADs) that interact with A/B (active/inactive) compartment-like structure. Furthermore, we discover that inactive compartment interactions are dependent on H3K9 methylation. ARC-C is a powerful new tool to interrogate genome architecture and regulatory interactions at high resolution.

Boophone disticha (B. disticha) has been used systemically in traditional medical practice in Zimbabwe and neighbouring countries for the management of various central nervous system conditions including hysteria. Abuse of the plant by teenagers in Zimbabwe for its claimed hallucinogenic effects has also been reported, with the advent of serious toxicity in some cases. In the present work, we describe the acute toxicity and neurotoxicological effects of a freeze dried hydro-ethanolic plant extract of the bulb of B. disticha. Thirty-three adult (6—12 weeks old), non-pregnant female Sprague Dawley rats were used for the oral LD50 estimation. Animals were given doses of 50, 120, 240, 360, 500 and 700 mg/kg and were observed using a modified Functional Observation Battery (FOB) for behavioural toxicity. The estimated oral LD50 of the plant extract was between 120 and 240 mg/kg. For doses of 240 mg/kg and less, signs of toxicity began approximately 10 minutes after gavage, and the most prominent initial signs were head tremors (at 50 mg/kg) and body tremors, severe body tremors(>360 mg/kg) followed by convulsions. Generally, symptoms of toxicity lasted approximately 2 hours for doses of 240 mg/kg and less; and 3 hours for doses over 240 mg/kg for animals that survived. These results point to a rapid gastrointestinal absorption of the active principles in the plant extract. The most prominent neurotoxicological effects were increased flaccid limb paralysis and spastic hind-limb paralysis. Tachypnoea was noted at low doses and higher doses produced laboured breathing. The retropulsion observed with higher doses could indicate the reported hallucinogenic effects of the plant extract.

TPS7592 Background: Consensus treatment guidelines are unavailable for patients with diffuse large B-cell lymphoma (DLBCL) whose disease progresses after first-line therapy. The receptor tyrosine kinase-like orphan receptor 1 (ROR1) is a transmembrane protein that is overexpressed in multiple cancers, including hematological malignancies. Zilovertamab vedotin (ZV; MK-2140, previously known as VLS-101), an antibody-drug conjugate comprising a humanized IgG1 monoclonal antibody, a proteolytically cleavable linker, and the antimicrotubule cytotoxic agent monomethyl auristatin E, targets ROR1 and has shown promising efficacy and tolerability in hematological malignancies, including DLBCL. This 2-part (part 1, dose confirmation; part 2, dose expansion), open-label, randomized, active-control, phase 2/3 study (NCT05139017) will assess safety and efficacy of ZV + standard of care in patients with relapsed/refractory (R/R) DLBCL. Methods: Eligible adult patients must have histologically confirmed DLBCL per WHO classification, ineligible for or have failed autologous stem cell transplantation and chimeric antigen receptor T cell therapy, R/R DLBCL after ≥1 line of prior therapy (cohort A) or ≥2 lines of prior therapy (cohort B), measurable disease per Lugano 2014 criteria, and Eastern Cooperative Oncology Group performance status ≤2. Approximately 420 patients will be enrolled in the study (cohort A, n = 230; cohort B, n = 190). In the part 1 dose confirmation phase, 30 patients from cohort A will receive ZV (at increasing doses: 1.5, 1.75, 2.0, 2.25, and 2.5 mg/kg; starting at 1.75 mg/kg) plus gemcitabine-oxaliplatin + rituximab (R-GemOx) to establish the recommended phase 2 dose using the mTPI design. A safety run-in phase of part 2 will include 30 patients from cohort B and will receive ZV + bendamustine and rituximab (BR). Approximately 360 patients will be included in the part 2 dose expansion phase (cohort A, n = 200; cohort B, n = 160). Patients from cohort A will be randomly assigned 1:1 to 6 cycles of either ZV + R-GemOx or R-GemOx. Patients from cohort B will be randomly assigned 1:1 to 6 cycles of either ZV + BR or BR. Disease response assessments by CT and PET scans will occur every 12 weeks until disease progression or study discontinuation. Adverse events (AEs) will be monitored throughout the study and graded per NCI CTCAE version 5.0. In part 1, the primary end point is safety, including DLTs, AEs, and discontinuation due to AEs. The primary end point for cohorts A and B in the dose expansion phase of part 2 will be progression-free survival by blinded independent central review per Lugano 2014 criteria. Key secondary end points in the dose expansion phase of part 2 for both cohorts include objective response rate (including complete response and partial response) and duration of response, both per Lugano 2014 criteria and overall survival. Clinical trial information: NCT05139017.

Gallium maltolate, tris(3-hydroxy-2-methyl-4H-pyran-4-onato)gallium (GaM), is an orally active gallium compound for therapeutic use. It is moderately soluble in water (10.7 ± 0.9 mg/mL at 25C∘) with an octanol partition coefficient of 0.41±0.08. The molecule is electrically neutral in aqueous solution at neutral pH; a dilute aqueous solution (2.5 ×10−5 M) showed little dissociation at pH 5.5-8.0. Single crystal X-ray diffraction analysis found the GaM molecule to consist of three maltolate ligands bidentately bound to a central gallium atom in a propeller-like arrangement, with one of the ligands disordered in two possible orientations. The compound is orthorhombic, space group Pbca, unit cell a = 16.675(3), b = 12.034(2), c = 18.435(2) A∘ at 158K. GaM was administered to healthy human volunteers at single doses of 100, 200, 300, and 500 mg (three subjects per dose). GaM was very well tolerated. Oral absorption of Ga into plasma was fairly rapid (absorption half life = 0.8-2.0h), with a central compartment excretion half life of 17-21h. Absorption appeared dose proportional over the dosage range studied. Estimated oral gallium bioavailability was approximately 25-57%, based on comparison with published data on intravenous gallium nitrate. Urinary Ga excretion following oral GaM administration was approximately 2% of the administered dose over 72h, in contrast to 49-94% urinary Ga excretion over 24h following i.v. gallium nitrate administration. We suggest that oral administration of GaM results in nearly all plasma gallium being bound to transferrin, whereas i.v. administration of gallium nitrate results in formation of considerable plasma gallate [Ga(OH)4−], which is rapidly excreted in the urine. These data support the continued investigation of GaM as an orally active therapeutic gallium compound.

During characterization of the surface antigens of serotype III group B streptococci (GBS), a protein with an apparent Mr~ 173 500 migrating on a SDS – polyacrylamide gel was found to have an N-terminal amino acid sequence identical to that of the plasmin receptor (Plr) of group A streptococci, a surface-localized glyceraldehyde-3-phosphate dehydrogenase (GAPDH). This work begins to characterize GBS GAPDH and to assess its functional activity on the cell surface. The 1.0-kb gapC gene of GBS was amplified by PCR. plr and gapC demonstrated 87% homology. An anti-Plr monoclonal antibody reacted with GBS whole cells, suggesting GBS GAPDH is surface localized. Multiple serotypes of GBS demonstrated functional GAPDH on their surfaces. The anti-Plr monoclonal antibody recognized GBS protein bands of approximately 41 and 173.5 kDa, by Western blot. Presumably, these represent monomeric and tetrameric forms of the GAPDH molecule. GBS GAPDH was demonstrated by Western blot analysis to interact with lys- and glu-plasminogens. Fluid-phase GBS GAPDH interacted, by means of ELISA, with immobilized lys-plasminogen, glu-plasminogen, actin, and fibrinogen. Enzymatically active GAPDH, capable of binding cytoskeletal and extracellular matrix proteins, is expressed on the surface of GBS.Key words: group B streptococci, glyceraldehyde-3-phosphate dehydrogenase.

The use of bacterial probiotic metabolite-based active-packaging and coatings is an innovative approach that has gained widespread attention worldwide. Additionally, its utilization can lead to improvements in qualities and properties of food products. This study was aimed to develop a food spoilage prevention system using active food packaging and coating material in preventing food spoilage while increasing its shelflife. The materials used were bacterial cellulose (BC) based bioplastics fortified with fermented soymilk extracts (FSME) using Lactobacillus acidophilus as the producer of the antimicrobial and antioxidant agents. Moreover, the applications of FSME containing probiotic bacterial metabolites are discussed to highlight their efficacy in enhancing the quality and shelf life of food products.The antimicrobial test showed that the FSME could inhibit the growth of pathogenic microbial cultures at minimum inhibitory concentration (MIC) of 10% (v/v) as shown by clear zones, around colonies of E. coli (14.33±0.58 mm), S. aureus (18.33±6.03 mm), S. Typhimurium (11.67±1.15 mm), L. monocytogenes (11.33±2.31 mm), and B. cereus (13.33±3.06 mm). Meanwhile the results of IC50 for antioxidant activity test (µg/mL) indicated that the FSME showed radical scavenging activity against DPPH at approximately 75.27±2.552 (2.5%, v/v), 55.00±0.791 (5.0%, v/v), 43.17±1.603 (7.5%, v/v) and 15.05±0.346 (10%, v/v), respectively. The shelflife of strawberries coated with the active food coating using the bioplastic fortified with FSME showed an increase in shelf life of 14 days at 4°C. The overall results indicated that the use of BC based bioplastics fortified with FSME can play an important role in preventing premature spoilage and increasing the shelf life of food products.

Abstract The effects of bacterial direct-fed microbial (DFM) mixtures on beef cattle feeding behavior were evaluated. Six ruminally cannulated crossbred Angus steers (BW = 520 ± 30 kg) were used in a replicated 3 × 3 Latin Square design. Steers were offered a steam-flaked corn-based finishing diet to ad libitum during three, 28 d periods (21-d adaptation and 7-d collection). Treatments assigned were: 1) Control (no DFM, lactose carrier only); 2) Treat-A [L. animalis, P. freudenreichii; B. subtilis; and B. licheniformis, at 1:1:1:3 ratio, respectively; totaling 6 × 109 CFU (50mg)/animal-daily minimum (Chr.Hansen]; and 3) Treat-B, the same DFM combination, but with doses at 1:1:3:1 ratio. Bacterial counts were approximately 30% greater than the minimum expected. The DFM diluted mixtures and carrier (Control) were pre-weighed and stored at -20°C until incorporated into the diet (2 g· animal-1· /day-1) 10 min prior to feeding. For mixing, approximately 1 kg (as is) of the diet being offered was removed from the individual recipient, DFM was mixed by hand, and the mixture was incorporated into the feed allocated for the day. The feeding behavior assessment consisted of a 24 h continuous visual observation (every 5 min) performed by trained personnel. Animals were assessed on d 26 of each period (no other management other than cleaning of the stall), while time spent eating, ruminating, resting, active, and drinking were taken. Time spent chewing was accounted for by adding time spent eating and ruminating. In addition, records allowed the count of the number of meals taken, as well as the time spent on each meal. Data were analyzed using the GLIMMIX procedure of SAS, with animal considered as the experimental unit, the fixed effects of treatment, and random effect of square, period, and animal (square). F-test protected pre-planned orthogonal contrasts was used to compare Control vs. Treat-A and Control vs. Treat-B. Meal number (16/d) and length (8 min/meal) were not affected by treatments (P ≥ 0.54). Main feeding behavior variables consisting of rumination (198 min/d), eating (117 min/d), chewing (315 min/d), drinking (22 min/d), resting (929 min/d), and active (174 min/d) were not affected (P ≥ 0.24) by treatments. A tendency was observed (P = 0.08) where animals offered the Treat-B spent numerically less time ruminating per unit of digestible DM and OM intake compared with Control (13.5 vs. 15.5, and 13.7 vs 15.6 min/kg, respectively). Other behaviors measured in min/kg of DM, NDF, and ADF intake were not affected (P ≥ 0.17) by treatments [rumination (11.5, 60.2, and 181), eating (6.5, 34.4, and 104), chewing (18, 94.6, and 285), and drinking (1.2, 6.6, and 19.8) min/kg, respectively]. The bacterial DFM mixtures offered appear not to directly affect the feeding behavior of beef cattle consuming a steam-flaked corn-based finishing diet.

TPS8614^ Background: Follicular lymphoma (FL) is the second most common non-Hodgkin lymphoma (NHL) and comprises approximately 22% of all NHL cases. Most patients treated eventually relapse and subsequent responses and duration of responses become shorter. Patients ultimately become resistant to chemoimmunotherapy and repeated treatment-related toxicity commonly outweighs the benefit of treatment. Ibrutinib is a potent inhibitor of BTK (downstream of the B-cell receptor, BCR) that binds covalently to Cys-481 in the active site, abrogating intrinsic survival pathways (eg, ERK1/2, NF-kB, AKT) as well as survival signals from the microenvironment (eg, TNF family members: BAFF, CD40L; cytokines from T-cells: IL4, IL6, IL10, TNFα). Irish et al (2010) have also shown that up to 60% of FL patients display BCR signaling addiction. Early indications from study PCYC-04753 suggest activity of the BTK inhibitor ibrutinib in FL. Three CR and 3 PR were observed in 11 patients at a dose of 2.5 mg/kg or higher that achieved full BTK occupancy (Fowler, ASH 2012). Methods: The DAWN study, PCI-32765FLR2002, is a phase II, single-arm study of ibrutinib in refractory FL. The study aims to enroll 110 patients with chemoimmunotherapy-resistant FL. Patients will receive an oral daily dose of 560 mg ibrutinib. Patients must have been treated with at least 2 prior lines of therapy, at least 1 rituximab-containing combination chemotherapy regimen, and the last prior line of therapy included an anti-CD20 monoclonal antibody-containing chemotherapy regimen. The primary objective of the study is to evaluate the ORR (CR + PR), with secondary objectives of duration of response, progression-free survival, overall survival, and safety. To better understand the mechanism of action of ibrutinib, blood and tumor samples will be collected and, where feasible, characterized by GEP, SMA, IHC, or other technology as applicable. These evaluations aim to identify biomarkers associated with response or resistance to ibrutinib in subjects with FL and results may assist in the development of this drug in this and potentially other indications. Approximately 64 sites in the US and Europe will enroll patients. Enrollment began in 1Q of 2013. Clinical trial information: NCT01779791.

IntroductionMultiple myeloma (MM) is a plasma cell tumour with over 5800 new cases each year in the UK. The introduction of biological therapies has improved outcomes for the majority of patients with MM, but in approximately 20% of patients the tumour is characterised by genetic changes which confer a significantly poorer prognosis, generally termed high-risk (HR) MM. It is important to diagnose these genetic changes early and identify more effective first-line treatment options for these patients.Methods and analysisThe Myeloma UK nine OPTIMUM trial (MUKnine) evaluates novel treatment strategies for patients with HRMM. Patients with suspected or newly diagnosed MM, fit for intensive therapy, are offered participation in a tumour genetic screening protocol (MUKnine a), with primary endpoint proportion of patients with molecular screening performed within 8 weeks. Patients identified as molecularly HR are invited into the phase II, single-arm, multicentre trial (MUKnine b) investigating an intensive treatment schedule comprising bortezomib, lenalidomide, daratumumab, low-dose cyclophosphamide and dexamethasone, with single high-dose melphalan and autologous stem cell transplantation (ASCT) followed by combination consolidation and maintenance therapy. MUKnine b primary endpoints are minimal residual disease (MRD) at day 100 post-ASCT and progression-free survival. Secondary endpoints include response, safety and quality of life. The trial uses a Bayesian decision rule to determine if this treatment strategy is sufficiently active for further study. Patients identified as not having HR disease receive standard treatment and are followed up in a cohort study. Exploratory studies include longitudinal whole-body diffusion-weighted MRI for imaging MRD testing.Ethics and disseminationEthics approval London South East Research Ethics Committee (Ref: 17/LO/0022, 17/LO/0023). Results of studies will be submitted for publication in a peer-reviewed journal.Trial registration numberISRCTN16847817, May 2017; Pre-results.

Dictyostelium alpha-actinin is a Ca(2+)-regulated F-actin cross-linking protein. To test the inhibitory function of the two EF hands, point mutations were introduced into either one or both Ca(2+)-binding sites. After mutations, the two EF hands were distinguishable with respect to their regulatory activities. Inactivation of EF hand I abolished completely the F-actin cross-linking activity of Dictyostelium discoideum alpha-actinin but Ca2+ binding by EF hand II was still observed in a 45Ca2+ overlay assay. In contrast, after mutation of EF hand II the molecule was still active and inhibited by Ca2+; however, approximately 500-fold more Ca2+ was necessary for inhibition and 45Ca2+ binding could not be detected in the overlay assay. These data indicate that EF hand I has a low affinity for Ca2+ and EF hand II a high affinity, implying a regulatory function of EF hand I in the inhibition of F-actin cross-linking activity. Biochemical data is presented which allows us to distinguish two functions of the EF hand domains in D. discoideum alpha-actinin: (a) at the level of the EF-hands, the Ca(2+)-binding affinity of EF hand I was increased by EF hand II in a cooperative manner, and (b) at the level of the two subunits, the EF hands acted as an on/off switch for actin-binding in the neighboring subunit. To corroborate in vitro observations in an in vivo system we tried to rescue the abnormal phenotype of a mutant (Witke, W., M. Schleicher, A. A. Noegel. 1992. Cell. 68:53-62) by introducing the mutated alpha-actinin cDNAs. In agreement with the biochemical data, only the molecule modified in EF hand II could rescue the abnormal phenotype. Considering the fact that the active construct is "always on" because it requires nonphysiological, high Ca2+ concentrations for inactivation, it is interesting to note that an unregulated alpha-actinin was able to rescue the mutant phenotype.

14076 Background: Enzastaurin (ENZ) targets the PKCβ and PI3K/AKT pathways to induce tumor cell apoptosis, reduce proliferation, and suppress tumor-induced angiogenesis. In vitro studies show ENZ solubility is highest at pH 2.0 and decreases with increasing pH. This study examined the effect of increased gastric pH by a proton pump inhibitor (lansoprazole) and the effect of food on the bioavailability of a single oral dose of ENZ in healthy subjects. Methods: In this open-label, three-period, fixed sequence, crossover study, healthy subjects received an oral, 500-mg single dose of ENZ in the fed state, alone (A) and with lansoprazole (B), and in the fasted state alone (C). Lansoprazole was dosed for 5 days, with ENZ administered 2 hours after the 5th dose. A two-week washout occurred between each period. Plasma samples were collected at predose and scheduled timepoints for up to 168 hours post-ENZ dose for pharmacokinetic (PK) characterization of ENZ and its active metabolites. Results: 22 subjects were enrolled in the study with 19 subjects completing all treatment periods. The table summarizes the PK results for enzastaurin and total analytes (enzastaurin + metabolites). ENZ Cmax and AUC(0–8) were unaffected by the administration of lansoprazole, when ENZ was then given in the fed state. Enzastaurin Cmax and AUC(0–8) increased significantly, respectively by 6.8 and 2.8 fold, when ENZ was administered after a standardized breakfast, compared to the fasted state. Median tmax increased from approximately 3 hours in the fed state to 6 hours in the fasted state, indicating slower absorption when ENZ was administered in the fasted state. Conclusions: No significant alterations in ENZ exposures were seen in the presence of lansoprazole, indicating that increased gastric pH did not decrease exposures of ENZ, when ENZ is administered in the fed state. Exposures of ENZ and its active metabolites were significantly enhanced by administration of ENZ after a meal. [Table: see text] No significant financial relationships to disclose.

TPS7572 Background: Approximately 1/3 of patients with DLBCL, the most common type of B-cell lymphoma, will become refractory to standard combination chemotherapy and have uniformly poor clinical outcomes (Crump, ASCO 2016). Axi-cel (autologous anti-CD19 chimeric antigen receptor [CAR] T cell therapy) has shown promising response rates in patients with refractory DLBCL compared with standard approaches, although some patients do not respond or progress after an initial response (Locke, Mol Ther 2016). Expression of PD-L1 on DLBCL cells and activation-dependent expression of PD-1 on CAR T cells after infusion led to the hypothesis that PD-1 pathway blockade may augment the activity of axi-cel and result in improved clinical outcomes. This study will evaluate safety and efficacy of axi-cel when given with atezolizumab (anti–PD-L1 antibody), delivered sequentially, in patients with refractory DLBCL. Methods: Phase 1 will enroll ~3-9 patients to estimate the incidence of dose-limiting toxicities. Phase 2 will enroll ~22 patients to evaluate safety and efficacy, with a primary endpoint of complete response (CR) rate (Cheson 2007). Secondary endpoints include key efficacy outcomes such as objective response rate (CR+partial response [PR]), duration of response, progression-free and overall survival, and safety and biomarker outcomes. Eligible adult patients will have received prior adequate therapy (including anti-CD20 monoclonal antibody and an anthracycline-based regimen) and have an ECOG PS of 0-1 and adequate bone marrow and organ function. Patients with a history of Richter transformation, transformed follicular lymphoma, CNS disease, or active infection are not eligible. Patients will receive fludarabine 30 mg/m2/d and cyclophosphamide 500 mg/m2/d × 3 d, followed by a single infusion of axi-cel (target dose, 2 × 106anti-CD19 CAR T cells/kg) followed by atezolizumab 1200 mg given every 21 d for 4 doses (phase 1, first dose to occur 21, 14, and 1 d after axi-cel infusion in cohorts 1, 2, and 3, respectively). The study opened to accrual in September 2016. Clinical trial information: NCT02926833.

TPS7068 Background: Despite the approval of multi-targeted protein kinase inhibitor midostaurin for use in combination with chemotherapy which improves 5-year survival in newly diagnosed (NDx) acute myeloid leukemia (AML) associated with FLT3 mutations; the cumulative incidence of relapse in FLT3 mutant AML remains high, with progression often characterized by secondary FLT3-TKD mutations. Crenolanib is a potent pan- FLT3 inhibitor that has shown promising efficacy and tolerability in combination with chemotherapy in Phase 1/2 trials for AML patients with FLT3-ITD or - TKD mutations. This is the first globally initiated, randomized Phase 3 trial comparing the efficacy of two FLT3-TKIs, crenolanib and midostaurin, combined with intensive chemotherapy in NDx FLT3-mutated AML patients. Methods: This Phase 3, randomized, multi-center trial will be conducted at multiple sites worldwide, with a target enrollment of 510 subjects. Patient inclusion was modified to match the midostaurin RATIFY criteria to enroll NDx FLT3-mutated AML (18 – 60 yo), who are eligible for intensive chemotherapy; with the addition of any FLT3-ITD and/or -TKD mutations being eligible. All subjects will receive TKI treatment and will be randomized in a 1:1 ratio to receive either crenolanib (arm A) or the active-control, midostaurin (arm B). All patients will be treated with 7+3 (100 mg/m2 IV cytarabine; 90 mg/m2 IV daunorubicin) and can initiate treatment while awaiting FLT3 results prior to randomization. Consolidation could include chemotherapy (3000 mg/m2 IV HiDAC) for up to 4 cycles and/or Allo-HSCT, depending on patient condition. During induction and consolidation patients on arm A will take crenolanib (100 mg TID) from d9 until 72h prior to the next cycle, and patients on arm B will take midostaurin (50 mg BID) on d8 to d21 of each cycle. Following consolidation or HSCT, patients may receive up to 12 months of FLT3-TKI maintenance. Maintenance efficacy will be evaluated over time using single-cell sequencing to assess MRD. Primary endpoint is event-free survival. Interim analyses will occur at approximately 178 and 267 events, and primary analysis at 356 events. Enrollment is underway as of January 31, 2019. Clinical trial information: NCT03258931.

Abstract The geological structures associated with the site of the 55 million m 3 Karameh embankment dam constructed in the Jordan Valley and the tectonic effects on dam foundation and reservoir margins were reviewed. The dam crosses the strike-slip fault of the Jordan Valley Rift Zone. Trace evidence of the fault indicates a displacement of 8 to 15 m over a rupture length of some 130 km, which probably took place several centuries ago. Earthquakes with Richter magnitudes as great as 7.8 have occurred along the Jordan Valley Fault. Deterministic studies by Tapponnier (1992) indicated that the dam design should incorporate the possibility of a 7.8 event, a maximum horizontal rupture displacement on the fault of 10 m and a design peak ground acceleration (PGA) of 0.74 g at the site of the dam. These values are consistent with those which would be used in the USA for a similar case. However, the dam was actually designed by a consultant and constructed for a PGA of about a quarter of this value, based on seismic hazard analysis following guidelines of the International Committee on Large Dams (ICOLD) (1989). Moreover, the dam was designed for displacements of 6 m horizontal and 2 m vertically. Liquefiable sand layers were found in the dam foundation. A PGA of 0.50 g will trigger liquefaction of the sand layers in the dam foundation which would be expected to result in a crest settlement of 4.4 m. Slope stability analysis indicated deep failure planes in the foundation zone. The excavation of loose materials from under the dam foundation has not precluded the possibility of liquefaction occurring under the expected earthquake. Field mapping of geological features during the dam foundation excavation and construction revealed that: a) the most likely location of the Jordan Valley fault is in the area where the Wadi Mallaha stream crosses the dam axis, b) zones of en echelon type open fissures have been defined in the laminates sub-parallel to the Jordan Valley Fault Zone, c) at the Wadi Mallaha stream bed a parallel zone of faulting and warping of the Lisan Formation was identified, and d) the alignment is clearly confirmed by the exposure immediately upstream of the core at Ch 1375. The main wrench fault zone crosses the embankment footprint (upstream to downstream approximately) and reaches the surface around Ch 1375. The critical safety elements of the embankment are the core, the downstream fine filter, the chimney drain and the drainage blanket. To resist large earthquake events safely, the following safety measures should be implemented: 1. A freeboard of 7.0 m instead of the 5.0 m constructed. 2. The foundation of the dam should be stabilized against liquefaction. 3. The embankment internal zoning should be designed to accommodate damage resulting from earthquake events with a magnitude of 7.8. 4. The foundation needs relief measures downstream to lower the pore pressure. This paper describes the measures taken during construction as overall defense against future fault movements through a wide plastic core, an extensive upstream blanket, a 5.0-m thick downstream chimney filter and drain zones, a 5-m freeboard and an upstream crack stopper zone which may be critical for normal faults with a lateral extension component. The geological determination of the main wrench fault alignment resulted in the addition of an extra 2-m width to each of the already wide chimney filter and drain zones. In order to reduce potential seepage, local cut-off trenches or slush grouting were used for treatment of any open fissures at the upstream edge of the external blanket and the right bank ridge. The scale and scope of this dam and inherent engineering geological hazards are unprecedented. The design is considered deficient. This paper documents serious safety issues with the dam. The constructed dam presents serious safety risks and represents a case history of a disaster waiting to happen.

Abstract Abstract 304 Background: BCL-2 is highly expressed in indolent non-Hodgkin lymphomas (NHL), mantle cell lymphoma (MCL) and other selected aggressive lymphomas, and is a promising target for therapeutic intervention. The first-generation BCL-2 inhibitor navitoclax showed some activity in indolent lymphoma, but its co-inhibition of BCL-xL resulted in dose-limiting thrombocytopenia, precluding the full exploration of the potential of BCL-2 inhibition with this drug in NHL. ABT-199 is an orally bioavailable, second-generation BH3-mimetic that inhibits BCL-2 (Ki<0.10 nM), but has 500-fold less activity for BCL-xL (Ki=48 nM). ABT-199 demonstrated antitumor activity against a variety of human cell lines and xenograft models that include B cell NHL, follicular lymphomas (FL), diffuse large B-cell Lymphoma (DLBCL) and MCL. Methods: This is a phase-I dose-escalation trial using a modified Fibonacci design in patients with relapsed/refractory NHL. The primary objectives of this study are to determine the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of ABT-199; to recommend a phase-2 dose; and to assess efficacy and biomarkers in patients with relapsed/refractory NHL. Adult patients requiring therapy, with ECOG performance status £1, and adequate marrow function received ABT-199 on Week 1 Day −7 (W1D-7), followed by continuous once-daily dosing from W1D1, until progressive disease (PD) or unacceptable toxicity. Due to concerns of potential tumor lysis, a strategy of commencing with a 2 to 3 week lead-in period with step-wise increases to the target cohort dose is being evaluated. In the first four cohorts, the starting dose increased from 50 to 200 mg (50, 100, 200, and 200 mg, respectively), with target cohort doses of 200 mg [n=3], 300 mg [n=3], 400 mg [n=4], and 600 mg [n=7]. Evaluations include: adverse events (AE; NCI-CTCAE-V4) and tumor response (IWG 2007 criteria). Results: To date, 17 patients (median age, 71 [35–85]) have been treated with ABT-199. Median prior therapies were 3 (range, 1–7) and 6 patients had bulky adenopathy (>5cm). Most common AEs (experienced by >2 patients) were nausea (41%), diarrhea (24%), dyspepsia (24%), fatigue (24%), extremity pain (24%); and anemia, constipation, upper respiratory tract infection and cough (18% each). Grade 3 or 4 AEs occurring in >1 patient were anemia (18%) and neutropenia (12%). Treatment-related thrombocytopenia has not been reported and no dose-limiting toxicities (DLTs) have been identified to date. After a single dose administration with a high-fat meal, ABT-199 reached Cmax at approximately 7 hrs with a terminal half-life of about 15 hrs. Food increased ABT-199 exposure by approximately 3-fold. With a median follow-up of 2.8 months (range, 1.2 to 10.8), 14 patients remain on study and 3 have discontinued due to PD. In patients who have completed at least a W6 assessment, reductions of >50% in target lesions have been observed in 8/15 patients (53%); 6/6 patients with MCL, 1/2 patients with WM and 1/2 patients with DLBCL. Additionally, 5 FL patients have been evaluated (3 with rituxan-refractory disease) with a median time on study of 6.4 months (range, 3.5 to 10.8). 4/5 FL patients had nodal disease reductions ranging from 18% to 40%. Conclusions: ABT-199 shows single agent anti-tumor activity in patients with NHL; particularly in MCL. Activity is also observed in DLBCL and WM. To date, no DLTs have been identified and tumor lysis syndrome related to ABT-199 has not been reported. Dose escalation is continuing to identify the optimal dosing regimen and MTD of ABT-199 in NHL. Updated results will be presented. Disclosures: Roberts: Abbott: Research Funding; Genentech: Research Funding. Anderson:Genentech: Research Funding; Abbott: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Kahl:Genentech: Consultancy, Research Funding; Abbott: Research Funding. Darden:Abbott: Employment, owner of Abbott stock Other. Nolan:Abbott: Employment, own Abbott stock Other. Gressick:Abbott: Employment, stock owner Other. Yang:Abbott: Employment, own Abbott stock Other. Chyla:Abbott: Employment, Stock owner Other. Busman:Abbott: Employment, Stock owner Other. Graham:Abbott: Employment, Stock owner Other. Cerri:Abbott: Employment, Stock owner Other. Enschede:Abbott: Employment, own Abbott stocks Other. Humerickhouse:Abbott: Employment, own Abbott stocks Other. Seymour:Roche: Advisory board member, Advisory board member Other, Consultancy; Genentech: Advisory board member, Advisory board member Other, Consultancy.

Background & Significance: Glanzmann thrombasthenia (GT) is a rare bleeding disorder resulting from a deficiency of integrin αIIbβ3 (also known as glycoprotein [GP] IIb/IIIa), a receptor for fibrinogen on platelets. Fibrinogen binding to αIIbβ3 bridges platelets and is a required step for normal platelet aggregation and hemostasis. GT is considered a severe bleeding disorder with approximately 50% of the patients reporting 1 bleed every day, and 13% reporting over 500 bleeds per year. The current standard of care for bleeding in patients with GT is reactive and on-demand, with no approved therapies for primary prophylaxis. HMB-001 is a bispecific antibody being developed by Hemab for prophylactic treatment to prevent and reduce bleeding events in patients with GT that can be administered subcutaneously. One arm of HMB-001 binds to and accumulates endogenous activated coagulation factor VII (FVIIa) while the second arm binds to the TREM-like transcript 1 receptor (TLT-1) on activated platelets. The combined effect of FVIIa accumulation and targeting to the surface of activated platelets via HMB-001 brings the activity of FVIIa to levels that are considered therapeutically effective based on clinical experience with recombinant FVIIa (rFVIIa). This is a first-in-human, Phase 1/2, dose escalation, safety, pharmacokinetic (PK), pharmacodynamic (PD), and preliminary efficacy study of HMB-001 in participants with Glanzmann thrombasthenia. Study Design and Methods: The study consists of 3 parts. Part A is a Phase 1, open-label, single ascending dose study, which will evaluate the safety, tolerability, PK, and PD of HMB-001 in participants with GT.Part B is a Phase 2, open-label, multiple ascending dose study evaluating the safety, tolerability, PK, PD, and preliminary effects on bleeding of repeat doses of HMB-001 monotherapy.Part C is a Phase 2, open-label, treatment extension portion study. It will be open to participants who have fulfilled the requirements of Part B and are considered eligible to continue by the Investigator. Part C will evaluate longer term safety, and preliminary efficacy of repeat doses of HMB-001 monotherapy for 9 months. Study Population: Male and female participants 18 to 65 years of age with GT. Part A is single-center, while Part B/C will be a multicenter study in various countries. Major inclusion and exclusion criteria include: Criteria for Inclusion: Part A and B Age 18 to 65Diagnosis of GTVital signs within normal rangeNegative pregnancy testMust meet the following baseline organ function eGFR &gt;45 mL/min/1.73m 2LFTs within normal rangeHgb &gt;85 g/L and platelet count &gt;150 x 10 9/L Part B only 2 bleeding events per week on average of any severity and type and at least 1 spontaneous or traumatic bleed within the last 12 months requiring treatment with rFVIIa, platelets, medical or surgical procedure Criteria for Exclusion: Part A Active severe infection or inflammationHistory of clinically significant hypersensitivity associated with monoclonal antibody therapies.Personal or family history of venous or arterial thrombosis or thromboembolic disease.Other risk factors that substantially increase risk of venous or arterial thrombosisCongenital or acquired bleeding disorders other than GTConcurrent disease, treatment, medications, or abnormality in clinical laboratory tests that may pose additional riskAddiction or other diseases that prevent the participant from appropriately assessing the nature and scope of the clinical study or participating in study procedures Participants included in Part B are eligible for Part C following completion of their dosing in Part B.

Abstract Abstract 3923 Background: BCL-2 is highly expressed in indolent non-Hodgkin lymphomas (NHL), mantle cell lymphoma (MCL) and other selected aggressive lymphomas, and is a promising target for therapeutic intervention. The first-generation BCL-2 inhibitor navitoclax showed some activity in indolent lymphoma, but its co-inhibition of BCL-xL resulted in dose-limiting thrombocytopenia, precluding the full exploration of the potential of BCL-2 inhibition with this drug in NHL. ABT-199 is an orally bioavailable, second-generation BH3-mimetic that inhibits BCL-2 (Ki<0.10 nM), but has 500-fold less activity for BCL-xL (Ki=48 nM). ABT-199 demonstrated antitumor activity against a variety of human cell lines and xenograft models that include B cell NHL, follicular lymphomas (FL), diffuse large B-cell Lymphoma (DLBCL) and MCL. Methods: This is a phase-I dose-escalation trial using a modified Fibonacci design in patients with relapsed/refractory NHL. The primary objectives of this study are to determine the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of ABT-199; to recommend a phase-2 dose; and to assess efficacy and biomarkers in patients with relapsed/refractory NHL. Adult patients requiring therapy, with ECOG performance status £1, and adequate marrow function received ABT-199 on Week 1 Day −7 (W1D-7), followed by continuous once-daily dosing from W1D1, until progressive disease (PD) or unacceptable toxicity. Due to concerns of potential tumor lysis, a strategy of commencing with a 2 to 3 week lead-in period with step-wise increases to the target cohort dose is being evaluated. In the first four cohorts, the starting dose increased from 50 to 200 mg (50, 100, 200, and 200 mg, respectively), with target cohort doses of 200 mg [n=3], 300 mg [n=3], 400 mg [n=4], and 600 mg [n=7]. Evaluations include: adverse events (AE; NCI-CTCAE-V4) and tumor response (IWG 2007 criteria). Results: To date, 17 patients (median age, 71 [35–85]) have been treated with ABT-199. Median prior therapies were 3 (range, 1–7) and 6 patients had bulky adenopathy (>5cm). Most common AEs (experienced by >2 patients) were nausea (41%), diarrhea (24%), dyspepsia (24%), fatigue (24%), extremity pain (24%); and anemia, constipation, upper respiratory tract infection and cough (18% each). Grade 3 or 4 AEs occurring in >1 patient were anemia (18%) and neutropenia (12%). Treatment-related thrombocytopenia has not been reported and no dose-limiting toxicities (DLTs) have been identified to date. After a single dose administration with a high-fat meal, ABT-199 reached Cmax at approximately 7 hrs with a terminal half-life of about 15 hrs. Food increased ABT-199 exposure by approximately 3-fold. With a median follow-up of 2.8 months (range, 1.2 to 10.8), 14 patients remain on study and 3 have discontinued due to PD. In patients who have completed at least a W6 assessment, reductions of >50% in target lesions have been observed in 8/15 patients (53%); 6/6 patients with MCL, 1/2 patients with WM and 1/2 patients with DLBCL. Additionally, 5 FL patients have been evaluated (3 with rituxan-refractory disease) with a median time on study of 6.4 months (range, 3.5 to 10.8). 4/5 FL patients had nodal disease reductions ranging from 18% to 40%. Conclusions: ABT-199 shows single agent anti-tumor activity in patients with NHL; particularly in MCL. Activity is also observed in DLBCL and WM. To date, no DLTs have been identified and tumor lysis syndrome related to ABT-199 has not been reported. Dose escalation is continuing to identify the optimal dosing regimen and MTD of ABT-199 in NHL. Updated results will be presented. Disclosures: Seymour: Roche: Advisory Board member Other, Consultancy; Genentech: Advisory Board member, Advisory Board member Other, Consultancy. Anderson:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Kipps:Abbott: Consultancy, Research Funding. Wierda:Abbott: Research Funding; Genentech: Consultancy, Research Funding; GlaxoSmithKline: Consultancy, Research Funding; AmGen: Research Funding; Merck: Consultancy; Celgene: Consultancy; Pharmacyclics: Consultancy; Genzyme: Consultancy. Kahl:Abbott: Research Funding; Genentech: Consultancy, Research Funding. Miller:Abbott: Research Funding; Genentech: Research Funding. Darden:Abbott: Employment, owner of Abbott stock Other. Nolan:Abbott: Employment, own Abbott stock Other. Gressick:Abbott: Employment, stock owner Other. Xiong:Abbott: Employment, own Abbott stock Other. Huang:Genentech: Research Funding; Abbott: Research Funding; Walter and Eliza Hall Institiute of Medical Research: Employment, receives commercial income related to ABT-199, receives commercial income related to ABT-199 Other. Chyla:Abbott: Employment, Stock owner Other. Busman:Abbott: Employment, Stock owner Other. Graham:Abbott: Employment, Stock owner Other. Cerri:Abbott: Employment, Stock owner Other. Enschede:Abbott: Employment, own Abbott stocks Other. Humerickhouse:Abbott: Employment, own Abbott stocks Other. Roberts:Abbott: Research Funding; Genentech: Research Funding; Walter and Eliza Hall Institute of Medical Research: Employment, Receives commercial income related to ABT-199, Receives commercial income related to ABT-199 Other.

TPS7589 Background: The preferred first-line regimen for DLBCL is rituximab with cyclophosphamide, doxorubicin, vincristine, and prednisone (R-CHOP); but novel therapies are needed. A recent phase 3 study showed that replacing vincristine with an antibody-drug conjugate (ADC) is a viable approach (Tilly H et al. N Engl J Med. 2022;386:351-363). Receptor tyrosine kinase–like orphan receptor 1 (ROR1) is an oncofetal protein that is minimally expressed in adult tissues and overexpressed in DLBCL. ZV is an ADC comprising a humanized IgG1 monoclonal anti-ROR1, a proteolytically cleavable linker, and the antimicrotubule agent, monomethyl auristatin E. The single-arm, open-label, phase 2 waveLINE-007 study (NCT05406401) will investigate ZV combined with R-CHP in patients with previously untreated DLBCL. Part 1 is being conducted to determine safety and tolerability and recommended phase 2 dose (RP2D) of ZV in combination with R-CHP. Part 2 will be conducted to investigate efficacy of ZV at the RP2D with R-CHP. Methods: Eligible patients will be ≥18 years of age and have previously untreated histologically confirmed DLBCL, positron emission tomography (PET)–positive disease verified by blinded independent central review (BICR), an ECOG PS of 0 or 1, and adequate organ function. Patients diagnosed with primary mediastinal B-cell lymphoma, with a history of transformation of indolent disease to DLBCL, or active central nervous system lymphoma will be excluded. Approximately 60 patients will be enrolled (part 1, n = 45; part 2, n = 15). Part 1 will use a modified toxicity probability interval design to establish the RP2D of ZV when administered with R-CHP. The starting dose of ZV will be 1.75 mg/kg (modified to 1.5, 2.0, 2.25, or 2.5 mg/kg) administered as an intravenous infusion every 3 weeks (Q3W) in combination with R-CHP. In part 2, an additional 15 patients will receive ZV at RP2D plus R-CHP Q3W for up to 6 cycles until disease progression per Lugano 2014 criteria, unacceptable toxicity, or withdrawal. Disease response assessment, computed tomography, and PET will occur at baseline and cycles 3 and 6. Adverse events (AEs) will be monitored up to 30 days after cessation of treatment (90 days for serious AEs, or 30 days if new anticancer therapy is initiated). AEs will be graded per National Cancer Institute Common Terminology Criteria for Adverse Events, version 5.0. Primary end points are safety and tolerability and RP2D for ZV in combination with R-CHP, and complete response rate per Lugano 2014 criteria as assessed by the investigator. Secondary end points are objective response rate and duration of response per Lugano 2014 criteria by investigator review. Exploratory end points include progression-free survival per Lugano 2014 criteria by BICR and overall survival. Recruitment is currently underway. Clinical trial information: NCT05406401 .

Abstract Abstract 3812 Background: Decitabine (DAC) is a hypomethylating agent indicated for the treatment of myelodysplastic syndromes (MDS). We hypothesized that low-dose subcutaneous (SC) schedules of DAC may be active and safe in patients with lower risk MDS. Methods: This randomized, open-label, multicenter phase II study evaluated 2 DAC regimens in patients with low- or intermediate-1 risk MDS: 20 mg/m2/day SC for 3 consecutive days every 28 days (Arm A) or 20 mg/m2/day SC every 7 days (on days 1, 8, and 15) for 21 days, followed by 7 days without DAC (Arm B). The aim was to determine clinical activity, safety, and tolerability of the 2 regimens. The primary objective was overall improvement rate (OIR), defined as complete remission (CR), partial remission (PR), marrow CR (mCR), or hematologic improvement (HI), measured at the end of each cycle using patient's best response according to International Working Group (IWG) 2006 criteria. Secondary objectives included safety, HI, transfusion independence, cytogenetic response, and overall survival (OS). The tertiary objective was change in DNA methylation. Treatment on study was for ≤1 year, but patients who had clinical benefit could continue DAC off protocol. Target enrollment was 80 patients. Categorical and continuous variables were compared using Fisher's exact test and 1-way ANOVA, respectively; survival was analyzed with Kaplan-Meier, Cox regression, and log-rank methods. Results: Sixty-seven patients were randomized at 5 sites when the trial terminated early after achievement of protocol-defined superiority. On Oct 12, 2009, the posterior probability of at least 95% was met that OIR to Arm A was superior to Arm B. Thus enrollment in Arm B was terminated on Oct 16, 2009. Arm A was terminated on Dec 2, 2009, based on sponsor review and confirmation of achievement of protocol-defined superiority. The mITT population comprised 65 patients (Arm A, n=43; Arm B, n=22). Overall mean age (SD) was 68 y (13), 69% men; 89% had de novo MDS, and median time since diagnosis was 3.6 months (range, 0, 118). 94% had ECOG performance status (PS) 0–1, 29% had IPSS low-risk classification, and 69% had normal baseline cytogenetics. Arms were balanced apart from having more men in Arm B (P =.01). Patients received a median 7.0 (range, 1, 13) cycles of therapy in Arm A and 5.5 (2, 16) in Arm B. At study end, OIR was 10/43 (23%; 7 CR, 3 HI) and 5/22 (23%; 1 mCR, 1 PR, 3 HI) for Arms A and B, respectively (95% CI of difference: -21.1, 22.1). For transfusion status, of patients who were RBC dependent at baseline, 6/17 (35%) in Arm A and 4/8 (50%) in Arm B became independent on study. Corresponding data for platelets were 3/4 (75%) and 1/4 (25%), respectively. Approximately 40% of patients in each arm who were RBC/platelet dependent at baseline became independent on study. Of patients who were RBC independent at baseline, 24/26 (92%) in Arm A and 11/14 (79%) in Arm B remained independent on study. Corresponding data for platelets were 34/39 (87%) and 17/18 (94%), respectively, and for patients who were RBC/platelet independent, 22/25 (88%) and 9/12 (75%), respectively. IPSS, age, time from MDS diagnosis, type of MDS, prior MDS therapy, baseline cytogenetics, and ECOG PS were similar and did not affect OIR, HI, or transfusion status. There were no cytogenetic responses. At 500 days follow-up, median OS had not been reached (Figure); there were 8 deaths (19%) in Arm A and 6 (27%) in Arm B (hazard ratio 1.5; 95% CI: 0.5, 4.5). Induction of hypomethylation was seen in patients in both arms. All patients experienced ≥1 treatment-emergent AE. The most frequent at least possibly drug-related AEs for Arms A and B, respectively, were neutropenia (28% vs 36%), anemia (23% vs 18%), thrombocytopenia (16% vs 32%), fatigue (19% vs 9%), and leukopenia (9% vs 27%). Drug-related AEs of grade ≥3 were mainly hematologic and reported in 17 patients (40%) in Arm A and 10 patients (46%) in Arm B. There were no deaths within the first 8 weeks on study. One patient (neutropenic sepsis) in Arm A and 2 patients (1 each of anemia and MDS) in Arm B died as a result of an AE; none were reported to be drug related. Conclusions: DAC 20 mg/m2/day SC is active and well tolerated in lower-risk MDS. The OIR was similar in both arms, but a 3-day regimen appears more favorable than a 3x per week regimen based on all efficacy and safety results and the statistical decision to terminate the study early based on Arm A superiority. Further studies with these regimens are warranted. Disclosures: Off Label Use: Dacogen is a nucleoside metabolic inhibitor indicated for treatment of patients with myelodysplastic syndromes (MDS) including previously treated and untreated, de novo and secondary MDS of all French-American-British subtypes (refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess blasts in transformation, and chronic myelomonocytic leukemia) and intermediate-1, intermediate-2, and high-risk International Prognostic Scoring System groups. Borthakur:Eisai: Research Funding. Faderl:Eisai: Research Funding. Stein:Eisai: Employment. Noble:Eisai: Employment. Kassalow:Eisai: Employment. Kantarjian:Eisai: Research Funding.

Abstract Abstract 2656 Poster Board II-632 NPM1 is a nucleolar phosphoprotein that forms oligomers and functions as a molecular chaperone for both proteins and nucleic acids. NPM1 normally shuttles between the nucleus and cytoplasm but is mutated and aberrantly localized to the cytoplasm in 35% of patients with AML, Mutant (m) NPM1 contains a 4-base insert that results in extra C-terminal residues encoding a nuclear export signal (NES), which causes mNPM1 to remain in the cytoplasm. In AML, the presence of mNPM1 is often co-detected with internal tandem duplications of FLT-3, which confers a poor prognosis. The effects of the cytosolic mNPM1 on the biology and drug-sensitivity of AML are unclear. In the present studies we determined the expression and biologic effects of mNPM1 in cultured and primary AML cells. Utilizing RT-PCR, immunoblot analysis and immunoflourescence microscopy (with a polyclonal anti-mNPM1 antibody, we confirmed that in the cultured AML OCI-AML3 cells (hetrozygous for NPM1 mutation) mNPM1 protein was localized in the cytosol, while the un-mutated NPM1 was nuclear. In AML HL-60 cells NPM1 was un-mutated and entirely nuclear. As compared to control siRNA, treatment with siRNA to NPM1 for 72 hours significantly induced p21 expression, decreased % of S phase cells in the cell cycle, as well as induced morphologic differentiation of OCI-AML3 but not of HL-60 cells. This was associated with marked induction of CEBPα in OCI-AML3 cells. NPM1 siRNA also attenuated the expression of HOXA9 and MEIS1 (which are known to be leukemogenic), associated with a marked loss of clonogenic survival of OCI-AML3 cells. NPM1 siRNA mediated depletion of Meis1, which is known to transactivate FLT3 tyrosine kinase in AML progenitor cells, was associated with down regulation of FLT-3 expression in OCI-AML3 cells. Importantly, treatment with NPM1 siRNA significantly sensitized OCI-AML3 more than HL-60 cells to all-trans retinoic acid (ATRA, 0.25 to 2.0 uM) and Ara-C (0.5 to 5.0 uM). Treatment with leptomycin B (5 nM for 24 hours), which is an inhibitor of the NES receptor CRM1/exportin-1, shifted the localization of mNPM1 from the cytosol to the nucleus, abrogated the levels of Meis1 and HoxA9 and induced apoptosis of OCI-AML3 but not HL-60 cells. Recently, treatment with NSC348884, a small molecule inhibitor of NPM1 oligomerzation, was shown to induce apoptosis of colon and prostate cancer cells. Our studies determined that NSC348884 (3.0 uM) was highly active in disrupting the oligomerization of mNPM1 and induce apoptosis of 90 % of OCI-AML but only 20% of HL-60 cells. Additionally, treatment with 5.0 uM of NSC34884 induced apoptosis of approximately 75% of primary AML cells with mNPM1, without inducing apoptosis of normal CD34+ progenitor cells. Collectively, these findings suggest that strategies targeting mNPM1 levels or oligomerization, or reversing the aberrant cytosolic localization of mNPM1, would induce relatively selective differentiation and apoptosis, as well as sensitize AML cells with mNPM1 to Ara-C and ATRA. Disclosures: No relevant conflicts of interest to declare.

Seawater electrolysis is expected to play an important role in the circular economy by reducing the hydrogen production cost due to the simplified process lines[1] and by saving scarce sources of safe and fresh water.[2] Seawater impurities especially chloride ions can cause the corrosion of the catalyst and be oxidized at high potential, competing with the oxygen evolution reaction (OER). Produced chlorine and hypochlorite are toxic to humans and the environment,[3] thus selective OER is desirable. Furthermore, impurity cations, such as Ca and Mg, may form hydroxide precipitates in a highly alkaline condition which is commonly used in the commercial electrolyzer. Therefore, selective OER in non-extreme pH (pH < 10) is highly desired, which requires additional challenges on the electrocatalyst and the electrolyte. In this study, we have effectively utilized the thermodynamic potential gap between chloride oxidation reaction (COR) and OER, and engineering of non-noble metal hydroxide electrocatalysts and buffer electrolyte successfully achieved the selective OER in the presence of chloride ion at commercially relevant operation conditions in non-extreme pH within the thermodynamic window. Firstly, the stability of non-noble metal materials in 1.0 mol kg− 1 potassium borate (K-borate, pH 9.2) buffer solution with 0.5 mol kg− 1 KCl was investigated. Ni foam and Ni hydroxide, which are commonly used in water splitting at extreme pH levels, were unstable, and Co, Fe, and Ti-based materials were found to be the candidates for seawater splitting. Among various combinations of these elements, the CoFeOxHy/TF (Ti felt) electrode showed better performance than CoOxHy/TF and FeOxHy/TF electrodes. The CoFeOxHy electrode possessed a three-hold larger double layer capacitance (C dl) which is the electrochemically active surface area than its counterparts. Our kinetic study revealed that Tafel slopes and apparent activation energies of CoFeOxHy resembled the control CoOxHy and FeOxHy electrodes, indicating that the performance difference was originated from the enlarged surface area caused by mixed Co and Fe. In addition to the development of electrocatalysts, electrolyte engineering plays a key role. By employing the CoFeOxHy/TF electrode, the OER performance was investigated in varied molality and counter cation borate electrolytes at pH 9.2. The molarity of K-borate above 1.0 mol kg− 1 was found to be essential to minimize the concentration overpotential, which helps to maintain the electrode potential below the thermodynamic window of COR. The OER performance was insensitive to cation identity except for the Na cation counterpart having a low solubility product. Furthermore, Cl−-containing electrolyte had a higher conductivity than that without Cl−, which can be an advantage to decrease the ohmic resistance in two electrode configurations for future application. The influence of the potential window on the stability and selectivity was investigated by using the developed electrode and 1.0 mol kg−1 K-borate (pH 9.2) with 0.5 mol kg−1 KCl. Below the redox potential of ClO− formation at 1.72 VRHE (V vs. Reversible hydrogen electrode), current density-potential relationships were insensitive to the Cl−. At 1.72 VRHE in the presence of Cl−, the faradaic efficiency (FE) of O2 (FEO2) reached 98±1%, whereas the FE of hypochlorite (FEHC) was merely 1±1%. Above this threshold potential, under chronopotentiometry (CP) testing at 50 mA cm−2 corresponding to approximately 1.82 VRHE, FEO2 decreased to 93±2% and FEHC increased to 7±2%. Critically, the potential of 1.82 VRHE was larger than 1.78 VRHE observed without Cl−, likely due to the blockage of the OER active site by Cl− or related species. Finally, aiming for the practical application, the OER performance over CoFeOxHy was examined at 353 K. The FEO2 reached 99±2% at 500 mA cm− 2 and 1.67 VRHE (Figure 1). Our calculations on thermodynamics revealed that the redox potential of OER decreases to 1.18 VRHE while that of ClO− formation reaction is 1.71 VRHE at 353 K, leaving a potential gap of 530 mV. High selectivity toward the OER was attributed to the consequence of fine engineering of electrocatalysts and electrolytes that successfully maintained the potential below the 1.71 VRHE where the OER is thermodynamically dominant. In addition, the electrode was stable for multiple on-off accelerated stability testing for 10 h at 500 mA cm− 2 and 353 K. Reference [1] S. Dresp, F. Dionigi, M. Klingenhof, P. Strasser, ACS Energy Lett. 2019, 4, 933–942. [2] C. J. Vörösmarty, P. B. McIntyre, M. O. Gessner, D. Dudgeon, A. Prusevich, P. Green, S. Glidden, S. E. Bunn, C. A. Sullivan, C. R. Liermann, Nature 2010, 467, 555–561. [3] J. G. Vos, M. T. M. Koper, J. Electroanal. Chem. 2018, 819, 260–268. Figure 1

Quantum Cascade Lasers (QCLs) [1] are important sources for coherent mid-wave infrared (MWIR) light that find widespread use in applications such as molecular spectroscopy, free-space optical communications, and defense countermeasures. These applications demand tunability of emission wavelength and high output powers. The conventional QCL is a two-terminal n-i-n device in which an externally applied bias establishes the necessary electric field in the cascade region for proper alignment of the quantum states, defines the emission wavelength, and also determines the current through the cascade structure. Because QCLs are 2-terminal devices, changing bias directly changes both wavefunction overlap and energy separation. The spectral location and magnitude of the gain peak thus has a strong dependence on applied voltage, impacting the capability to modulate. To overcome these limitations, the transistor-injected quantum-cascade laser (TI-QCL) has been proposed [2]. The TI-QCL is a three-terminal device in which the cascade region is placed within a heterojunction bipolar transistor. In forward-active mode the current injection is controlled by a forward-biased emitter-base junction, and the reverse bias on the base-collector junction is used to control the electric field for alignment of the quantum states. This novel structure enables the decoupling of emission wavelength and output power. This extra control positions the TI-QCL to impact a wide range of applications such as spectroscopy with its wide tunability, and free-space communication with its capability to separately modulate power and frequency. Previously, transistor characteristics and near-infrared (NIR) band-to-band base emission was demonstrated in a GaAs design [3, 4] before moving to an InP-based design [5]. We will review the progress made to push the TI-QCL concept towards full functionality, focusing on InP-based epitaxial designs. A self-consistent Schrodinger-Poisson solver and non-equilibrium Green’s function method is used to aid in the active-region design of the structure, which is optimized for a peak emission wavelength of 8.27 μm as shown in Figure 1 [6]. A wide wavelength tunability from approximately 7.8 μm to 8.8 μm is projected based on computational analysis. The InP-based structure is grown using molecular beam epitaxy by IntelliEpi; a cross-section of the material is depicted in Figure 2. It contains an active-region composed of 30 repeated periods of In0.41Ga0.59As/In0.36Al0.64As strain-balanced superlattice. The devices are fabricated using standard III-V processing. An image of a fabricated TI-QCL bar consisting of a sloped, 20 μm wide emitter mesa and evaporated SiO2 as the passivation layer is depicted in Figure 3. The fabricated devices are diced and cleaved into 2 to 4 mm x 500 μm die and mounted onto In-plated copper mounts followed by wire bonding. The devices are electrically characterized at room temperature, 77 K, and 18 K. Under cryogenic temperatures, the common-base characteristics exhibit negative differential resistance (NDR) in collector current at all emitter injection currents as shown in Figure 4, which signifies tunneling transport through the quantum cascade superlattice region. The base current also exhibits corresponding NDR behavior and switching in the band-to-band NIR light (1.57 m) output from the In0.53Ga0.47As base is experimentally observed when biased in a region where transport through the cascade region is rejected due to quantum state misalignment. References: [1] J. Faist, et al., "Quantum cascade laser," Science, vol. 264, no. 5158, pp. 553-556, 1994. [2] K. Chen and J. Dallesasse, "Design and modeling of mid-infrared transistor-injected quantum cascade lasers," Proceedings of CS MANTECH 2014, pp. 75-78, 2014. [3] J. M. Dallesasse, et al., “Progress on the transistor-injected quantum-cascade laser,” in Quantum Sensing and Nano Electronics and Photonics XV, 2018, vol. 10540, p. 105401P. [4] K. Chen, et al., “Control of radiative base recombination in the quantum cascade light-emitting transistor using quantum state overlap,” Applied Physics B, vol. 124, no. 7, pp. 1–8, 2018. [5] R. B. Kaufman, et al., “Design and fabrication considerations for transistor-injected quantum cascade lasers for compact, efficient, and controllable mid-wave infrared lasing,” Proceedings of CS MANTECH 2020, pp. 275–278, 2020. [6] Z. Lin, et al., "Modeling of the electrically-tunable transistor-injected quantum cascade laser," Journal of Applied Physics, vol. 122, no. 23, p. 235701, 2017. Figure 1

Abstract Abstract 1665 Poster Board I-691 Mantle cell lymphoma (MCL) is an aggressive, incurable B-cell malignancy that represents approximately 6% of lymphoma cases. Patients with MCL who relapse after conventional therapy or stem cell transplantation have a poor prognosis and are candidates for treatment with novel agents. The characteristic feature of MCL is overexpression of cyclin D1. Cyclin D1 is under the control of the phosphatidylinositol-3 kinase signal-transduction pathway and is downstream of the mammalian target of rapamycin kinase (mTOR). We have previously shown that temsirolimus (CCI-779), a dihydroester of the selective mTOR inhibitor rapamycin, is an active antitumor agent in MCL. In previous phase 2 trials of temsirolimus as a single agent, we observed a 40% overall response rate (ORR) with a median duration of response (DR) of 6 months in patients with relapsed or refractory disease (J Clin Oncol 23:5347-56, 2005; Cancer 113:508-514, 2008). Due to the fact that rituximab has improved the response rate and overall outcome of lymphoma patients when combined with chemotherapy, we tested the efficacy and toxicity of temsirolimus in combination with rituximab in patients with MCL. Eligible patients with confirmed relapsed or refractory MCL based on morphologic and phenotypic features received temsirolimus 25 mg intravenously every week and four weekly doses of rituximab with the first cycle and then one dose of rituximab every other cycle. Patients with a tumor response after six cycles were eligible to continue treatment for a total of 12 cycles or two cycles after complete remission, and were then observed without maintenance. Seventy-one patients were enrolled between May 2005 and March 2009. The median age was 67 years (range, 44 to 86 years), with 51 males and 20 females enrolled. Patients had received a median of two prior therapies (range, 1-9), 31% had received a prior stem cell transplant and 28% were rituximab refractory. The ORR was 48% (34/71 patients) with 20% (14/71) complete responses and 28% (20/71) partial responses. The median DR was 9.5 months (95% CI, 4.5 to 11.3 months). The median DR in the rituximab sensitive patients was 9.5 months (CI: 4.5-11.6) and 7.15 months (CI: 4.1-11.3) in the rituximab refractory patients (p= 0.73). While the combination was generally well tolerated, 12 patients experienced grade 4 toxicity and 2 patients died while on therapy – both from progressive disease. Hematologic toxicities were the most common, with 5 patients having grade 4 thrombocytopenia and 3 having grade 4 neutropenia. This study demonstrates that temsirolimus in combination with rituximab is well tolerated and has substantial antitumor activity in relapsed MCL. Further comparative studies of this combination in MCL are therefore warranted. Disclosures Witzig: Wyeth: Patents & Royalties.

Protonic ceramic electrolysis cells (PCECs) have been attracting attention due to their ability to operate in the intermediate temperature (300 - 500 ˚C) sweet-spot. These temperatures enable fast kinetics, inexpensive materials (no precious metals; stainless interconnects), and lower operating voltage for producing green hydrogen, without the accelerated degradation that may be observed at higher temperatures. The steam electrodes often support triple conduction and thereby host the proton exchange / oxygen evolution process at the surface, where steam is split. Steam electrode materials development is at a relatively early stage, with open questions around the relationships among bulk composition, evolving surface chemistry, kinetic quantities, and degradation mechanisms. We are investigating the proton exchange surface coefficient (k) and its evolution over time on model thin film electrodes in order to ultimately develop highly active and long-term stable steam electrodes for PCECs. ABO3 perovskites with Ba on the A-site and nominally multivalent ions on the B-site are candidates for steam electrodes given the varied compositions in this class that can transport oxide ions, protons, and electronic species, and the lowering of hydration enthalpies by the basicity introduced by Ba. However, very few measurements of k exist for these materials, and the evolution of surface chemistry and thus k is poorly understood. In this work, we fabricated and compared two perovskite triple conducting oxides by growing geometrically well-defined films of Ba(Pr,Y)O3-x (BPY) and Ba(Co,Fe,Zr,Y)O3-x (BCFZY) with pulsed laser deposition. We examined the proton surface exchange coefficients (k-values) using simultaneous in-situ optical transmission relaxation and electrical conductivity relaxation measurements upon switching pH2O at a temperature of 400 ˚C, and continued these switches as a function of time to assess stability. To our knowledge, this is the first use of an optical transmission method to probe elevated temperature steam splitting kinetics, and we interpret the optical (and electrical) changes as indicative of redox during the hydrogenation process. The k-values of BPY thin films were approximately ten times higher than those of the BCFZY thin films, and BPY also retained higher k-values over time. The bulk and surface chemical compositions of BPY and BCFZY thin films before and after relaxation measurements were compared using Rutherford backscattering spectrometry, angle-resolved X-ray photoelectron spectroscopy, and scanning transmission electron microscopy with spectroscopy. Pronounced Ba enrichment and Si contamination (from the quartz tube) were evident at the surface of BPY by both S/TEM-EDS and XPS, whereas for BCFZY these surface chemical features were only detectable by XPS and absent in S/TEM-EDS. Implications for materials design of steam electrodes will be discussed, considering efficiency and lifetime.

BackgroundHospitalizations due to relapse or disease complications are major concerns during follow-up of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV).ObjectivesTo determine rates of hospitalization in a large cohort of patients with AAV compared with the national general population, and to describe features and associated primary discharge diagnoses.MethodsBetween 2007 and 2018, we examined the hospitalization records of AAV patients from 13 Italian hospitals. Hospitalization dates, features, length of stay, primary discharge diagnoses and patient data were abstracted from charts. Age- and sex-standardized hospitalization rates (SHR) were calculated by an indirect method, per year and for the study period, using the 2007–2018 hospitalization data provided by the Italian Ministry of Health. Multivariable and survival models were used to explore associations between these outcomes, clinical parameters at diagnosis, and pre-existing comorbidities.ResultsA total of 610 hospitalizations occurred in 635 patients with AAV (19.4% microscopic polyangiitis, MPA; 34.6% granulomatosis with polyangiitis, GPA; 46.0% eosinophilic GPA, EGPA) during a 12-year observation; in 19.8% for life-threatening conditions and leading to death in 2.3%. The median time to first hospitalization was 504 days (25-75%IQR, 95-1497), and the median hospitalization length was 8 days (25-75%IQR, 8-14).The 2018 SHR (95%CI) was 1.14 (0.91, 1.43) for all AAV combined, 1.13 (0.68, 1.76) for MPA, 1.48 (1.02, 2.08) for GPA, and 0.90 (0.60, 1.31) for EGPA. These rates tended to a gradual increase from 2007 to 2018 in the whole AAV cohort of patients and in every disease subset (Figure 1A).The main causes of hospitalization in patients with AAV were infectious diseases (18.7%), followed by major relapse and diagnostic re-evaluation (17.2% each), and cardiovascular diseases (10.8%). Among those due to infections, the main site was the respiratory system (44.6%), followed by urinary tract (9.6%) and sepsis (6.3%).Among AAV patients hospitalized during follow-up (47.1%), 55.5% had only 1 hospitalization, 18.7% had 2, and 25.6% had 3 or more hospitalizations. Patients with a diagnosis of GPA or MPA (versus EGPA), higher vasculitis activity (assessed by BVAS), ANCA positivity at diagnosis, and hospitalization at diagnosis (all p<0.001), more pre-existing comorbidities and older age (both p<0.05), were more likely to be hospitalized during follow-up (Figure 1B).ConclusionPatients with AAV have a significant burden of hospitalization during the disease course. Approximately half of the patients is hospitalized during follow-up, with infections, relapses and cardiovascular diseases as the main causes of hospitalizations. Our findings showed the existence of risk profiles of patients more likely to be hospitalized, requiring more active vigilance.References[1]Wallace, Z. et al. ‘Nationwide Trends in Hospitalizations and In-Hospital Mortality of Granulomatosis with Polyangiitis’, Arthritis Care Res. 2016[2]Mohammad, AJ et al. Severe Infection in Antineutrophil Cytoplasmic Antibody-associated Vasculitis. The Journal of Rheumatology, 2017Figure 1.Age- and sex-SHR by year for patients with AAV, MPA, GPA and EGPA during 2007-2018 (A). Kaplan-Meier Plots of the probability of hospitalization after AAV diagnosis (B).Acknowledgements:NIL.Disclosure of InterestsAlvise Berti Speakers bureau: GSK, Marta Ottone: None declared, Silvia Sartorelli Employee of: S. Sartorelli worked at the IRCCS San Raffaele Scientific Institute and San Raffaele University at the time of the study and is now an employee of Bristol Myers Squibb., Elena Treppo: None declared, Alessandra Bettiol: None declared, Roberto Padoan: None declared, Francesca Regola: None declared, Sara Monti: None declared, Chiara Marvisi: None declared, Alessandro Giollo: None declared, Lorenza Maria Argolini: None declared, Matteo Righini: None declared, angelica gattamelata: None declared, Giulia Cassone: None declared, Laura Sottini: None declared, Matteo Maule: None declared, Paola Toniati: None declared, Bianca Lucia Palermo: None declared, Federica Bello: None declared, Silvia Guella: None declared, raffaella izzo: None declared, Francesco Muratore: None declared, Maria Grazia Catanoso: None declared, Angelo Fassio: None declared, Pierluigi Cataleta: None declared, Andrea Buscaroli: None declared, Paolo Giorgi Rossi: None declared, Franco Franceschini: None declared, Roberto Caporali Speakers bureau: AbbVie, Amgen, BMS, Celltrion, Fresenius, Galapagos, Janssen, Lilly, Novartis, Pfizer, and UCB, Consultant of: AbbVie, Fresenius, Galapagos, Lilly, Novartis, Pfizer, and UCB, Carlomaurizio Montecucco: None declared, Fabrizio Conti: None declared, Giacomo Emmi: None declared, Luca Quartuccio: None declared, Giuseppe Paolazzi: None declared, Lorenzo Dagna: None declared, Franco Schiavon: None declared, Carlo Salvarani: None declared, Roberto Bortolotti: None declared.

Abstract Background: Imexon (AOP 99.0001 (4-imino-1,3-diazobicyclo-(3, 1, 0)-hexan-2-one) exerts antiproliferative activity on a range of tumor cells including myeloma cells and was found to inhibit tumor development in murine models of B-cell lymphoma. The antitumor activity of Imexon is caused by a sequence of molecular events starting with alkylation of thiol groups, reduction of the intracellular antioxidative defence mechanism, increase of reactive oxygen species (ROS) in mitochondria, and mitochondrial damage and induction of apoptosis. Methods/Patients: 36 patients with relapsed or refractory myeloma who had been pretreated with at least two lines of prior therapy were included. Imexon was applied as a 15-min infusion on 5 consecutive days for 2 weeks (d1–5 and d8–12) with a rest period of one week (1 cycle). Escalation of the dose of Imexon was started with 50 mg/m2 during the phase I part of the study. Results: The plasma half life of the parent compound and its active metabolite Imexon was found to be approximately 1.2 hrs and 2.6 hrs, respectively. The mean duration of treatment with Imexon was 6.8 weeks in a dose range between 50 and 1000 mg/m2 without reaching dose limiting toxicity. Drug related grade 1–2 AEs occurring with a frequency of &gt;10 % were fatigue, nausea, constipation, headache, anorexia, muscle/bone pain, anemia, thrombocytopenia, leucopenia and transient elevation of GGT Grade 3–4 AEs related to study drug were leucopenia (n=3), anemia (n=2), thrombocytopenia (n=4), osteonecrosis (n=1), creatinine increase (n=1). In two patients dose reductions due to AEs were required. A total of 7 SAEs occured in 4 patients. All SAEs occurred at the 400 mg/m2 DL 1 SAE (increase in creatinine) was considered to be related to study drug. There was no correlation between the dose of the study drug and AEs. In addition, no mortality was encountered while patients were on treatment with Imexon. Imexon treatment resulted in a minimal response in one patient at the DL of 500 mg/m2. In addition, a significant improvement of the preexisting distal polyneuropathy was noted in this patient during these first 3 therapy cycles. After discontinuation of Imexon, reappearance of the polyneuropathy was noted within 4 months. This led to retreatment of the patient at a dose level of 600 mg/m2, which resulted in a gradual and finally complete resolution of the polyneuropathy with maintenance of the MR, but without further reduction in the mIg. No other objective response was observed in this study. Conclusion: Overall, Imexon was safe and well tolerated in the dose range investigated. Imexon demonstrated only minor clinical activity in one patient. These results do not support the further development of Imexon as single agent in multiple myeloma. Due to its unique mechanism of action and its favourable toxicity profile it may, however, qualify for use and evaluation in combination with other agents in multiple myeloma and other malignancies.

Abstract Introduction: Vyxeos® is a liposomal formulation employing a 1:5 molar ratio of daunorubicin:cytarabine. Clinical trials in high risk acute myeloid leukemias demonstrated a significant benefit in CR rates and median OS, culminating in FDA approval August 2017. Emerging pediatric data suggest this benefit may extend to acute lymphoblastic leukemia (ALL). The following pilot trial was performed to better understand the activity and toxicity profile in adults with relapsed and refractory ALL. Methods: Adults with ALL or mixed phenotype leukemia were eligible if &gt;5% lymphoblasts and/or extramedullary disease &gt;1x1cm. Induction consisted of Vyxeos 100 units/m2 on days 1, 3 and 5. Those with clinical benefit could receive up to 3 cycles of consolidation delivered at 65 units/m2 on days 1 & 3 after recovery of neutrophils (&gt;500 cells/uL) and platelets (&gt;50,000 cells/uL). Response: 11 patients (pts) have been treated to date, with median age of 39y (22-74y), 9 male, and 3 Caucasian. Six pts had B-ALL, including 1 B-myeloid. Four of 5 T-ALL pts had early T-cell precursor (ETP) phenotype. NGS was available in 9 pts, and included TP53 mutation (n=4), and PH-like changes (n=2). Median prior lines of therapy was 3, with 7 pts showing primary refractory disease. Prior blinatumomab, inotuzumab, CAR-T cell therapy, and allogeneic transplant were noted in 5, 2, 1, and 3 pts. Pancytopenia was uniform during induction, with febrile neutropenia noted in 9 pts. One pt passed from pneumonia after moving to comfort measures in lieu of intubation 20 days after Vyxeos, and is non-evaluable for response. A second pt developed grade 3 sepsis. The remainder of infections were grade 1-2. One pt had grade 3 gastrointestinal bleed, and 3 pts had grade 1 spontaneous subdural bleeding. One pt developed recurrent pericarditis in setting of anterior mediastinal mass. One case of veno-occlusive disease was observed in a pt with prior allogeneic transplant and inotuzumab. Median time to ANC recovery was 33.5 days among 10 evaluable pts. Two pts with refractory disease failed to recover platelets; among the remaining pts, median time to platelet recovery was 30.5 days. Adverse events were uncommon during consolidation, and include foot cellulitis and myopericarditis, each in 1 pt. Among 10 evaluable pts, 3 achieved CR/CRi, including 2 ETP T-ALL and one B-ALL. Two pts with TP53 mutation demonstrated &gt;50% blast reduction with hematologic recovery, allowing for prolonged time to subsequent therapy. Four pts received 1-3 cycles consolidation. One pt was bridged to donor leukocyte infusion. Responses were noted in only 2 pts after progression following Vyxeos, highlighting refractory status of those enrolled. Median PFS was 57 days (95%CI: 10, 105), time to next therapy 76 days (95%CI: 47, 105), and OS 223 days (95%CI: 144, 302). Conclusions Vyxeos in high-risk refractory adult ALL was overall well tolerated and active, with median OS of approximately 7.5 months in this pilot trial. Confirmation of benefit in a larger study is warranted, incorporating a second induction course and/or the addition of novel agents to further improve on remission rate and duration of response. Disclosures Shah: Incyte: Research Funding; Jazz Pharmaceuticals: Research Funding; Servier Genetics: Other; BeiGene: Consultancy, Honoraria; Acrotech/Spectrum: Honoraria; Pharmacyclics/Janssen: Honoraria, Other: Expenses; Kite, a Gilead Company: Consultancy, Honoraria, Other: Expenses, Research Funding; Precision Biosciences: Consultancy; Amgen: Consultancy; Novartis: Consultancy, Other: Expenses; Pfizer: Consultancy, Other: Expenses; Bristol-Myers Squibb/Celgene: Consultancy, Other: Expenses; Adaptive Biotechnologies: Consultancy. Chavez: Astra Zeneca: Research Funding; Novartis: Consultancy; Merck: Research Funding; Morphosys: Speakers Bureau; Adaptive Biotech: Research Funding; ADC Therapeutics: Consultancy, Research Funding; Beigene: Speakers Bureau; Kite/Gilead: Consultancy; karyopharm: Consultancy; Epizyme: Speakers Bureau; Abbvie: Consultancy. Sokol: Dren Bio: Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin: Membership on an entity's Board of Directors or advisory committees. Lancet: Celgene/BMS: Consultancy; Millenium Pharma/Takeda: Consultancy; Daiichi Sankyo: Consultancy; AbbVie: Consultancy; BerGenBio: Consultancy; ElevateBio Management: Consultancy; Agios: Consultancy; Astellas: Consultancy; Jazz: Consultancy. OffLabel Disclosure: Vyxeos is being evaluated in the described trial for the treatment of relapsed or refractory acute lymphoblastic leukemia in adults.

The microbial diversity of atmospheric bioaerosols involves microorganisms that can cause allergic and infectious diseases or toxic effects. They include bacteria of the Bacillus cereus group (B. cereus, B. thuringiensis, B. anthracis, B. mycoides, B. pseudomycoides, etc.), which can result in diarrhea, pneumonia, meningitis, septicemia, and other infectious diseases. Accordingly, monitoring the presence of Bacillus cereus group bacteria in aerosols is critical. However, practically no data exist on Bacillus cereus and other cereus-group bacteria in southwestern Siberia’s poorly investigated atmospheric aerosol environment. Bacteria of the cereus group are capable of effective production of various biologically active compounds, with important implications for biotechnology; microorganism strains with new capabilities are being investigated. This study aimed to determine the occurrence and characteristics of B. cereus group bacteria in ground-level and high-altitude atmospheric aerosols in Novosibirsk region of southwestern Siberia, and to evaluate the biotechnological potential of the obtained microbial isolates. High-altitude atmospheric samples were collected over Karakan Pine Forest, approximately 50 km south of Novosibirsk, at altitudes of 7000, 5500, 4000, 2000, 1500, 1000, and 500 m, by aircraft sounding. Вoundaries of the aircraft flight path: 54° 26'38'' N, 82° 30'47'' E; 54°10'55'' N, 81° 44'00'' E. Ground-level samples were collected at various sites in Koltsovo settlement, Novosibirsk region. Impingers with a flow rate of 50 L/min containing 50 ml of Hanks’ solution were used for air sampling. The obtained aerosol samples were sown on a set of nutrient media and incubated at 28–30°C and 6–10 °C. The titers of microorganisms in high-altitude and ground-level samples were determined in terms of 1 m3 of atmospheric air. Standard microbiological methods were employed to study the phenotypic characteristics of the identified microbial isolates. Lipolytic activity was determined on yolk agar and LB agarized medium containing fatty acid esters with 0.01% CaCl2 . The substrates used were 1.0% monolaurate (tween-20) and monooleate (tween-80). Amylolytic activity of the cultures was determined by their isolation on starch-ammonia agar, and proteolytic activity by their ability to hydrolyse milk gelatin and casein (Maniatis T et al., 1984). The ability to hemolysis was taken into account when cultures were plated on LB medium with the addition of ram’s blood. Nuclease activity was studied on LB medium with the addition of Sigma DNA (USA) (Maniatis T et al., 1984). The content of plasmid DNA in the isolates was determined by screening according to Maniatis T et al. (1984). The capacity for RNAase secretion in culture medium (peptone - 9.27 g/l, yeast extract - 5 g/l, NaCl - 3.00; 10 ml of 50% glycerin, 2 ml of 20% glucose; pH 7.07.2) during cultivation of bacteria at 30 °С, for 18-24 h, was determined by the accumulation of acid-soluble products, formed upon hydrolysis of high-polymer RNA of yeast (1 mg/ml). Antibiotic activity of the studied strains was determined by cross-strics (Yasuda T et al., 1992) on LB medium at 37 °C. The following pathogenic test strains were used: Staphylococcus aureus ATCC 6538, Bacillus subtilis ATCC 6633, Candida albicans 620, Klebsiella pneumoniae B-4894, Escherichia coli ATCC 25922, Salmonella typhimurium 2606, and Shigella sonnei 32 (collection of FSBI State Research Centre Vektor of the Rospotrebnadzor). The genetic analysis of bacterial isolates was performed using PCR with specific primers on 16S rRNA. The calculation of the number of cultivated microorganisms in the samples was carried out according to the Kerber method [Bottone EJ, 2010], and the number of microorganisms was averaged over three parallels of the inoculated samples. The annual average numbers of cultivated microorganisms were calculated as a mean ± the confidence interval at a significance level of 95% of t-Student’s (p < 0.05). Percentages of spore-forming cultured bacteria in aerosol samples varied significantly across the years of observation (1998–present): in high-altitude samples, the minimum and maximum were 0.5% (in 2005) and 55% (in 2011), respectively, and, in ground-level samples, the minimum and maximum were 0.1% (in 2002) and 83% (in 2016), respectively (See Fig. 1 and 2). Annual averages of total concentration of microorganisms ranged from < 1 to 5×105 CFU/m3 . The number of cereus-group bacteria also varied significantly from sample to sample, with averages ranging from 0.01% to 6.5% of the total number of isolated microorganisms. A total of 2.025 bacterial isolates, of which 62 formed endospores, were isolated from ground-level and highaltitude aerosol samples collected during the predominance of south-westerly winds from Kazakhstan in autumn 2016, and were characterized by increased dust-component content. Spore-forming bacteria were identified as belonging to the genera Bacillus, Paenibacillus, Brevibacillus, Lysinibacillus, and some others. Both high-altitude and ground-level aerosol samples were shown to contain bacteria of the cereus group: Bacillus cereus (Bc) and Bacillus thuringiensis (Bt), Bt ssp. kurstaki, Bt ssp. galleriae subspecies; Bt strains with indefinite serotype were also found. Notably, Bacillus anthracis species were not found (See Table 1). Screening for enzyme secretion revealed Bt and Bc strains with pronounced proteolytic, phosphatase, lipolytic, and amylolytic activities in a medium pH range from 5.0 to 9.0 (See Table 2). An atypical strain of B. thuringiensis Cb-527, which demonstrates high production of RNase, was isolated. All strains demonstrated hemolysis capability, were multi-resistant to antibiotics (resistant to 6-15 drugs (See Table 3), and suppressed the growth of the pathogenic yeast, Candida albicans, to varying degrees. The Bt Cb-527 strain, as well as several Bc strains, also effectively inhibited the growth of Gram-positive test strains of Staphylococcus aureus and Bacillus subtilis. Gram-negative bacterial test strains were low-sensitive to the action of metabolites of the studied Bc and Bt strains (See Table 4). In high-altitude and ground-level samples of the studied atmospheric aerosols, bacteria of the cereus group belonging to Bacillus cereus and Bacillus thuringiensis species were found in amounts ranging from 0.01 to 6.5% of the total number of cultured microorganisms isolated under experimental conditions. The presence of aggression enzymes such as phospholipases, hemolysins, proteases, and nucleolytic enzymes typical of representatives of these taxa, was found. We isolated Bc and Bt strains with high levels of secretion of enzymes and metabolites that possess antibiotic activity; these strains are promising as producers. The Bacillus thuringiensis Cb-527 strain (with a pronounced secretion of the RNase complex) can be used for the development of anti-RNA-containing virus drugs. The isolated Bc and Bt strains demonstrated multiple antibiotic resistance, which confirms literature data on the increasing prevalence of polyresistance among the identified natural microbial isolates. The paper contains 4 Figures, 4 Tables, and 41 References.

With the increased demand on electric vehicles, portable devices and energy storage systems, a great attention is being drawn to improve the energy density of lithium-ion batteries (LiBs) [1]. The commercial LiBs anodes are usually consisted of graphite-based material, which limits the energy density due to its relatively low theoretical capacities. Silicon (Si) has been recognized as an alternative anode material with its high theoretical capacity, natural abundance, and low discharge potential. Despite the above-mentioned advantages, Si undergoes a substantial volume expansion (300% approximately) during the battery operation [2]. There are some approaches to mitigate the adverse of expansion, whereas the cost and scalability of these techniques are the bottlenecks to solve this issue [3]. In this work, we focus on maintaining the structural integrity, along with the coordination of Li as functional binder which could be an effective solution to the abovementioned challenges. In addition to the energy density and capacity targets, considering more environmentally friendly approaches is also one of the key topics for battery manufacturing. Aqueous processing of the anodes is already state-of-the-art whilst, the cathode has its own challenges to be overcome. Ni-rich transition oxide active materials, such as LiNi0.8MN0.1Co0.1 (NMC811), are attractive for high-capacity target applications. Due to the high reactive surface of the active material, during the slurry processing, NMC811 reacts with water, which leads to Li+/H+ exchange [4]. Thus, the slurry results in high pH which can corrode the aluminum foil current collector and cause poor capacity due to Li loss. The advantage of using polyacrylic acid (PAA) is to employ COOH, in which the leached Li+ can bond to the backbone. Hence, the pH of slurry is maintained on desirable coating range and the capacity loss can be reduced due to the proposed aqueous processing. In this study, we explore the utilization of a well-known polymer, polyacrylic acid, as binder for both negative and positive electrodes. On the anode, PAA is functionalized with Li+ and used LiPAA prior to electrode production. It is highly crucial to observe how the dynamics of Si and LiPAA impacts the anode performance during the cycling. To comprehend this, post mortem, scanning electron microscopy (SEM) was performed to detect the positioning and concentration of Si on pristine and cycled anodes. On the cathode side, the focus is to utilize the leached Li+ during the slurry processing by having PAA as binder. This technique has been already successfully implemented by our group on the research pilot scale [5]. The idea is to make pH stable slurry without using any additional acid, in conjunction with creating in situ functional binder (LiPAA). Deep understanding of functional binders is to serve as a guide for the rational design of Si-electrode and aqueous NMC811 cathodes for LiBs. References [1] T. Kim, W. Song, D. Y. Son, L. K. Ono, and Y. Qi, “Lithium-ion batteries: outlook on present, future, and hybridized technologies,” Journal of Materials Chemistry A, vol. 7, no. 7. Royal Society of Chemistry, pp. 2942–2964, 2019. doi: 10.1039/C8TA10513H. [2] P. Li, H. Kim, S. T. Myung, and Y. K. Sun, “Diverting Exploration of Silicon Anode into Practical Way: A Review Focused on Silicon-Graphite Composite for Lithium Ion Batteries,” Energy Storage Materials, vol. 35. Elsevier B.V., pp. 550–576, Mar. 01, 2021. doi: 10.1016/j.ensm.2020.11.028. [3] X. Zhao and V.-P. Lehto, “Challenges and prospects of nanosized silicon anodes in lithium-ion batteries,” Nanotechnology, vol. 32, no. 4, p. 042002, Jan. 2021, doi: 10.1088/1361-6528/abb850. [4] M. Wood et al., “Chemical stability and long-term cell performance of low-cobalt, Ni-Rich cathodes prepared by aqueous processing for high-energy Li-Ion batteries,” Energy Storage Mater, vol. 24, pp. 188–197, Jan. 2020, doi: 10.1016/j.ensm.2019.08.020. [5] B. Boz, M. Vuksanovic, L. Neidhart, M. Höchtl, K. Fröhlich, and M. Jahn, “Aqueous Manufacturing of Ni-Rich Cathodes Using Polyacrylic Acid As Binder for Lithium-Ion Batteries,” ECS Meeting Abstracts, vol. MA2022-02, no. 7, pp. 2542–2542, Oct. 2022, doi: 10.1149/MA2022-0272542mtgabs. Acknowledgment This project has received funding from European Union’s Horizon EUROPE Research and Innovation Programme under Grant Agreement N° 101069612 (NoVOC).

Abstract CD19-specific CAR-T cells are highly successful against B-cell non-Hodgkin lymphomas and acute lymphoblastic leukemia but targets for other lymphoproliferative disorders have been harder to define. Almost all HL and some NHL express the CD30 antigen both at diagnosis and relapse, and monoclonal antibodies (mAb) targeting CD30 (e.g. brentuximab) produce objective antitumor responses. However, mAb have limited bio-distribution and their benefits may be short-lived. We therefore expressed the antigen binding domain of a CD30 mAb as part of a chimeric antigen receptor (CAR) on T cells, coupled to the CD28 and z chain endodomains. We have previously published results of a phase 1 study of activated autologous CD30.CAR-T cells (CD30.CARTs) infused in patients with relapsed/refractory CD30+ HL or NHL without preceding chemotherapy (Ramos et al., J Clin Invest 2017). Of 6 patients with relapsed active HL, 1 entered complete remission (CR) that has lasted more than 3 years, and 3 had transient stable disease. No significant toxicities were observed. In order to boost the in vivo expansion and potentially the efficacy of these CD30.CARTs we are now infusing them after lymphodepleting chemotherapy (RELY-30, NCT02917083). We report here preliminary results of that study, which suggest a substantial improvement in efficacy. We have manufactured CD30.CARTs for 15 patients using retroviral transduction. Culture duration was 15±3 days, with a final transduction efficiency of 97.6%±1.8%. The cell products comprised >98% T cells, with a majority of them being effector T cells (CD45RO+ 95.5%±6.0%). 51Cr-release cytotoxicity assays confirmed that patients' CD30.CARTs lysed a CD30+ tumor line, HDLM-2 (45.9%±15.4% killing at a 20:1 effector:target ratio), with negligible effects on CD30− target cells (<5% killing). During cell manufacture, 1 patient became ineligible due to rapid worsening of his performance status and liver function. Five patients are awaiting treatment on trial. Nine relapsed/refractory HL patients have received CD30.CARTs under the RELY-30 trial. Six of these had relapsed or progressed after treatment with brentuximab. Three patients have been treated on dose level (DL) 1 (2×107 CD30.CAR+ T cells/m2) and 6 patients on DL2 (1×108). All patients received lymphodepleting chemotherapy (cyclophosphamide 500 mg/m2 and fludarabine 30 mg/m2 daily for 3 days) before CART infusion. CART infusions were associated with grade 1 cytokine release syndrome in 4 of the patients, and a transient maculopapular rash in 6 of the patients, starting approximately one week after administration. The molecular signal from CARTs, assessed by Q-PCR in peripheral blood, peaked at 1-2 weeks following infusion, but dropped progressively after 4 weeks and decreased to near the limit of detection level by 6 months post infusion. The signal level was dose dependent, with a peak average of 19,371 copies/mg DNA in patients treated on DL2 versus 7,132 copies/mg for DL1. Compared to patients who received the same CART dose but who were not given lymphodepleting chemotherapy in our previous trial, expansion levels were 45 and 119-fold higher, respectively. Eight patients have been evaluated at 6 weeks after infusion. Six have had a CR lasting from >6 weeks to >6 months, while 2 patients had disease progression. In conclusion, our data indicate that infusion of T cells carrying a CD30.CAR containing a CD28 endodomain after lymphodepleting chemotherapy is safe, with limited toxicities at the dose levels tested. CD30.CAR expansion is improved with inclusion of pre-infusion standard lymphodepleting chemotherapy and appears to be associated with improved efficacy in relapsed patients (6/8 CR versus 1/6 CR, P = 0.03). Disclosures Rooney: Marker: Equity Ownership. Heslop:Marker: Equity Ownership; Cytosen: Membership on an entity's Board of Directors or advisory committees; Tessa Therapeutics: Research Funding; Cell Medica: Research Funding; Gilead Biosciences: Membership on an entity's Board of Directors or advisory committees; Viracyte: Equity Ownership. Brenner:Marker: Equity Ownership.

Abstract Background: We recently characterized a novel proteasome inhibitor NPI-0052, a small molecule derived from the fermentation of a marine gram-positive actinomycete Salinispora tropica. NPI-0052 induces apoptosis in multiple myeloma (MM) cells resistant to conventional and bortezomib therapies. Importantly, NPI-0052 is distinct from bortezomib in its chemical structure, proteasome inhibition profiles, and mechanisms of action. In the present study, we utilized a human plasmacytoma xenograft mouse model to examine the effect of NPI-0052 on proteasome activity profiles in selected organs and tumors. Our results demonstrate that NPI-0052 rapidly leaves the vascular compartment in an active form after intravenous (IV) administration and inhibits the proteasome in extra-vascular tumors and other organs, excluding brain. NPI-0052 triggers a more sustained proteasome inhibition in tumors than in other organs examined. Importantly, we also confirmed the anti-tumor efficacy of NPI-0052. Methods and Model: Animal studies were approved by the DFCI Institutional Animal Care and Use Committee. Sixty CB-17 SCID-male mice were inoculated with 5.0 × 106 MM.1S cells in 100ul of serum free RPMI-1640 medium. The mice were divided into three different groups: Groups 1 and 2 (25 mice EA group) for pharmacodynamic studies (time course) and Group 3 (10 mice) for drug efficacy study. Tumor size was measured every third day in two dimensions using calipers, and tumor volume was calculated using the formula V = 0.5 a × b2, where a and b are the long and short diameter of the tumor respectively. When tumors were ~250 mm3 (~three-four weeks after injection), mice were treated with 0.15 mg/kg of NPI-0052 (IV) or vehicle control. Proteasome inhibition was assessed after either single NPI-0052 treatment (given at Day1) or three treatments (given at Day1, Day4 and Day8). Mice were euthanized at 10 mins, 1h, 4h, and 24h; and packed whole blood (PWB), liver, spleen, lung, kidney, brain and tumors were analyzed for chymotrypsin-like (CT-L), Caspase-like (C-L), and Trypsin-like (T-L) proteasome activities. For efficacy studies mice were treated with NPI-0052 twice a week for three weeks. Mice were sacrificed when their tumors reached ~1.5 cm3. NPI-0052 was dissolved in 100% DMSO to generate a 10 mg/ml stock solution, aliquoted, and stored frozen at − 80°C. The stock solution was serially diluted with 100% DMSO and for injection with 5% Solutol (Solutol HS, polyethylene glycol 660, 12 hydroxystearate; BASF, Shreveport, LA) yielding a final concentration of 2% DMSO and 98% (5% Solutol). The vehicle control was 2% DMSO and 98% (5% Solutol). The pH of the dosing solutions is between 6–7. Results: Inhibition of all three proteasome activities after a single treatment of NPI-0052 was detectable as early as 10 mins in the liver, lung, spleen, kidney and PWB; Within 24h after either a single or three IV treatments of NPI-0052, proteasome activity recovered in liver, lung, spleen and kidney, but not in tumor or PWB; No significant proteasome inhibition was noted in brain up to 24h after either a single or three IV treatments with NPI-0052; CT-L activity was inhibited within 1h post first dose, and 24h exposure triggered marked inhibition of CT-L, C-L and T-L activities in vivo in the xenografted MM.1S tumors. For example, in 1h CT-L activity was inhibited 34%, T-L activity 6% and C-L activity 16%. After 24h hours, CT-L activity was inhibited 60%, T-L activity 24% and C-L activity 49%; and finally, 6) Inhibition of CT-L, C-L and T-L activities increased in the tumor after the third NPI-0052 treatment compared to the first treatment. For example, at 1h post third dose all three activities were inhibited approximately 70–80%. Additionally, the anti-MM activity of NPI-0052 was associated with significant proteasome inhibition in tumors (P < 0.005). Conclusions: Our findings show that NPI-0052 induces a prolonged inhibition (>24h) of all three-20S proteasome activities in established MM.1S tumor xenografts which correlated with marked anti-tumor activity. In contrast, proteasome inhibition in normal tissues including liver, spleen, kidney and lung markedly recovered within 24h of administration after a single or three treatments with NPI-0052. In addition, no significant inhibition of proteasome activities was detectable in the brain after either treatment schedule.

Sonadrill Lands Contract for Drillship Seadrill confirmed a new contract has been secured by Sonadrill Holding, Seadrill’s 50:50 joint venture with an affiliate of Sonangol for the drillship West Gemini. Sonadrill has secured a 10‑well contract with options for up to eight additional wells in Angola for an unknown operator. Total contract value for the firm portion of the deal is expected to be around $161 million, with further revenue potential from a performance bonus. The rig is expected to begin the work in the fourth quarter of this year with a firm term of about 18 months, in direct continuation of the West Gemini’s existing contract. The West Gemini is the third drillship to be bareboat chartered into Sonadrill, along with two Sonangol‑owned units, the Sonangol Quenguela and Sonangol Libongos. Seadrill will manage and operate the units on behalf of Sonadrill. Together, the three units position the Seadrill joint venture as an active rig operator in Angola, furthering the goal of building an ultradeepwater franchise in the Golden Triangle and driving efficiencies from rig clustering in the region. Petrobras Receives TotalEnergies, Shell Payments for Atapu TotalEnergies and Shell have formalized payments to Petrobras for separate, minority stakes in the pre‑salt Atapu field in the Santos Basin. TotalEnergies paid $4.7 billion reais ($940 million) while Shell paid closer to $1.1 billion. The Atapu block was acquired by the consortium comprising Petrobras (52.5%), Shell (25%), and TotalEnergies (22.5%) in the Second Bidding Round for the Transfer of Rights auction held 17 December 2021. The payments are compensation for monies spent thus far by Petrobras, which was granted contractual rights to produce 550 million BOE from Atapu in 2010. The partners will now work together to produce additional volumes from the field. Production at Atapu started in June 2020 via the P-70 FPSO. The unit is in about 2000 m of water and has the capacity to produce 150,000 BOED. CNOOC Brings New Bohai Sea Discoveries On Stream CNOOC Limited has kicked off production from its Luda 5‑2 oil field North Phase I project and Kenli 6‑1 oil field 4‑1 Block development project. Luda 5‑2 is in the Liaodong Bay of Bohai Sea, with average water depth of about 32 m and utilizes a thermal recovery wellhead platform and production platform tied into the Suizhong 36‑1 oil field. A total of 28 development wells are planned, including 26 production wells and two water‑source wells. The project is expected to reach its peak production of 8,200 B/D of oil in 2024. Kenli 6‑1 is in the south of Bohai Sea, with average water depth of about 17 m. The resource is being developed by a wellhead platform in addition to fully utilizing the existing processing facilities of the Bozhong 34‑9 oil field. A total of 12 development wells are planned, including seven production wells and five water‑injection wells. The field is expected to reach its peak production of 4,000 B/D of oil later this year. CNOOC Limited is operator and sole owner of the Luda 5‑2 oil field North and the Kenli 6‑1 oil field 4‑1 Block. Stabroek Block Bounty Off Guyana Gets Bigger The partners in the prolific Stabroek Block have again increased the gross discovered recoverable resource estimate for the area offshore Guyana. The owners now believe they have discovered reserves of at least 11 billion BOE, up from the previous estimate of more than 10 billion BOE. The updated resource estimate includes three new discoveries on the block at Barreleye, Lukanani, and Patwa in addition to the Fangtooth and Lau Lau discoveries announced earlier this year. The Barreleye‑1 well encountered approximately 70 m of hydrocarbon‑bearing sandstone reservoirs of which 16 m is high‑quality oil‑bearing. The well was drilled in 1170 m of water and is located 32 km southeast of the Liza field. The Lukanani‑1 well encountered 35 m of hydrocarbon‑bearing sandstone reservoirs of which approximately 23 m is high‑quality oil‑ bearing. The well was drilled in water depth of 1240 m and is in the southeastern part of the block, approximately 3 km west of the Pluma discovery. The Patwa‑1 well encountered 33 m of hydrocarbon‑bearing sandstone reservoirs. The well was drilled in 1925 m of water and is located approximately 5 km northwest of the Cataback‑1 discovery. “These new discoveries further demonstrate the extraordinary resource density of the Stabroek Block and will underpin our queue of future development opportunities,” said John Hess, chief executive of Hess and a partner in Stabroek. The co‑venturers have sanctioned four developments to date on Stabroek with both Liza and Liza Phase 2 on stream. The third planned development at Payara is ahead of schedule and is now expected to come on line in late 2023; it will utilize the Prosperity FPSO with a production capacity of 220,000 BOPD. The fourth development, Yellowtail, is expected to come on line in 2025, utilizing the ONE GUYANA FPSO with a production capacity of 250,000 BOPD of oil. At least six FPSOs with a production capacity of more than 1 million gross BOPD are expected to be on line on the Stabroek Block in 2027, with the potential for up to ten FPSOs to develop gross discovered recoverable resources. The Stabroek Block is 6.6 million acres. ExxonMobil affiliate Esso Exploration and Production Guyana Limited is operator and holds 45% interest; Hess Guyana Exploration holds 30% interest; and CNOOC Petroleum Guyana Limited holds 25%. ConocoPhillips Gets Ekofisk License Extension Norway’s Ministry of Petroleum and Energy (MPE) has extended production licenses in the Greater Ekofisk Area from 2028 to 2048 with ConocoPhillips as operator. The company said the license extension provides long‑term operations and resource management aligned with the company’s long‑term perspective on the Norwegian continental shelf. Fields on the shelf are required to operate with a valid production license where the operator and licensees enter into an agreement with the authorities, including relevant field activities. The authorities may require commitments, leading to increased oil recovery. The existing production licenses 018, 018 B, and 275 in the Greater Ekofisk Area were set to expire on 31 December 2028; however, the MPE approved an extension through 2048. The new terms provide a potential for extending Ekofisk’s lifetime to nearly 80 years. The license partners are ConocoPhillips (operator, 35.11%), TotalEnergies EP Norge (39.896%), Vår Energi (12.388%), Equinor (7.604%), and Petoro (5%). BHP’s Wasabi Disappoints in US GOM Australian operator BHP encountered noncommercial hydrocarbons with its Wasabi‑2 well in the US Gulf of Mexico. BHP said the well in Green Canyon Block 124 was plugged and abandoned following the disappointing results. “This completes the Wasabi exploration program, with results under evaluation to determine next steps,” the company said. The well was targeting oil in an early Miocene reservoir. Transocean drillship Deepwater Invictus spudded the well in 764 m of water in November 2021. The previous Wasabi‑1 well had a mechanical problem and was plugged and abandoned 4 days earlier, prior to reaching its prospective targets. BHP operates Wasabi with a 75% interest. Lukoil Says Titonskaya Holds 150 Million BOE Russia’s Lukoil believes it has discovered around 150 million BOE following analysis of the two wells it drilled at the Titonskaya structure on the Caspian Sea shelf. Work is now underway to refine the seismic models of productive deposits and study deep samples of formation fluids. The results of the assessment will be submitted to the State Reserves Commission of the Russian Federation. The structure is in the central part of the Caspian Sea, not far from the Khazri field. Lukoil drilled the first well at the Titonskaya structure in 2020 and announced the new discovery in April 2021. According to that assessment, the probable geological resources of the Titonskaya are 130.4 million tons. In 2021, drilling of the second prospecting and appraisal well began to identify oil and gas deposits in the terrigenous‑carbonate deposits of the Jurassic‑ Cretaceous age. The well was drilled using the Neptune jackup drilling rig. The new find at Titonskaya will likely be tied into Khazri infrastructure. Petrobras’ Roncador IOR Project Comes On Line Petrobras has successfully started production from the first two wells of the improved oil recovery (IOR) project at the Roncador field in the Campos Basin offshore Brazil. The two wells are the first of a series of IOR wells to reach production. Startup is almost 5 months ahead of schedule and at half of the planned cost, according to partner Equinor. The wells will add a combined 20,000 BOED to Roncador, bringing daily production to around 150,000 bbl and reducing the carbon intensity (emissions per barrel produced) of the field. Through this first IOR project, the partnership will drill 18 wells that are expected to provide additional recoverable resources of 160 million bbl. Improvements in well design and the partners’ combined technological experience are the main drivers behind the 50% cost reduction across the first six wells, including the two in production. Roncador is Brazil’s fifth‑largest producing asset and has been in production since 1999. Petrobras operates the field and holds a 75% stake. In 2018, Equinor entered the project as a strategic partner with the remaining 25% interest. In addition to the planned 18 IOR wells, the partnership believes it can further improve recovery and aims to increase recoverable resources by a total of 1 billion BOE. The field has more than 10 billion BOE in place under a license lasting until 2052. The strategic alliance agreement also includes an energy‑efficiency and CO2‑emissions‑reduction program for Roncador. Gazania-1 To Spud Off South Africa Africa Energy will move ahead with its planned Gazania‑1 wildcat well offshore South Africa after securing partner Eco Atlantic’s $20 million in capital requirements for its portion of the probe. The well will be drilled in Block 2B. Island Drilling semisubmersible Island Innovator has been contracted for the work and is expected to mobilize from its current location in the North Sea for the 45‑day trip to South Africa. The Block 2B joint venture plans to spud the well by October with drilling expected to last 30 days, including a full set of logs if the well is successful. The block has significant contingent and prospective resources in relatively shallow water and contains the A‑J1 discovery that flowed light sweet crude oil to surface. Gazania‑1 will target two large prospects 7 km updip from A‑J1 in the same region as the recent Venus and Graff discoveries. Block 2B is located offshore South Africa in the Orange Basin where both TotalEnergies and Shell recently announced significant oil and gas discoveries offshore Namibia. The block covers 3062 km2 approximately 25 km off the west coast of South Africa near the border with Namibia in water depths ranging from 50 m to 200 m. The Southern Oil Exploration Corp. (Soekor) discovered and tested oil on Block 2B in 1988 with the A‑J1 borehole, which intersected thick reservoir sandstones between 2985 m and 3350 m. The well flowed 191 B/D of 36 °API oil from a 10‑m sandstone interval at around 3250 m. Africa Energy has a 27.5% interest in Block 2B offshore South Africa. The block is operated by a subsidiary of Eco Atlantic which holds a 50% interest. A subsidiary of Panoro Energy holds a 12.5% stake, and Crown Energy AB indirectly holds the remaining 10%. Brazil Grants New Exploration Blocks Brazil’s National Agency of Petroleum, Natural Gas, and Biofuels (ANP) has granted 59 exploratory blocks of oil and natural gas to 13 companies, including Shell, TotalEnergies, and 3R Petroleum. The awards were part of a permanent bid offer round held in Rio de Janiero in April. The auction totaled 422.4 million reais in signature bonuses with leases granted in six Brazilian states: Rio Grande do Norte, Alagoas, Bahia, Espírito Santo, Santa Catarina, and Paraná. The awards will result in investments of 406.3 million reais in the exploratory phase of the contracts. Shell Brazil (70%) was granted six blocks in the Santos Basin in a consortium with the Colombian Ecopetrol (30%). The blocks leases were SM‑1599, SM‑1601, SM‑1713, SM‑1817, SM‑1908, and SM‑1910. TotalEnergies won two areas in the same basin while Brazilian company 3R Petroleum received six areas in the Potiguar Basin. Petro‑Victory was also awarded 19 new blocks in Potiguar, increasing its holdings in Brazil to 38 blocks (37 in Potiguar). The new blocks are nearby Petro‑Victory infrastructure at the Andorinha, Alto Alegre, and Trapia oil fields. Eni Finds More Oil in Egypt’s Western Desert Eni struck new oil and gas reserves with a trio of discoveries in the Meleiha concessions of Egypt’s Western Desert. The finds have already been tied into existing infrastructure in the region and have added around 8,500 BOED to overall production from the area. The operator drilled the Nada E Deep 1X well, which encountered 60 m of net hydrocarbon pay in the Cretaceous‑Jurassic Alam El Bueib and Khatatba formations Meleiha SE Deep 1X well, which found 30 m of net hydrocarbon pay in the Cretaceous‑Jurassic sands of the Matruh Khatatba formations, and the Emry Deep 21 well, which encountered 35 m of net hydrocarbon pay in the massive cretaceous sandstones of Alam El Bueib. The results, added to the discoveries of 2021 for a total of eight exploration wells, give Eni a 75% success rate in the region. The company added that additional exploration activities in the concession are ongoing with “promising indications.” With these discoveries, Eni, through AGIBA, a joint venture between Eni and EGPC, continues to pursue its near‑field strategy in the mature basin of the Western Desert, aimed at maximizing production by containing development costs and minimizing time to market. Eni is planning a new high‑resolution 3D seismic survey in the Meleiha concession this year to investigate the gas potential of the area. Eni is currently the leading producer in Egypt with an equity production of around 360,000 BOED.

Abstract Introduction MCL is an aggressive B-cell lymphoma with a poor overall prognosis. For patients who fail initial therapy, conventional chemotherapy achieves only short-term remissions. Ibrutinib is a first-in-class, once-daily, oral, covalent inhibitor of Bruton's tyrosine kinase that has been shown to be highly active for previously treated MCL patients (overall response rate [ORR] ~65%; complete response [CR] ~20%) in single-arm phase 2 studies. Temsirolimus has demonstrated significantly longer progression-free survival (PFS) vs investigator's choice. In this phase 3, randomized, open-label study (MCL3001 [RAY]), ibrutinib was compared with temsirolimus in patients with relapsed or refractory (R/R) MCL who had received ≥1 prior rituximab-containing therapy. Methods Patients were randomized at a 1:1 ratio to receive oral ibrutinib (560 mg once-daily) or intravenous temsirolimus (175 mg: Days 1, 8, and 15 of Cycle 1; 75 mg: Days 1, 8, and 15 of subsequent cycles). Stratification factors were number of prior lines of therapy (1-2 vs ³3) and simplified MCL international prognostic index (sMIPI) risk (low [0-3] vs intermediate [4-5] vs high [6-11]). Patients in both arms received treatment until disease progression or unacceptable toxicity. Primary end point of the study was PFS, as assessed by an independent review committee (IRC). Secondary end points included ORR, overall survival (OS), time to next treatment (TTNT), time to worsening of lymphoma symptoms (measured by FACT-Lym lymphoma subscale), and safety. Results Overall, 280 patients were randomized to ibrutinib (n = 139) or temsirolimus (n = 141). Baseline disease characteristics and demographics were generally well balanced. Median age (range) was 68 years (34-88) and median number (range) of prior lines of therapy was 2 (1-9). Approximately two-thirds of the patients had intermediate- or high-risk disease. At the time of this analysis, median follow-up was 20.0 months. Ibrutinib was superior to temsirolimus for the primary end point of IRC-assessed PFS, with a statistically significant 57% reduction in the risk of progression or death (Figure 1). The median PFS was 14.6 months for the ibrutinib arm and 6.2 months for the temsirolimus arm, respectively. At a landmark of 2 years, the PFS rate is 41% in the ibrutinib arm vs 7% in the temsirolimus arm. PFS results were consistent across most assessed subgroups. Investigator-assessed PFS was consistent with the IRC results. IRC-assessed ORR was significantly higher for ibrutinib vs temsirolimus (71.9% vs 40.4%; p < 0.0001) with a CR rate of 18.7% vs 1.4%, respectively. Median OS was not reached with ibrutinib vs 21.3 months with temsirolimus, showing a positive trend toward patients in the ibrutinib arm (reduced risk of death by 24% [HR, 0.76; 95% CI, 0.53-1.09]). However, these results may have been confounded by 23% of patients that initially received temsirolimus crossing over to receive ibrutinib. A greater proportion of patients treated with ibrutinib vs temsirolimus avoided worsening of lymphoma symptoms throughout the study; 27% of ibrutinib patients had worsening vs 52% of temsirolimus patients. Median TTNT was not reached with ibrutinib vs 11.6 months with temsirolimus. Median treatment duration was 14.4 months for ibrutinib and 3.0 months for temsirolimus. Overall, 6.5% of subjects discontinued treatment due to AEs in the ibrutinib arm and 25.5% of subjects discontinued treatment due to AEs in the temsirolimus arm. The most common TEAEs with ibrutinib were diarrhea, fatigue, and cough, whereas with temsirolimus, thrombocytopenia, anemia, and diarrhea were most commonly observed (Table 1). Grade ³3 TEAEs were reported for 67.6% of ibrutinib patients vs 87.1% of temsirolimus patients; most frequent were thrombocytopenia, anemia, and neutropenia. When adjusted for exposure, TEAE incidence was consistently lower for the ibrutinib arm vs the temsirolimus arm. Conclusions Ibrutinib is superior to temsirolimus for PFS and ORR, and showed preferable tolerability. The results of this phase 3 trial confirm the efficacy and favorable safety profile of ibrutinib shown in phase 2 studies. Future concepts will investigate ibrutinib-based combination approaches for patients with R/R MCL. Disclosures Rule: Gilead: Research Funding; Celgene: Consultancy, Other: Travel reimbursement; J&J: Consultancy, Other: Travel reimbursement, Research Funding; Roche: Consultancy, Other: Travel reimbursement. Rusconi:Roche: Honoraria. Joao:Celgene, Novartis: Consultancy; Janssen, Roche: Membership on an entity's Board of Directors or advisory committees. Witzens-Harig:Pfizer: Honoraria, Research Funding; Roche: Honoraria. Hess:Pfizer, Janssen, Roche, Mundipharma: Honoraria, Research Funding; Janssen, Roche, , Celgene, Novartis: Consultancy. Balasubramanian:Janssen: Employment, Equity Ownership; Pharmacyclics LLC, an AbbVie Company: Equity Ownership. Bandyopadhyay:Janssen: Employment. Sun:Janssen/J&J: Employment, Equity Ownership. Goldberg:Janssen: Employment. Bothos:Janssen: Employment. Traina:Janssen: Employment. Enny:Janssen: Employment. Rizo:Janssen: Employment. Vermeulen:Janssen: Employment.

Abstract Acute myeloid leukemia (AML) is a devastating hematopoietic malignancy. With current therapies, only approximately 30% of patients achieve long-term survival. Therefore, novel, more active and less toxic treatments are urgently needed. Programmed death-1 (PD-1) is a cell surface receptor that functions as a T cell checkpoint and plays a central role in regulating T cell exhaustion. Binding of PD-1 to its ligand, programmed death-ligand 1 (PD-L1), activates downstream signaling pathways and inhibits T cell activation. Abnormally high PD-L1 expression on tumor cells and antigen-presenting cells in the tumor microenvironment mediates tumor immune escape, and PD-1/PD-L1 immune checkpoint blockade has showed promising results in cancer patients. Recently, PD-1 expressed on melanoma cells was also shown to play a pivotal role in tumor growth. To date, in AML, the function of PD-1 has been mainly studied in the host T cells, while little is known regarding the role of PD-1 in AML cells. Herein, we examined the level and role of PD-1 in AML cells using AML murine model and patient samples. We used MLL PTD/WT/Flt3 ITD/ITD knock-in mouse in B6 background, a well characterized AML model, to study the expression and function of PD-1 in AML. Flow cytometric analysis of LSK (Lin -Sca-l1 +c-kit +) cells from the bone marrow (BM) of wild-type (WT, n=5) and AML (n=10) mice showed that 20.9%-61.9% of AML LSKs versus (vs) &lt;5.0% of normal LSKs are PD-1 positive (P&lt; 0.0001). Western blot and Q-RT-PCR analysis confirmed higher levels of PD-1 in AML LSKs than in normal LSKs. The PD-1 levels on LSKs increased over time and associated with disease progression. Similar results were obtained in AML patients, showing PD-1 + in 0.2%-14.6% of AML CD34 + cells vs &lt; 1.0% of normal CD34 + cells (P&lt; 0.05). Data analysis based on TCGA showed that higher PD-1 levels are associated with shorter survival in AML patients (P=0.0125). To assess the functional role of PD-1 in AML, we sorted PD-1 + and PD-1 - fractions fromAML LSKs and observed a lower frequency of quiescent cells (G0, 16.60% vs 44.87%, P&lt; 0.05) and a higher cell growth rate in the PD-1 + vs PD-1 - cells. Further in vivo study showed that PD-1 + AML LSKs (CD45.2) generated higher white blood cell (WBC) counts (P&lt; 0.0001), higher AML engraftment (P&lt; 0.0001) and a shorter survival (median survival 57.5 vs &gt;75 days, P&lt; 0.001) in recipient mice (CD45.1) compared with PD-1 - AML LSKs. Similar results were observed in human samples. Compared to PD-1 - CD34 + cells, PD-1 + CD34 + cells are less quiescent and more proliferative (P&lt; 0.01). PD-1 + AML blasts had higher engraftment rate (13.18% vs 2.68%, p=0.0002) and shorter survival (median survival: 27 vs 45 days, P= 0.0008) in NSGS mice than PD-1 - AML blasts. To evaluate if these in vivo differences observed in PD-1 + vs PD-1 - AML LSKs were mediated by interactions between PD-1 + AML and T cells, PD-1 + and PD-1 -LSKs from AML mouse were transplanted into T-, B- cell-deficient NSG mice. Recipient mice transplanted with PD-1 + AML LSKs had higher WBC counts (P&lt; 0.01), higher AML engraftment (P&lt; 0.0001) and a shorter survival (median survival: 76 vs &gt;130 days, P&lt; 0.0001) than recipients with PD-1 - AML LSKs, suggesting that these differences were T cell-independent. Next, we examined whether blocking PD-1 could affect leukemic cell growth. We sorted LSK cells from AML mice and performed colony forming cell (CFC) assay in the presence of anti-PD1 antibody or isotype antibody. Blocking PD-1 with anti-PD-1 antibody significantly suppressed CFC and cell growth in vitro but did not induce apoptosis compared to isotype control antibody. To explore the molecular mechanism by which PD-1 contributes to AML growth, we then sorted PD-1 + and PD-1 - LSKs from AML mice for molecular studies. Western blot assays revealed higher levels of SHP-2 and phosphorylated (p) -ERK in PD-1 + vs PD-1 - AML LSKs. We validated these results in primary human AML cells by immunofluorescence staining. Confocal microscopy of PD-1 + and PD-1 - human AML CD34 +cells demonstrated that PD-1 localized at the cell membrane and in the cytoplasm and p-ERK was markedly enhanced in the PD-1 + CD34 + cells. In conclusion, we showed here that a subpopulation of murine and human AML blasts expresses high levels of PD-1 which mediated disease initiation and growth through activation of the MAPK/ERK signaling pathways. PD-1 blocking antibody reversed these activities and might contribute to the clinical efficacy of anti-PD-1 therapy in AML. Disclosures Marcucci: Novartis: Other: Speaker and advisory scientific board meetings; Agios: Other: Speaker and advisory scientific board meetings; Abbvie: Other: Speaker and advisory scientific board meetings.

Introduction: Daratumumab (DARA) monotherapy is effective and well tolerated in heavily pretreated relapsed/refractory multiple myeloma (RRMM) patients. However, approximately 70% of patients do not respond and eventually all patients will develop progressive disease. DARA treatment results in depletion of CD38+ immune suppressor cells and thereby increased T cell frequencies. A partner drug with immune stimulating activity through a different mechanism of action could further improve the efficacy of DARA. As a single agent, the Programmed Death (PD)-1 checkpoint inhibitor nivolumab induced only stable disease in 67% of RRMM. Immune modulation through targeting CD38 combined with blocking the PD-1/PD-L1 axis may lead to improved T and NK cell activity and therefore better anti-MM efficacy. Preclinical studies showed that cyclophosphamide has synergistic activity with both DARA and PD-1 inhibitors. In this study, we investigate the efficacy and safety of DARA combined with nivolumab, with or without low-dose cyclophosphamide, in RRMM. This trial is registered at ClinicalTrials.gov as NCT03184194. Methods: In part A of this prospective multicenter phase 2 trial, we treated 6 patients with nivolumab-daratumumab (ND), and subsequently 6 patients with nivolumab-daratumumab-cyclophosphamide (NDc) as safety run-in. Next, 28 patients were randomized between both treatment arms at a 1:1 ratio. Twenty additional patients will be treated with either ND or NDc in part B, based on safety and efficacy data as derived in part A. Patients were treated with 28-day cycles until progressive disease. Daratumumab 16 mg/kg i.v. was administered weekly in cycles 1-2, biweekly in cycles 3-6 and every 4 weeks from cycle 7. Nivolumab was administered biweekly (240mg i.v) in cycles 1-6 (in cycle 1 on day 2 and 16) and every 4 weeks (480mg i.v ) thereafter. In the NDc arm, low-dose oral cyclophosphamide (50mg once daily) was given continuously. Inclusion criteria were age ≥18 years, WHO performance score of 0-2, ≥2 prior therapies, lenalidomide-refractory disease, and prior treatment with a proteasome-inhibitor-containing regimen for ≥2 consecutive cycles. Main exclusion criteria were platelet count <75x109/L, absolute neutrophil count <1.0x109/L, FEV1 <50%, significant hepatic or renal dysfunction (CrCl <30 mL/min), or active autoimmune disease or inflammatory disorder. All patients gave written informed consent. The study was conducted in accordance with the principles of the Declaration of Helsinki. In this first planned interim analysis we report on efficacy (overall response rate (ORR)) and safety of part A of the study. Results: Between February 2018 and January 2019, 40 patients were enrolled in part A of this study. The demographics are described in Table 1. At data cut-off (July 1st 2019), 13 patients were still on treatment. Median follow-up of surviving patients is 8.6 months (range 5.0-16.1). The ORR was 50% in both treatment groups (Figure 1); the disease control rate (≥ stable disease) was 85% for ND and 80% for NDc. Ten patients (25%) died due to progressive disease, which was equally distributed over treatment arms. Two patients died during NDc treatment: one (2.5%) due to a cardiac arrest and one (2.5%) due to an Aspergillus fumigatus infection. Non-hematologic toxicity was manageable: daratumumab-associated infusion related reactions (IRRs) occurred in 8 (20%) patients, all during the first administration and all grade ≤3. No IRRs related to nivolumab were reported. Two immune-mediated adverse events occurred: both concerned grade 2 hypothyroidism. The infection rate was higher in patients treated with NDc (24 infections in 12 patients; CTC grade ≥3 in 25% of infections), compared to ND treatment (13 infections in 9 patients; CTC grade ≥3 in 8%). A higher need for supportive care in the form of granulocyte-colony stimulating factor, erythrocyte- and/or platelet transfusion was found in the NDc arm (n=10; 50%), compared to ND treatment (n=6; 30%). Conclusion: Here we show for the first time that, although follow-up is still short, the combination of daratumumab and nivolumab may be a new therapeutic regimen with an acceptable safety profile in RRMM. Addition of low-dose cyclophosphamide did not improve ORR, but increased the frequency of infections and hematologic toxicity, when compared to ND alone. Therefore, the nivolumab-daratumumab regimen was selected for further evaluation in part B. Disclosures Minnema: Gilead: Honoraria; Amgen: Honoraria; Jansen Cilag: Honoraria; Servier: Honoraria; Celgene Corporation: Honoraria, Research Funding. Bos:Celgene: Research Funding; Kiadis Pharma: Other: Shareholder (of Cytosen, acquired by Kiadis). Mutis:Genmab: Research Funding; Janssen Research and Development: Research Funding; Celgene: Research Funding; Onkimmune: Research Funding. Broyl:Celgene, amgen, Janssen,Takeda: Honoraria. Sonneveld:Amgen: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; BMS: Honoraria; SkylineDx: Research Funding; Takeda: Honoraria, Research Funding; Karyopharm: Honoraria, Research Funding. Zweegman:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees, Research Funding; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding. Levin:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: Educational Grant; Roche: Membership on an entity's Board of Directors or advisory committees, Other: Educational Grant; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: Educational Grant; Amgen: Membership on an entity's Board of Directors or advisory committees, Other: Educational grant ; Takeda: Membership on an entity's Board of Directors or advisory committees, Other: Educational grant . Van De Donk:Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees, Research Funding; Servier: Membership on an entity's Board of Directors or advisory committees; Bayer: Membership on an entity's Board of Directors or advisory committees; Janssen Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees, Research Funding; Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AMGEN: Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: Membership on an entity's Board of Directors or advisory committees. OffLabel Disclosure: Nivolumab: off-label treatment for Multiple Myeloma

BackgroundThe preferential Janus kinase-1 inhibitor FIL is approved for treatment of moderate to severe active RA in Europe and Japan.ObjectivesEfficacy and safety of FIL were assessed in pts with IR to MTX who completed a Phase 3 trial (NCT02889796)1 and enrolled in an LTE (NCT03025308).MethodsPts completing the PS1 on study drug were eligible to enter the LTE (data cutoff: June 1, 2020). Median exposure: 2.2 years (y). Efficacy data to W48 are reported for 4 treatment groups (all with background MTX): pts receiving FIL 200 mg (FIL200) or FIL 100 mg (FIL100) in the PS and continuing their dose in LTE (FIL200/FIL200, FIL100/FIL100) and ADA pts rerandomized, double blind, to FIL200 or FIL100 for LTE (ADA/FIL200, ADA/FIL100); safety data are reported.ResultsAs of June 1, 2020, 522/571 (91%) FIL200/FIL200, 502/570 (88%) FIL100/FIL100, 118/128 (92%) ADA/FIL200, and 115/130 (89%) ADA/FIL100 pts remained on study drug. LTE baseline disease characteristics were similar between groups: mean duration of RA approximately 8.7 y; DAS28(CRP) 2.55, and mean concurrent MTX dosage was 15.0 mg/week. Proportions of pts achieving ACR20/50/70, DAS28(CRP) ≤3.2, <2.6, and CDAI ≤10, ≤2.8 were generally maintained in all LTE groups through W48 (Figure 1). Numerically greater proportions of pts met response criteria at W48 in the FIL200 groups vs FIL100, regardless of PS treatment. Treatment-emergent AEs (TEAE), serious AEs, and AEs Grade ≥3 were largely comparable between groups and lowest in ADA/FIL100. There were 10 deaths (Table 1). Exposure-adjusted incidence rates (EAIRs)/100 pt-y of exposure for deaths were lower for FIL/FIL vs ADA/FIL.Table 1.EAIRs of TEAEs in LTE, as of June 1, 20201TEAE, n (%)3FIL200+MTX → FIL200+MTX6ADA+MTX → FIL200+MTX9FIL100+MTX → FIL100+MTX12ADA+MTX → FIL100+MTX2EAIR (95% CI)4n=5717n=12810n=57013n=1305PYE=859.48PYE=197.811PYE=852.314PYE=192.6TEAE429 (75.1)91 (71.1)443 (77.7)88 (67.7)49.9 (45.4, 54.9)46.0 (37.5, 56.5)52.0 (47.4, 57.0)45.7 (37.1, 56.3)TEAE Grade ≥364 (11.2)15 (11.7)72 (12.6)7 (5.4)7.4 (5.8, 9.5)7.6 (4.6, 12.6)8.4 (6.7, 10.6)3.6 (1.7, 7.6)TE serious AE52 (9.1)13 (10.2)60 (10.5)9 (6.9)6.1 (4.6, 7.9)6.6 (3.8, 11.3)7.0 (5.5, 9.1)4.7 (2.4, 9.0)Death3 (0.5)2 (1.6)3 (0.5)2 (1.5)0.3 (0.1, 1.1)1.0 (0.3, 4.0)0.4 (0.1, 1.1)1.0 (0.3, 4.2)TE infections243 (42.6)52 (40.6)249 (43.7)43 (33.1)28.3 (24.9, 32.1)26.3 (20.0, 34.5)29.2 (25.8, 33.1)22.3 (16.6, 30.1)TE serious infections7 (1.2)2 (1.6)13 (2.3)1 (0.8)0.8 (0.4, 1.7)1.0 (0.3, 4.0)1.5 (0.9, 2.6)0.5 (0.1, 3.7)Opportunistic infections2 (0.4)02 (0.4)00.2 (0, 0.8)0 (0, 1.9)0.2 (0, 0.8)0 (0, 1.9)TE herpes zoster16 (2.8)5 (3.9)13 (2.3)1 (0.8)1.9 (1.1, 3.0)2.5 (1.1,6.1)1.5 (0.9, 2.6)0.5 (0.1, 3.7)TE MACE (adjudicated)1 (0.2)03 (0.5)3 (2.3)01 (0, 0.6)0 (0, 1.9)0.4 (0.1, 1.1)1.6 (0.5, 4.8)TE DVT/PE (adjudicated)3 (0.5)03 (0.5)00.3 (0.1, 1.0)0 (0, 1.9)0.4 (0.1, 1.0)0 (0, 1.9)Malignancies (excluding NMSC)5 (0.9)3 (2.3)4 (0.7)00.6 (0.2, 1.4)1.5 (0.5, 4.7)0.5 (0.1, 1.2)0 (0, 1.9)NMSC3 (0.5)02 (0.4)00.3 (0.1, 1.0)0 (0, 1.9)0.2 (0, 0.8)0 (0, 1.9)DVT, deep vein thrombosis; MACE, major adverse cardiovascular event; NMSC, nonmelanoma skin cancer; PE, pulmonary embolism; TE, treatment-emergentFigure 1.ConclusionDuring the LTE through W48, response rates generally were maintained for FIL/FIL and ADA/FIL pts. Though there were differences between LTE groups, safety was largely comparable and consistent with PS observations1 and previously reported results from 7 trials2: rates of AEs of special interest were low; all confidence intervals were overlapping. Limitation: the LTE was not formally randomized for comparison between FIL/FIL and ADA/FIL treatment groups, the groups were of unequal size, and the switch from ADA to FIL for LTE was by design, rather than based on disease activity.References[1]Combe B et al. Ann Rheum Dis 2021;80:848–58.[2]Winthrop K et al. Arthritis Rheumatol 2020;72(suppl 10); abstract 0229.AcknowledgementsThis study was funded by Gilead Sciences, Inc., Foster City, CA. Medical writing support was provided by Claudine Bitel, PhD, of AlphaScientia, LLC, San Francisco, CA; and funded by Gilead Sciences, Inc., Foster City, CA.Disclosure of InterestsBernard Combe Speakers bureau: BMS, Eli Lilly & Co., Gilead Sciences, Inc., MSD, Pfizer, Roche-Chugai, and UCB, Consultant of: AbbVie, Eli Lilly & Co., Gilead Sciences, Inc., Janssen, Pfizer, Roche-Chugai, and Sanofi, Grant/research support from: Novartis, Pfizer, and Roche-Chugai, Yoshiya Tanaka Speakers bureau: AbbVie, Asahi-Kasei, Astellas, Bristol-Myers, Chugai, Daiichi- Sankyo, Eli Lilly, Eisai, Gilead, GSK, Janssen, Mitsubishi-Tanabe, Novartis, Pfizer, Sanofi, and YL Biologics, Consultant of: AbbVie, Ayumi, Daiichi- Sankyo, Eli Lilly, GSK, Sanofi, and Taisho, Grant/research support from: AbbVie, Asahi-Kasei, Chugai, Daiichi-Sankyo, Eisai, Mitsubishi-Tanabe, and Takeda, Paul Emery Consultant of: AbbVie, BMS, Celltrion, Gilead, Lilly, Novartis, Roche, Samsung, and Sandoz, Grant/research support from: AbbVie, BMS, Lilly, and Samsung, Alena Pechonkina Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Albert Kuo Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Qi Gong Shareholder of: Gilead Sciences, Inc., Employee of: Gilead Sciences, Inc., Katrien Van Beneden Shareholder of: Galapagos NV, Employee of: Galapagos NV, Vijay Rajendran Shareholder of: Galapagos NV, Employee of: Galapagos NV, Hendrik Schulze-Koops Speakers bureau: AbbVie, Amgen, BMS, Celgene, Celltrion, Chugai, Gilead, Janssen, Eli Lilly and Company, Merck Sharp & Dohme, Novartis-Sandoz, Pfizer, Roche, and Sanofi, Consultant of: AbbVie, Amgen, BMS, Celgene, Celltrion, Chugai, Gilead, Janssen, Eli Lilly and Company, Merck Sharp & Dohme, Novartis-Sandoz, Pfizer, Roche, and Sanofi, Grant/research support from: AbbVie and Novartis

The paper presents the results of studies related to the study of photosynthetic gas exchange at the leaf level in situ of three-year-old seedlings of Hopea odorata Roxb. during the dry season (South Vietnam). The results obtained will contribute to a better theoretical understanding of the growth and development of plants of this species. The obtained quantitative values of the daily fluxes of photosynthetic gas exchange, as well as the physiological reactions of the plant to environmental conditions, will allow a more qualitative approach to the assessment of carbon fluxes in the corresponding ecosystems. OBJECTS AND METHODS OF RESEARCH The research was conducted from January to April 2020 on the territory of the Cat Tien National Park (South Vietnam) (11.41530 s.w., 107.42460 v.d.) during the dry season. Three-year-old H. odorata seedlings planted in mid-January 2020 were selected as the object of the study. 25 seedlings were selected for observation. The average height of seedlings is 110.0 0.5 cm (SD = 14.4 cm), and their average diameter at a height of ~10 cm is 8.3 0.1 mm (SD = 0.6 mm). According to the illumination conditions of the site and the location of the seedlings, the site was divided into three experimental sites (SA1, SA2, SA3), Fig. 1. The SA1 site (seedlings № 1-16) was located in a relatively open space. The total value of photosynthetically active radiation (FAR) per seedling of this site was 25.71.2 molm-2. The SA2 site (seedlings № 17-20) was located under the crowns of adult trees. The total value of FAR is 10.80.5 molm-2. The SA3 site (seedlings № 21-25) was adjacent to an untouched part of the forest. The total value of FAR is 9.20.4 molm-2. During planting, as well as on 12.02 and 19.03, the seedlings were watered. On 17.02 there was heavy rain at the site. To clarify the question of the effect of the moisture content in the soil on the condition of the studied plants, seedlings № 4-9 were watered from 26.03 to 5.04. The processes of photosynthesis were considered from the standpoint of CO2 gas exchange. Photosynthesis was measured in situ using the Portable Photosynthesis System LI-6800 (Li-Cor, USA). The formed intact leaves in the upper part of the crowns were used for the study. The moisture content in the soil was determined in a 12 cm surface layer using the HydroSense II soil moisture meter (Campbell Scientific, Inc. USA). Soil moisture below 10% corresponded to withering humidity. To study the growth of seedlings in thickness, the stem diameters were measured at a height of 10 cm. The MichaelisMunten equation was used as a basis for the mathematical description of the dependence of photosynthesis on FAR. We used this equation in a modified form [Kaibeyainen, 2009]: A = AmQ/(Q + KM) + Ad. (1) To evaluate the efficiency of photosynthesis, we used the angular coefficient of the tangent (a) to the curve of the function (1) at the point corresponding to KM. From a physical point of view, this coefficient reflects the rate of change in photosynthesis when the headlights change by one unit. RESULTS Figures 3, 4, 5 show graphs showing the daily dynamics of photosynthesis and FAR of seedlings growing on SA1, SA2, SA3. Figure 6 shows graphs showing the daily dynamics of photosynthesis and FAR of watered and non-watered seedlings. The soil moisture under the watered seedlings was ~25.1%, under the non-watered ones - ~8.0%. Photosynthesis of watered seedlings was well associated with FAR, r = 0.84 (for non-watered seedlings, r = -0.34). Fig. 7 shows the values of photosynthesis depending on the FAR, the curves approximating these values obtained according to (1), and tangents to these curves at points corresponding to KM. The indicators characterizing the photosynthetic features of the leaves of seedlings obtained according to (1) are summarized in Table.1. Figure 8 (a) shows the dynamics of the growth of the studied plants by the diameter of the trunk, and Figure 8 (b) shows its derivative, showing the rate of growth per day. DISCUSSION In the morning, the daily dynamics, Fig. 3-5, were characterized by a high degree of association of photosynthesis with FAR, as well as a rapid increase in the values of photosynthesis to maximum values. At the same time, saplings growing on SA1 had a high degree of association of photosynthesis with FAR and in the evening hours, with the decline of FAR. In the midday hours, except for SA2 seedlings, the values under consideration were not associated, and the midday depression of photosynthesis was clearly traced on the daily curves. At the same time, on SA3 seedlings, midday depression was traced until the end of the day. In SA2 seedlings, photosynthesis was well associated with FAR throughout the day. It should be noted that the maximum values of the FAR on SA2 were ~ 640 mmolm2s-1, whereas on other sites - 1600 mmolm-2s-1 and more. The analysis of environmental factors showed that in our case, the main factors that could have inhibitory effects on the photosynthesis of seedlings are FAR and their water supply, determined by soil moisture. To find out which of these factors had the greatest inhibitory effect on photosynthesis, an experiment was conducted with additional watering of seedlings. As can be seen from Fig. 6, FAR did not have any noticeable inhibitory effect on the photosynthesis of the watered seedlings. However, as follows from the comparison of the dynamics of photosynthesis with the dynamics of soil moisture, with a decrease in soil moisture, there is an increasingly depression of photosynthesis. The depression of photosynthesis caused by lack of water has a close effect on its net productivity. Net productivity can be estimated by the increase in plant biomass. Indirectly, the increase in plant biomass can be estimated by the growth of the plant in the thickness of the trunk. When comparing the dynamics of the growth of the studied seedlings in thickness with the dynamics of soil moisture, it can be seen that with a decrease in soil moisture, the increase also decreases, up to a negative value observed in plants at SA3, which we associate with a certain shrinkage of wood. The maximum values of photosynthesis for saplings on SA1 and SA3, Table 1, were approximately the same and were limited by insufficient moisture content in the soil. Nevertheless, saplings on SA1, in comparison with SA3, were characterized by better parameters of leaf growth and development, and, accordingly, biomass growth, Fig. 8. For SA2 seedlings, Table 1, the maximum values of photosynthesis intensity were 5.0 mmolm2s-1 and were mainly limited by a limited FAR. Photosynthesis indicators of these plants were better than those of others, which suggests that SA2 seedlings had a certain shade tolerance. The watered seedlings were not subjected to any noticeable inhibitory effects from environmental factors. The maximum value of photosynthesis for them was 10.5 mmolm2s-1, and the values of photosynthesis efficiency indicators showed that the leaves of these plants were still in the development stage. Based on the analysis, we can make an assumption explaining the high degree of association of photosynthesis of seedlings with FAR, as well as the rapid growth of photosynthesis to the maximum values observed in the morning. This assumption is that during the dark time of the day, seedlings could restore their water balance. At the same time, the restoration of the water balance was possible until the moisture content in the soil corresponding to the withering humidity was reached. Thus, the depression of photosynthesis of H. odorata seedlings revealed during the study was a consequence of their insufficient water supply, which was regulated by the moisture content in the soil. The greatest net productivity of photosynthesis was distinguished by seedlings growing in more illuminated conditions. Seedlings growing in shaded conditions were less exposed to lack of moisture. Seedlings growing in competitive relationships were subjected to the greatest depression of photosynthesis.

Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane). Biosurfactants can reduce the stress between solids and liquids on the surface and at the end. Biosurfactants have several properties, i.e. they are stable, less harmful, as well as readily degradable, and extremely eco-friendly. Biosurfactants also have a wide range of industrial uses because they are a versatile category of chemical substances. The principal justification for conducting such research was the isolation of possible biosurfactants containing bacteria. Sampling was performed for the isolation of bacteria producing biosurfactants from different oil-polluted sites That is to say, experiment for emulsification, test for oil spreading, test for drop collapse, and measure for hemolysis. The capability to produce biosurfactants was seen in 22 different isolates from polluted sites B1, B2, and B3. Through different biochemical tests and Gram staining, it was identified that isolated bacterial strains are Pseudomonas spp and that is Pseudomonas aeruginosa. The procedure used as characterizing biosurfactants was the TLC plate’s procedure, by using TLC plates process yellow dots emerged after spraying on silica gel plates with an throne and ninhydrin reagents. These yellow spots confirmed the presence and production of rhamnolipid in the biosurfactant. Hence, it was concluded that identified strains in the study can be helpful in the heavy metals, pesticides, and hydrocarbons bio-degradation and bioremediation. These may also be used as biological control agents to protect plants from various pathogens, resulting in improved crop yields. Introduction Biosurfactants are natural substances produced by several bacterial and fungal organisms that are amphiphilic and are extracellular (a part of the cell membrane) (Chen et al., 2007; Ghayyomiet al., 2012). Main purpose of the bio-surfactantsgeneration or production is a consequence of financial availability (Van Dyke et al., 1993 It is reported that almost 50 percent of the world's surfactants are used because of the need for cleaning agents as well as the rate of growth grows every day (Deleu and Paquot, 2004). Appropriate use of bio-surfactants will control environmental emissions what these are the most dangerous, constantly rising gradually and disrupting the routine maintenance of life every day. Awareness campaign initiatives have been introduced and also increase for environmental laws, various innovative approaches need to be implemented and even the issue of pollution focused entirely. Developing appropriate advanced technologies to help clear up chemicals and toxins from the ecosystem, like hydrocarbons (both inorganic and organic). Studies on biosurfactants are being launched by scholars and researchers with significant health issues like adverse environmental effects, air contamination, environmental change, and waste management (Makkar and Cameotra, 2002 Biosurfactants contribute to expanded demand for such microbial products as alternatives to chemical surfactants (Benatet al., 2000). Microbes seem to have the capability to degrade contaminants, but their biodegradation is limited leading to hydrophobicity, low solubility in water, and inadequate bioavailability, of such pollutants (Patil, et al., 2012). GhayyomiJazeh, Mishraet. al (2001) those bacteria that produce biosurfactants were isolated from the site of petroleum spills and afterward, 160 strains and as well as 59 strains were able to produce biosurfactants have shown better performance in a test for hemolysis of blood, and 45 strains with positive findings within oil spread experiment were applied in the laboratory to isolate and segregate the media cultured Banat process (Rahman et al., 2002) These were observed and researched that biosurfactants of Pseudomonas aeruginosa spp are most likely to disrupt the bonding of hydrocarbons like nonadecane, octa, Hexa, and hepta, in marine Water contaminated with oil spills up To approximately 47%, 53%, 73% and 60%(Abrar et al., 2020). Current study concluded that the isolated strain having the ability to degrade hydrocarbon as well as the ability to degrade the heavy metal. The strain also can protect the plant from various diseases. The present research found that the isolated strain is capable of degrading hydrocarbon while also being capable of degrading the heavy metal. As well as the strain does have the capability to defend plants from different diseases. Material And Methods Area of Study The investigation was conducted at HazaraUniversity(HU) Microbiology Laboratory, MansehraPakistan. Assemblage of Samples Thehomestay area of the city Mansehra Pakistan which is named as a township, where oil spills arose, oil spills soil samples were obtained as well as sampling from various Mansehra automobile workshops were also done. Sterilized bags of polythene were being used to collect samples of the soil, after thatthe sample was taken towards the Hazara University (HU) Mansehra Microbiology Laboratory to examine and extract bacterial strains that could develop biosurfactants. The soil temperature at the time of sample selection was around 30 ° C. The pH was also verified by Galvano science companies at the time of selection by pH meter, and the pH being reported was 7. Preparation of Media 15 x 100 mm Petri dishes were being used to prepare the media. Agar plates were thoroughly cleaned with water from the tap and then carefully covered in aluminum foil following cleaning then placed within autoclave at 121°C for about 15 min at 15 psi for sterilization. The nutrient agar which contains 0.5% NaCl, 0.3% beef extract, 0.5% peptone, and 1.5% agar, in 500 ml of distilled water, 14 g of the nutrient agar media (Merck) were dissolved. The nutrient level used mainly for the production of non-fastidious species. Nutrient agar is widely known as it's capable of growing a variety of bacteria types and provides nutrients required for the growth of bacteria. Upon sufficient dissolution of such nutrient agar in distilled water, these were then sterilized by autoclaving for 15 min at 15 psi in the autoclave and held at 121 °C Upon autoclaving, pouring of the media was done in laminar flow hood, and then packed and placed for yet more use in a fridge at 4°C. 2.4 Preparation of serial dilution The bacteria are isolated using the serial dilution process. During this process, 10 test tubes were taken and distilled water (9ml) was added in each tube. After that tubes were put for 15 minutes in the autoclave machine at 121°C. After that 1gm of a crude oil sample from the soil was added in a test tube containing distilled water. Further, 1 ml of the solution was taken from the first test tube and poured to the adjoining tubes for the preparation dilution as under . Afterward, 10μl of the solution was pipetted from both the dilution of and shifted for spread culture techniques, then incubated the plates at 37°C for 48hrs. Biosurfactants extraction Firstly, in nutrient broth solution theculture of bacteria was added and inoculated with oil, the bacterial colony was then incubated at the temperature of 25°C in a shaking incubator just for 7 days. Incubation after seven days of trembling. Thebacterial Crop was then taken and centrifuged at 5000rpm at temperature 4°C for 20minutes. Following centrifugation, the supernatant was collected and then mixed in the equivalent amount in Methanol: Chloroform. White sediment was then retained and collected for further use . Bacterial Colonies Isolation 1 g of the soil polluted with oil was diluted serially up to 106 dilutions.10 μl of 104 and 106 dilutions for spread culture were transferred to the MSM agar plates and nutrient agar. The plates were then incubated at 37°C for 48hrs. Twenty-two morphologically separate colonies were separated for further specific examination just after the incubation and processed by using the technique of streak plate. Screening of Isolates’ Biosurfactants Behavior To check the activity of biosurfactants produced by the bacterial species the following methods of screening were done. Hemolytic Activity of Biosurfactants for Erythrocytes Blood agar containing 5% of blood was prepared as after the fresh isolates were added and inoculated on blood agar plates, then the plates were taken and placed in the incubator at temperature 37°C for 48hrs (Rashediet al., 2005). Thereafter the observation of clear zone in the colonies indicated the existence of bacterial species that produce biosurfactants. This experiment was undertaken to control the ability of isolated bacteria to induce blood agar hemolysis. Three forms of hemolysis usually involve; alpha, beta, and hemolysis of the gamma. The agar underneath the species is dark greenish, then it is Alpha, the yellowish color produced in beta hemolysis and gamma hemolysis does not affect the bacterial sppwhichadded on the plates (Anandaraj and Thivakaran, 2010). Bio-surfactant identification with process of CTAB MSM (Mineral salt agar medium) with (2%) of glucose serving both as carbon source, (0.5 mg / ml) acetyl-tri-methyl-ammonium-bromide (CTAB), and methylene blue (MB: 0.2 mg/ml) are used to detect anionic bio-surfactants (Satpute et al., 2008). For this method, thirty microliters (30μl) of cell-free supernatant were added to each of the wells of the methylene blue agar plate that comprises of borer (4 mm in diameter). after that, the incubation of the plates was done for 48-72 hrs at 37°C. Just after incubation in each of the wells, a dark blue halo zone was being used to show the successful anionic bio-surfactant production. Table 1: Composition of MSM Media S. No Ingredients Amount (gm/L) I Potassium dihydrogen phosphate (KH2PO4) II Magnesium Sulfate (MgSO4) III Iron Sulfate (FeSO4) IV Sodium Nitrate (NaNO3) V Calcium Chloride (CaCl2) VI Ammonium Sulfate (NH4)2SO4 Technique for Spreading of Oil A sufficient number of isolated bacteria were inoculated into a solution of 100ml nutrient broth. Over 3 days, the culture was incubated at 37 ° C in a rotating shaker incubator (150 rpm). After that biosurfactants synthesis was checked in culture suspensions (Priya and Usharani, 2009; Anandaraj and Thivakaran, 2010). For this process, thirty milliliters (30ml) of distilled water was added in a Petri dish. In the middle of the distilled water, 1 milliliter (1ml) of diesel oil was added, and then a centrifuged twenty microliter (20μl) culture was introduced to the middle of a plate, which was isolated from oil spilled soil or local oily groundwater. The species producing the bio-surfactant displace the hydrocarbons and disperse it even in the water. Then it was calculated and analyzed within 1 mint (Ali et al., 2013). Technique for Drop collapse In this process, 96-wellsformed in each of the plates of nutrient agar. Afterwards, all the 96-wells of microliter plates was then filled withmineral oil of about 2ml. Then stabilized the plate at 37oC for 1 hour, after which the oil surface was filled with 5μl of supernatant culture. Therefore, the drop shape was taken to be observed on the oil surface after 1min. The drop which was collapsed, generated by the supernatant culture which is used to signify positive(+ive) outcome and the drops which stayed the same and displayed no changeindicates negative(-ive) outcome. And was taking distilled water as a control(Plaza et al., 2006). Emulsification index The emulsification index was calculated, as stated by the process followed by Cooper and Goldenberg (1981) In this process, 2 ml of kerosene oil was taken and inserted in each of the test tubes to the same amount of cell-free supernatant, and then homogenized for 2 min in a vortex at high speed and allowed for 24 hours to stand. The emulsification steadiness was then determined after the 24 hours, and the emulsification value was estimated by measuring the emulsified layer height by the total liquid layer height, then multiplied by 100. Quantification for the Dry weight of Biosurfactants The bacterial colony was inserted and inoculated in the nutrient broth medium, followed by oil and centrifuged at 5000rpm and after that, the supernatant was clutched and treated with chloroform and methanol and mixed. The white colored deposits were taken and used for the furtherprocess of dry weight. Afterwards, took the clean Petri plate and determined the empty plate weight. Next, the sediment was poured onto Petri plates. Now, for the drying process the hot air oven was used and set the 100ºC of temperature for 30minutes and the plates were put in the oven. After the drying process, the plates were weighted again. The dry weight was calculated for the biosurfactants using the formula which described below: Selected strains Identification and their characterization Instead, various basic biochemical methods were used to identify the isolated bacterial strains. Various biochemical tests, such as Gram staining, Oxidase test, Urease test.Catalase test, Methyl red test, Motility test, Indole test, Starch hydrolysis, Citrate test, Spore staining, Gelatin hydrolysis. Then afterwards, for the preliminary characterization of the biosurfactant, the thin layer chromatography process was used. Physical characterization of the strains selected Gram staining First, on the slide, using the wire loop the bacterial pure culture was taken, and smear was prepared on the slide, and then a drop of purified water was applied. Then, the sterile loop or needle was correctly mixed the bacterial colony and purified water, then mixed up until it is somewhat turbid. Then, spirit lamp was used to fixed the bacterial smear on slide and cooled to room temperature. With this glass slide was loaded with solution of crystal violet and stood for 1minute anddistilled water was applied on slide. Meanwhile the slide was submerged for 1 minute with the iodine solution, and then flushed and rinsed with water. Therefore, decolorizer of about 1 to 2 drops(5 percent acetone and 95 percent alcohol) were added to the slide’s smear and stand for 30seconds, and then treated with water. After then slide was rinsed with safranin for 60seconds, and then treated with water anddry in air. Microscopic analysis was done with 100x objective lenses using emersion oil on smear. Cell morphology The isolates of the bacterial cell were gram stained on slides and then the slides were observed under the light microscope, showing the shape and color of the cells. Biochemical characterization of the selected strains Catalase test Aim of this study is to identify, evaluate and examine that, whether or not the microbes are capable of producing catalase enzymes, while catalase is a protective enzyme, i.e. catalase has the potential to protect against the lethal chemicals known as (H2O2). In this study a bacterial culture that was clarified overnight was used. This culture has been smeared on a glass slide, and 3 percent hydrogen peroxide (H2O2) has been applied and observed on smear. Effects have been observed for bubble formation. Citrate test This study was performed to check the amount or ingest the citrate as the carbon and energy supply for growth and metabolism. Medium containing bromothymol blue and sodium citrate as pH indicator, bacterial was introduced. Ammonium chloride is also present in this medium used as a nitrogen source. Results were noted with variations of color from green to blue. Urease test The capability of urease enzyme for degrading urea was calculated in this bacterial capacity test. Bacterial culture was taken and inoculated for 48 hours at 37 ° C in urease broth, and then color was observed. Methyl red test Through using the process known as mixed acid fermentation which is used to evaluate the bacteria's acid production. The bacterial culture was taken and introduced in the broth of MR-VP and then incubated for 3days at a temperature of 37°C. Two (2) to three (3) drops of Methyl red were added in the broth medium after the incubation period. The change in broth color was observed for final results after a few seconds. Indole test Through using the process to assess the bacteria 's capability to crash indole from tryptophane molecules. After the 24 hours of incubated, taken the fresh inoculum of bacteria and then inserted into the tryptone medium, 24 hours of incubation of about 30oC, 2ml of the tryptone broth medium was added into a sterile test tube. Kovac's reagent was taken to be added (few drops) in sterile test tube and stimulated for a few minutes, and variations of color were detected. Gelatin test It is the approach assess to figure out the use of enzymes known as gelatins from bacterial organisms that precipitate the gelatin. Fresh inoculum of bacteria was taken after 24 hours, and inserted into the media of gelatin agar. This was incubated for around 24 hours, so the temperature did not exceed 30 ° C. Media was observed after incubation time. Starch hydrolysis Several of the micro-organisms that use the starch as a carbon energysource. Therefore, this method has been used to assess whether or not bacteria may use starch as a source of carbon. The bacterial fresh inoculum was spread on the petri starch agar plates, and after that the plate was incubated for 24 hours andmaintained the temperature at 30 to 35 ° C, then gradually applying the supplements of iodine to the plates to flow the change, and then examining the plates. Preliminary characterization of the strains selected Experimental characterization of the bio-surfactant was performed by using the process of TLC (Anandaraj et al., 2010). On a silica gel plate, crude portion of the rudimentary bio-surfactant was separated using Methanol: Chloroform: water (CH3OH: CHCl3: H2O) in the ratio of as an eluent with a different color producing reagents. Ninhydrin reagent (0.5 g ninhydrin in 100ml anhydrous acetone) was used to find bio-surfactant lipopeptide as red spots and anthrone reagent (1 g anthrone in 5ml sulfuric acid combined with 95ml ethanol) as yellow spots to identify rhamnolipid bio-surfactant (Yin et al., 2008). Results and Discussion Isolation of bacteria At first, twenty-two (22) strains from a polluted soil sample were isolated from nutrient agar media.Mixed culture provided by these colonies, so they were taken and smeared on the plates of nutrient agar and then fresh inoculum was collected and stored at temperature of 4oC for the further analysis. Bio-surfactants (surface-active compounds)are formed by a variety of amphiphilic bacterial and fungal organisms that are extracellular (a part of the cellular membrane) (Chen et al., 2007). Screening of Isolated strains for biosurfactant producing colonies Different experiments were carried out to identify, isolate and screen bacteria that are capable of generating bio-surfactants and that is Oil spreading technique(OST), blood hemolysis test(BHT), CTAB test, Emulsification operation. There were twenty-two distinct isolates observed in the current research. And the B1, B2 and B3culture were taken and selected from the twenty-two (22) strains isolated from the polluted spot, which were found to produce biosurfactant. And the oil spreading technique showed promising results for these strains. And strain B2 showed a greater displacement of oil and this is 4 mm. Oil spreading method is quick and often easy to handle, and this technique requires no particular equipment, only a very small amount of sample is used. This approach can be applied when the production and quantity of biosurfactant is small (Plaza et al., 2006) and (Youssef et al., 2004) Only bacterial cultures have been allocated and screened for bacterial species that can generate or use biosurfactants. Just three (3) strainsamong them presented the best results.Those 3 strain,s (B1, B2 and B3) were selected as an additional analysis. Blood hemolysis test On the petri plates of blood agar, the . Isolated bacteriaof B1, B2 and B3 were taken andstreak at the temperature about 37°C for 48 hours. Strain B1 demonstrated β (Beta) hemolysis after the incubation cycle and B2 and B3strains demonstrated γ (Gamma) hemolysis. The B1 strain had an emulsification index of about 74 percent and that was very high as compared to 70 percent for B2 and about 53 percent for B3 respectively. Around the same time, B1 strain showed β (Beta) hemolysis and γ (Gamma) hemolysis was shown bystrains B2 and B3 on the platesof bloodagar. The β hemolysisshowed by the strain B1 in the blood agar test, and the strain B2 and B3 showed γ (Gamma) hemolysis. It is determined that 20 percent strains that are the bestproducer of rhamnolipid have not fully lysed the blood, because the ability of the producer strains capacity not be responsible for the hemolytic activity. According to many researchers, who have shown that this is not such an effective tool for biosurfactant detection due to many bioproducts that may also induce red blood cell lysis, that is not so sufficient to be the surface-active molecule (Youssef et al., 2004). (Rashedi and others, 2005). Table2 Blood Hemolysis Test CTAB agar plate test This test confirms the anionic biosurfactants development. After plate incubation at a temperature of 37 ° C for 72 hours, dark blue hollow zone was existedaround each of the B1 strains wells, which clearly indicated the positive (+ive) development of anionic Biofactant. In addition, the B1 and B2 strains showed positive (+ I ve) results and, in the CTAB analysis, the B3 strain was found to be negative (-ive). The growing microorganisms when secreted the anionic biosurfactants on the plates of CTAB (cetyl-tri-methyl-ammonium-bromide) and methylene blue, then as a result the dark blue-purple insoluble ion pairs formed on the plates. The halo zone around each of the colonies was developed that can recognize rhamnolipid production and that was dark blue in colour, and could correlate with production of rhamnolipid (Siegmund et al., 1991). As indicated in (Fig1) Fig1: B1 positive on CTAB agar plate Oil Spreading Technique The oil was displaced by B1, B2and B3 strains in this test strain and showed a zone that was so clear. The bacterial strains capable of developing biosurfactant were tested and separated from the sample of soil which was oil spilled and brought from the District of Mansehra, Pakistan and from automobile workshops of Mansehra. As shown in (Fig.2). Fig.2: Results of Oil Spreading by B1, B2 and B3Table 3;.Test for oil spreads Bacterial culture Formation of zone (mm) Readings B,1 B,2 B,3 Drop-collapse technique During this process the drop shape was observed at the oil surface. As seen in Fig 3, the collapsed drop was provided by the supernatant culture B1 , B2 and B3.. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Emulsification index Emulsification stability was measured with the use of kerosene oilin this test, and then observed the results. Since this emulsification index was calculated by dividing the height of the emulsion layer by the total height of the liquid layer and then multiplying by 100, as shown in the formulation below. Fig 3: Result of Drop-collapse test Table 4: The activity of Biosurfactant emulsification Dry weight of bio-surfactants In this examination, white-colored sediment was collected. Then measured the weight of the sterile Petri plate which was empty in the first step. Then, the sediment was poured into plates. The plates were taken and weighted after 30 minutes of drying on a hot air oven, following the process of drying. The weight of biosurfactants (dry weight) was measured using the following formulations: Fig 4: Dry weight of biosurfactants Table: 5: Dry weight of the biosurfactants Bacterial Culture Weight of the plate (g) biosurfactant in The plate after drying (g) Dry weight of Biosurfactant (g) B,1 B,2 B,3 Identification of selected strains and their characterization Gram staining For structural applications, and stroke analysis gram staining method was used.(Fig.5) shows findings from the process of gram staining. Fig 5: Microscopic view of Gram staining Biochemical identification of bacterial strains and their characterization Specific biochemical studies were performed to identify the species for further recognition and characterization. The bio-surfactant producing microorganism was found to be Pseudomonas aeruginosa after conducting various characterizations and the biochemical tests(Eric Deziel et al., 1996), Which can be used to further analyze and study the industrial development of the biosurfactant. Rhamnolipid is also isolated and produced from the Pseudomonas aeruginosa species on the silica gel plate (Rashedi et al., 2005), a form of biosurfactants highly recommended for processes of bioremediation. All the findings collected from biochemical testing were labeled as Berge 's Manual and it revealed that the protected microorganism was (Pseudomonas aeruginosa). Results of biochemical test were tabulated in (Table.5) Table 6: Bacterial strain identification Tests B1 B2 B3 Gram staining Negative Negative Negative Oxidases Positive e Positive Positive Catalase Positive Positive Positive Indole Positive Negative Negative Citrate Positive Negative Negative Urease Negative Positive Negative Nitrate Positive Positive Positive Motility Positive Positive Positive Gelatin hydrolysis Positive Negative Negative Lactose Negative Positive Positive Methyl red Negative Positive Positive Voges Proskauer Negative Negative Negative Fig 6: Results of biochemical tests(A) Methyl red and Voges Proskauer tests (b) catalase tests (c) oxidase tests (d) indole tests (e) citrate tests (g) lactose tests (h) urease tests Preliminary bacterial strain’s characterization The plates showed yellow dots, when sprayed with anthrone reagent. It indicated the existence of biosurfactants of rhamnolipid in the organism on the plate of TLC as seen in theFig.7 Fig 7: Biosurfactant characterization by TLC Conclusion Biosurfactant development is exciting and perceptible across industries to clean up oil waste and pollutants, particularly in the ecosystem.Compared with chemical surfactants, the biosurfactants are less harmful. It plays an important role in defining the advantages and the importance of industrial applications. Therefore, it is not possible to disregard the growing role and importance of biosurfactants in environmental sustainability.Biosurfactant formulations which can be used for bacterial, fungal, and viral organisms as growth inhibitors. Such biosurfactant inhibition properties can make them components that are applicable to Numerous illnesses that are used as medicinal agents. Therefore it was decided that the described strain could be used as a potential source for heavy metal bioremediation pesticide and hydrocarbon polluted sites. And also used as shielding the plant from different pathogens, contributing to improved crop yields. There is no doubt that the biosurfactants are a multifunctional, advanced, versatile, long-lasting and updated type not only for the twenty-first century but beyond. Conflict of interest The authors declared that they have no conflict of interest and the paper presents their own work which does not been infringe any third-party rights, especially authorship of any part of the article is an original contribution, not published before and not being under consideration for publication elsewhere. References Ali, S.R.; Chowdhury, B.R.; Mondal, P. and Rajak, S. “Screening and characterization of biosurfactants producing microorganism from natural environment (Whey spilled soil)”. Nat. Sci. Res. 2013, 3(13), 34–64. 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Environments containing significant concentration of NaCl such as salt lakes harbor extremophiles microorganisms which have a great biotechnology interest. To explore the diversity of Bacteria in Chott Tinsilt (Algeria), an isolation program was performed. Water samples were collected from the saltern during the pre-salt harvesting phase. This Chott is high in salt (22.47% (w/v). Seven halophiles Bacteria were selected for further characterization. The isolated strains were able to grow optimally in media with 10–25% (w/v) total salts. Molecular identification of the isolates was performed by sequencing the 16S rRNA gene. It showed that these cultured isolates included members belonging to the Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus genera with less than 98% of similarity with their closest phylogenetic relative. The halophilic bacterial isolates were also characterized for the production of biosurfactant and industrially important enzymes. Most isolates produced hydrolases and biosurfactants at high salt concentration. In fact, this is the first report on bacterial strains (A4 and B4) which were a good biosurfactant and coagulase producer at 20% and 25% ((w/v)) NaCl. In addition, the biosurfactant produced by the strain B4 at high salinity (25%) was also stable at high temperature (30-100°C) and high alkalinity (pH 11).Key word: Salt Lake, Bacteria, biosurfactant, Chott, halophiles, hydrolases, 16S rRNAINTRODUCTIONSaline lakes cover approximately 10% of the Earth’s surface area. The microbial populations of many hypersaline environments have already been studied in different geographical regions such as Great Salt Lake (USA), Dead Sea (Israel), Wadi Natrun Lake (Egypt), Lake Magadi (Kenya), Soda Lake (Antarctica) and Big Soda Lake and Mono Lake (California). Hypersaline regions differ from each other in terms of geographical location, salt concentration and chemical composition, which determine the nature of inhabitant microorganisms (Gupta et al., 2015). Then low taxonomic diversity is common to all these saline environments (Oren et al., 1993). Halophiles are found in nearly all major microbial clades, including prokaryotic (Bacteria and Archaea) and eukaryotic forms (DasSarma and Arora, 2001). They are classified as slight halophiles when they grow optimally at 0.2–0.85 M (2–5%) NaCl, as moderate halophiles when they grow at 0.85–3.4 M (5–20%) NaCl, and as extreme halophiles when they grow at 3.4–5.1 M (20–30%) NaCl. Hyper saline environments are inhabited by extremely halophilic and halotolerant microorganisms such as Halobacillus sp, Halobacterium sp., Haloarcula sp., Salinibacter ruber , Haloferax sp and Bacillus spp. (Solomon and Viswalingam, 2013). There is a tremendous demand for halophilic bacteria due to their biotechnological importance as sources of halophilic enzymes. Enzymes derived from halophiles are endowed with unique structural features and catalytic power to sustain the metabolic and physiological processes under high salt conditions. Some of these enzymes have been reported to be active and stable under more than one extreme condition (Karan and Khare, 2010). Applications are being considered in a range of industries such as food processing, washing, biosynthetic processes and environmental bioremediation. Halophilic proteases are widely used in the detergent and food industries (DasSarma and Arora, 2001). However, esterases and lipases have also been useful in laundry detergents for the removal of oil stains and are widely used as biocatalysts because of their ability to produce pure compounds. Likewise, amylases are used industrially in the first step of the production of high fructose corn syrup (hydrolysis of corn starch). They are also used in the textile industry in the de-sizing process and added to laundry detergents. Furthermore, for the environmental applications, the use of halophiles for bioremediation and biodegradation of various materials from industrial effluents to soil contaminants and accidental spills are being widely explored. In addition to enzymes, halophilic / halotolerants microorganisms living in saline environments, offer another potential applications in various fields of biotechnology like the production of biosurfactant. Biosurfactants are amphiphilic compounds synthesized from plants and microorganisms. They reduce surface tension and interfacial tension between individual molecules at the surface and interface respectively (Akbari et al., 2018). Comparing to the chemical surfactant, biosurfactant are promising alternative molecules due to their low toxicity, high biodegradability, environmental capability, mild production conditions, lower critical micelle concentration, higher selectivity, availability of resources and ability to function in wide ranges of pH, temperature and salinity (Rocha et al., 1992). They are used in various industries which include pharmaceuticals, petroleum, food, detergents, cosmetics, paints, paper products and water treatment (Akbari et al., 2018). The search for biosurfactants in extremophiles is particularly promising since these biomolecules can adapt and be stable in the harsh environments in which they are to be applied in biotechnology.OBJECTIVESEastern Algeria features numerous ecosystems including hypersaline environments, which are an important source of salt for food. The microbial diversity in Chott Tinsilt, a shallow Salt Lake with more than 200g/L salt concentration and a superficies of 2.154 Ha, has never yet been studied. The purpose of this research was to chemically analyse water samples collected from the Chott, isolate novel extremely or moderate halophilic Bacteria, and examine their phenotypic and phylogenetic characteristics with a view to screening for biosurfactants and enzymes of industrial interest.MATERIALS AND METHODSStudy area: The area is at 5 km of the Commune of Souk-Naâmane and 17 km in the South of the town of Aïn-Melila. This area skirts the trunk road 3 serving Constantine and Batna and the railway Constantine-Biskra. It is part the administrative jurisdiction of the Wilaya of Oum El Bouaghi. The Chott belongs to the wetlands of the High Plains of Constantine with a depth varying rather regularly without never exceeding 0.5 meter. Its length extends on 4 km with a width of 2.5 km (figure 1).Water samples and physico-chemical analysis: In February 2013, water samples were collected from various places at the Chott Tinsilt using Global Positioning System (GPS) coordinates of 35°53’14” N lat. and 06°28’44”E long. Samples were collected randomly in sterile polythene bags and transported immediately to the laboratory for isolation of halophilic microorganisms. All samples were treated within 24 h after collection. Temperature, pH and salinity were measured in situ using a multi-parameter probe (Hanna Instruments, Smithfield, RI, USA). The analytical methods used in this study to measure ions concentration (Ca2+, Mg2+, Fe2+, Na+, K+, Cl−, HCO3−, SO42−) were based on 4500-S-2 F standard methods described elsewhere (Association et al., 1920).Isolation of halophilic bacteria from water sample: The media (M1) used in the present study contain (g/L): 2.0 g of KCl, 100.0/200.0 g of NaCl, 1.0 g of MgSO4.7HO2, 3.0 g of Sodium Citrate, 0.36 g of MnCl2, 10.0 g of yeast extract and 15.0 g agar. The pH was adjusted to 8.0. Different dilutions of water samples were added to the above medium and incubated at 30°C during 2–7 days or more depending on growth. Appearance and growth of halophilic bacteria were monitored regularly. The growth was diluted 10 times and plated on complete medium agar (g/L): glucose 10.0; peptone 5.0; yeast extract 5.0; KH2PO4 5.0; agar 30.0; and NaCl 100.0/200.0. Resultant colonies were purified by repeated streaking on complete media agar. The pure cultures were preserved in 20% glycerol vials and stored at −80°C for long-term preservation.Biochemical characterisation of halophilic bacterial isolates: Bacterial isolates were studied for Gram’s reaction, cell morphology and pigmentation. Enzymatic assays (catalase, oxidase, nitrate reductase and urease), and assays for fermentation of lactose and mannitol were done as described by Smibert (1994).Optimization of growth conditions: Temperature, pH, and salt concentration were optimized for the growth of halophilic bacterial isolates. These growth parameters were studied quantitatively by growing the bacterial isolates in M1 medium with shaking at 200 rpm and measuring the cell density at 600 nm after 8 days of incubation. To study the effect of NaCl on the growth, bacterial isolates were inoculated on M1 medium supplemented with different concentration of NaCl: 1%-35% (w/v). The effect of pH on the growth of halophilic bacterial strains was studied by inoculating isolates on above described growth media containing NaCl and adjusted to acidic pH of 5 and 6 by using 1N HCl and alkaline pH of 8, 9, 10, 11 and 12 using 5N NaOH. The effect of temperature was studied by culturing the bacterial isolates in M1 medium at different temperatures of incubation (4°C–55°C).Screening of halophilic bacteria for hydrolytic enzymes: Hydrolase producing bacteria among the isolates were screened by plate assay on starch, tributyrin, gelatin and DNA agar plates respectively for amylase, lipase, protease and DNAse activities. Amylolytic activity of the cultures was screened on starch nutrient agar plates containing g/L: starch 10.0; peptone 5.0; yeast extract 3.0; agar 30.0; NaCl 100.0/250.0. The pH was 7.0. After incubation at 30 ºC for 7 days, the zone of clearance was determined by flooding the plates with iodine solution. The potential amylase producers were selected based on ratio of zone of clearance diameter to colony diameter. Lipase activity of the cultures was screened on tributyrin nutrient agar plates containing 1% (v/v) of tributyrin. Isolates that showed clear zones of tributyrin hydrolysis were identified as lipase producing bacteria. Proteolytic activity of the isolates was similarly screened on gelatin nutrient agar plates containing 10.0 g/L of gelatin. The isolates showing zones of gelatin clearance upon treatment with acidic mercuric chloride were selected and designated as protease producing bacteria. The presence of DNAse activity on plates was determined on DNAse test agar (BBL) containing 10%-25% (w/v) total salt. After incubation for 7days, the plates were flooded with 1N HCl solution. Clear halos around the colonies indicated DNAse activity (Jeffries et al., 1957).Milk clotting activity (coagulase activity) of the isolates was also determined following the procedure described (Berridge, 1952). Skim milk powder was reconstituted in 10 mM aqueous CaCl2 (pH 6.5) to a final concentration of 0.12 kg/L. Enzyme extracts were added at a rate of 0.1 mL per mL of milk. The coagulation point was determined by manual rotating of the test tube periodically, at short time intervals, and checking for visible clot formation.Screening of halophilic bacteria for biosurfactant production. Oil spread Assay: The Petridis base was filled with 50 mL of distilled water. On the water surface, 20μL of diesel and 10μl of culture were added respectively. The culture was introduced at different spots on the diesel, which is coated on the water surface. The occurrence of a clear zone was an indicator of positive result (Morikawa et al., 2000). The diameter of the oil expelling circles was measured by slide caliber (with a degree of accuracy of 0.02 mm).Surface tension and emulsification index (E24): Isolates were cultivated at 30 °C for 7 days on the enrichment medium containing 10-25% NaCl and diesel oil as the sole carbon source. The medium was centrifuged (7000 rpm for 20 min) and the surface tension of the cell-free culture broth was measured with a TS90000 surface tensiometer (Nima, Coventry, England) as a qualitative indicator of biosurfactant production. The culture broth was collected with a Pasteur pipette to remove the non-emulsified hydrocarbons. The emulsifying capacity was evaluated by an emulsification index (E24). The E24 of culture samples was determined by adding 2 mL of diesel oil to the same amount of culture, mixed for 2 min with a vortex, and allowed to stand for 24 h. E24 index is defined as the percentage of height of emulsified layer (mm) divided by the total height of the liquid column (mm).Biosurfactant stability studies : After growth on diesel oil as sole source of carbone, cultures supernatant obtained after centrifugation at 6,000 rpm for 15 min were considered as the source of crude biosurfactant. Its stability was determined by subjecting the culture supernatant to various temperature ranges (30, 40, 50, 60, 70, 80 and 100 °C) for 30 min then cooled to room temperature. Similarly, the effect of different pH (2–11) on the activity of the biosurfactant was tested. The activity of the biosurfactant was investigated by measuring the emulsification index (El-Sersy, 2012).Molecular identification of potential strains. DNA extraction and PCR amplification of 16S rDNA: Total cellular DNA was extracted from strains and purified as described by Sambrook et al. (1989). DNA was purified using Geneclean® Turbo (Q-BIO gene, Carlsbad, CA, USA) before use as a template in polymerase chain reaction (PCR) amplification. For the 16S rDNA gene sequence, the purified DNA was amplified using a universal primer set, forward primer (27f; 5′-AGA GTT TGA TCM TGG CTC AG) and a reverse primer (1492r; 5′-TAC GGY TAC CTT GTT ACG ACT T) (Lane, 1991). Agarose gel electrophoresis confirmed the amplification product as a 1400-bp DNA fragment.16S rDNA sequencing and Phylogenic analysis: Amplicons generated using primer pair 27f-1492r was sequenced using an automatic sequencer system at Macrogene Company (Seoul, Korea). The sequences were compared with those of the NCBI BLAST GenBank nucleotide sequence databases. Phylogenetic trees were constructed by the neighbor-joining method using MEGA version 5.05 software (Tamura et al., 2011). Bootstrap resembling analysis for 1,000 replicates was performed to estimate the confidence of tree topologies.Nucleotide sequence accession numbers: The nucleotide sequences reported in this work have been deposited in the EMBL Nucleotide Sequence Database. The accession numbers are represented in table 5.Statistics: All experiments were conducted in triplicates. Results were evaluated for statistical significance using ANOVA.RESULTSPhysico-chemical parameters of the collected water samples: The physicochemical properties of the collected water samples are reported in table 1. At the time of sampling, the temperature was 10.6°C and pH 7.89. The salinity of the sample, as determined in situ, was 224.70 g/L (22,47% (w/v)). Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions (table 1). SO4-2 and Mg+2 was present in much smaller amounts compared to Na +and Cl- concentration. Low levels of calcium, potassium and bicarbonate were also detected, often at less than 1 g/L.Characterization of isolates. Morphological and biochemical characteristic feature of halophilic bacterial isolates: Among 52 strains isolated from water of Chott Tinsilt, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for further characterization (table 2). The colour of the isolates varied from beige, pale yellow, yellowish and orange. The bacterial isolates A1, A2, A4, B1 and B5 were rod shaped and gram negative (except B5), whereas A3 and B4 were cocci and gram positive. All strains were oxidase and catalase positive except for B1. Nitrate reductase and urease activities were observed in all the bacterial isolates, except B4. All the bacterial isolates were negative for H2S formation. B5 was the only strain positive for mannitol fermentation (table 2).We isolated halophilic bacteria on growth medium with NaCl supplementation at pH 7 and temperature of 30°C. We studied the effect of NaCl, temperature and pH on the growth of bacterial isolates. All the isolates exhibited growth only in the presence of NaCl indicating that these strains are halophilic. The optimum growth of isolates A3 and B1 was observed in the presence of 10% NaCl, whereas it was 15% NaCl for A1, A2 and B5. A4 and B4 showed optimum growth in the presence of 20% and 25% NaCl respectively. A4, B4 and B5 strains can tolerate up to 35% NaCl.The isolate B1 showed growth in medium supplemented with 10% NaCl and pH range of 7–10. The optimum pH for the growth B1 was 9 and they did not show any detectable growth at or below pH 6 (table 2), which indicates the alkaliphilic nature of B1 isolate. The bacterial isolates A1, A2 and A4 exhibited growth in the range of pH 6–10, while A3 and B4 did not show any growth at pH greater than 8. The optimum pH for growth of all strains (except B1) was pH 7.0 (table 2). These results indicate that A1, A2, A3, A4, B4 and B5 are neutrophilic in nature. All the bacterial isolates exhibited optimal growth at 30°C and no detectable growth at 55°C. Also, detectable growth of isolates A1, A2 and A4 was observed at 4°C. However, none of the bacterial strains could grow below 4°C and above 50°C (table 2).Screening of the halophilic enzymes: To characterize the diversity of halophiles able to produce hydrolytic enzymes among the population of microorganisms inhabiting the hypersaline habitats of East Algeria (Chott Tinsilt), a screening was performed. As described in Materials and Methods, samples were plated on solid media containing 10%-25% (w/v) of total salts and different substrates for the detection of amylase, protease, lipase and DNAse activities. However, coagulase activity was determined in liquid medium using milk as substrate (figure 3). Distributions of hydrolytic activity among the isolates are summarized in table 4.From the seven bacterial isolates, four strains A1, A2, A4 and B5 showed combined hydrolytic activities. They were positive for gelatinase, lipase and coagulase. A3 strain showed gelatinase and lipase activities. DNAse activities were detected with A1, A4, B1 and B5 isolates. B4 presented lipase and coagulase activity. Surprisingly, no amylase activity was detected among all the isolates.Screening for biosurfactant producing isolates: Oil spread assay: The results showed that all the strains could produce notable (>4 cm diameter) oil expelling circles (ranging from 4.11 cm to 4.67 cm). The average diameter for strain B5 was 4.67 cm, significantly (P < 0.05) higher than for the other strains.Surface tension and emulsification index (E24): The assimilation of hydrocarbons as the sole sources of carbon by the isolate strains led to the production of biosurfactants indicated by the emulsification index and the lowering of the surface tension of cell-free supernatant. Based on rapid growth on media containing diesel oil as sole carbon source, the seven isolates were tested for biosurfactant production and emulsification activity. The obtained values of the surface tension measurements as well as the emulsification index (E24) are shown in table 3. The highest reduction of surface tension was achieved with B5 and A3 isolates with values of 25.3 mN m−1 and 28.1 mN m−1 respectively. The emulsifying capacity evaluated by the E24 emulsification index was highest in the culture of isolate B4 (78%), B5 (77%) and A3 (76%) as shown in table 3 and figure 2. These emulsions were stable even after 4 months. The bacteria with emulsification indices higher than 50 % and/or reduction in the surface tension (under 30 mN/m) have been defined as potential biosurfactant producers. Based on surface tension and the E24 index results, isolates B5, B4, A3 and A4 are the best candidates for biosurfactant production. It is important to note that, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was choosen for futher analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4.biosurfactant in medium containing respectively 25% and 20% (w/v) NaCl.Stability of biosurfactant activities: The applicability of biosurfactants in several biotechnological fields depends on their stability at different environmental conditions (temperatures, pH and NaCl). For this study, the strain B4 appear very interesting (It can produce biosurfactant at 25 % NaCl) and was chosen for further analysis for biosurfactant stability. The effects of temperature and pH on the biosurfactant production by the strain B4 are shown in figure 4. The biosurfactant produced by this strain was shown to be thermostable giving an E-24 Index value greater than 78% (figure 4A). Heating of the biosurfactant to 100 °C caused no significant effect on the biosurfactant performance. Therefore, the surface activity of the crude biosurfactant supernatant remained relatively stable to pH changes between pH 6 and 11. At pH 11, the value of E24 showed almost 76% activity, whereas below pH 6 the activity was decreased up to 40% (figure 4A). The decreases of the emulsification activity by decreasing the pH value from basic to an acidic region; may be due to partial precipitation of the biosurfactant. This result indicated that biosurfactant produced by strain B4 show higher stability at alkaline than in acidic conditions.Molecular identification and phylogenies of potential isolates: To identify halophilic bacterial isolates, the 16S rDNA gene was amplified using gene-specific primers. A PCR product of ≈ 1.3 kb was detected in all the seven isolates. The 16S rDNA amplicons of each bacterial isolate was sequenced on both strands using 27F and 1492R primers. The complete nucleotide sequence of 1336,1374, 1377,1313, 1305,1308 and 1273 bp sequences were obtained from A1, A2, A3, A4, B1, B4 and B5 isolates respectively, and subjected to BLAST analysis. The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus as shown in table 5. The halophilic isolates A2 and A4 showed 97% similarity with the Halomonas variabilis strain GSP3 (accession no. AY505527) and the Halomonas sp. M59 (accession no. AM229319), respectively. As for A1, it showed 96% similarity with the Halomonas venusta strain GSP24 (accession no. AY553074). B1 and B4 showed for their part 96% similarity with the Salinivibrio costicola subsp. alcaliphilus strain 18AG DSM4743 (accession no. NR_042255) and the Planococcus citreus (accession no. JX122551), respectively. The bacterial isolate B5 showed 98% sequence similarity with the Halobacillus trueperi (accession no. HG931926), As for A3, it showed only 95% similarity with the Staphylococcus arlettae (accession no. KR047785). The 16S rDNA nucleotide sequences of all the seven halophilic bacterial strains have been submitted to the NCBI GenBank database under the accession number presented in table 5. The phylogenetic association of the isolates is shown in figure 5.DICUSSIONThe physicochemical properties of the collected water samples indicated that this water was relatively neutral (pH 7.89) similar to the Dead Sea and the Great Salt Lake (USA) and in contrast to the more basic lakes such as Lake Wadi Natrun (Egypt) (pH 11) and El Golea Salt Lake (Algeria) (pH 9). The salinity of the sample was 224.70 g/L (22,47% (w/v). This range of salinity (20-30%) for Chott Tinsilt is comparable to a number of well characterized hypersaline ecosystems including both natural and man-made habitats, such as the Great Salt Lake (USA) and solar salterns of Puerto Rico. Thus, Chott Tinsilt is a hypersaline environment, i.e. environments with salt concentrations well above that of seawater. Chemical analysis of water sample indicated that Na +and Cl- were the most abundant ions, as in most hypersaline ecosystems (with some exceptions such as the Dead Sea). These chemical water characteristics were consistent with the previously reported data in other hypersaline ecosystems (DasSarma and Arora, 2001; Oren, 2002; Hacěne et al., 2004). Among 52 strains isolated from this Chott, seven distinct bacteria (A1, A2, A3, A4, B1, B4 and B5) were chosen for phenotypique, genotypique and phylogenetique characterization.The 16S rDNA sequence analysis showed that the isolated strains belong to the genera Halomonas, Staphylococcus, Salinivibrio, Planococcus and Halobacillus. Genera obtained in the present study are commonly occurring in various saline habitats across the globe. Staphylococci have the ability to grow in a wide range of salt concentrations (Graham and Wilkinson, 1992; Morikawa et al., 2009; Roohi et al., 2014). For example, in Pakistan, Staphylococcus strains were isolated from various salt samples during the study conducted by Roohi et al. (2014) and these results agreed with previous reports. Halomonas, halophilic and/or halotolerant Gram-negative bacteria are typically found in saline environments (Kim et al., 2013). The presence of Planococcus and Halobacillus has been reported in studies about hypersaline lakes; like La Sal del Rey (USA) (Phillips et al., 2012) and Great Salt Lake (Spring et al., 1996), respectively. The Salinivibrio costicola was a representative model for studies on osmoregulatory and other physiological mechanisms of moderately halophilic bacteria (Oren, 2006).However, it is interesting to note that all strains shared less than 98.7% identity (the usual species cut-off proposed by Yarza et al. (2014) with their closest phylogenetic relative, suggesting that they could be considered as new species. Phenotypic, genetic and phylogenetic analyses have been suggested for the complete identification of these strains. Theses bacterial strains were tested for the production of industrially important enzymes (Amylase, protease, lipase, DNAse and coagulase). These isolates are good candidates as sources of novel enzymes with biotechnological potential as they can be used in different industrial processes at high salt concentration (up to 25% NaCl for B4). Prominent amylase, lipase, protease and DNAase activities have been reported from different hypersaline environments across the globe; e.g., Spain (Sánchez‐Porro et al., 2003), Iran (Rohban et al., 2009), Tunisia (Baati et al., 2010) and India (Gupta et al., 2016). However, to the best of our knowledge, the coagulase activity has never been detected in extreme halophilic bacteria. Isolation and characterization of crude enzymes (especially coagulase) to investigate their properties and stability are in progress.The finding of novel enzymes with optimal activities at various ranges of salt concentrations is of great importance. Besides being intrinsically stable and active at high salt concentrations, halophilic and halotolerant enzymes offer great opportunities in biotechnological applications, such as environmental bioremediation (marine, oilfiel) and food processing. The bacterial isolates were also characterized for production of biosurfactants by oil-spread assay, measurement of surface tension and emulsification index (E24). There are few reports on biosurfactant producers in hypersaline environments and in recent years, there has been a greater increase in interest and importance in halophilic bacteria for biomolecules (Donio et al., 2013; Sarafin et al., 2014). Halophiles, which have a unique lipid composition, may have an important role to play as surface-active agents. The archae bacterial ether-linked phytanyl membrane lipid of the extremely halophilic bacteria has been shown to have surfactant properties (Post and Collins, 1982). Yakimov et al. (1995) reported the production of biosurfactant by a halotolerant Bacillus licheniformis strain BAS 50 which was able to produce a lipopeptide surfactant when cultured at salinities up to 13% NaCl. From solar salt, Halomonas sp. BS4 and Kocuria marina BS-15 were found to be able to produce biosurfactant when cultured at salinities of 8% and 10% NaCl respectively (Donio et al., 2013; Sarafin et al., 2014). In the present work, strains B4 and A4 produce biosurfactant in medium containing respectively 25% and 20% NaCl. To our knowledge, this is the first report on biosurfactant production by bacteria under such salt concentration. Biosurfactants have a wide variety of industrial and environmental applications (Akbari et al., 2018) but their applicability depends on their stability at different environmental conditions. The strain B4 which can produce biosurfactant at 25% NaCl showed good stability in alkaline pH and at a temperature range of 30°C-100°C. Due to the enormous utilization of biosurfactant in detergent manufacture the choice of alkaline biosurfactant is researched (Elazzazy et al., 2015). On the other hand, the interesting finding was the thermostability of the produced biosurfactant even after heat treatment (100°C for 30 min) which suggests the use of this biosurfactant in industries where heating is of a paramount importance (Khopade et al., 2012). To date, more attention has been focused on biosurfactant producing bacteria under extreme conditions for industrial and commercial usefulness. In fact, the biosurfactant produce by strain B4 have promising usefulness in pharmaceutical, cosmetics and food industries and for bioremediation in marine environment and Microbial enhanced oil recovery (MEOR) where the salinity, temperature and pH are high.CONCLUSIONThis is the first study on the culturable halophilic bacteria community inhabiting Chott Tinsilt in Eastern Algeria. Different genera of halotolerant bacteria with different phylogeneticaly characteristics have been isolated from this Chott. Culturing of bacteria and their molecular analysis provides an opportunity to have a wide range of cultured microorganisms from extreme habitats like hypersaline environments. Enzymes produced by halophilic bacteria show interesting properties like their ability to remain functional in extreme conditions, such as high temperatures, wide range of pH, and high salt concentrations. These enzymes have great economical potential in industrial, agricultural, chemical, pharmaceutical, and biotechnological applications. Thus, the halophiles isolated from Chott Tinsilt offer an important potential for application in microbial and enzyme biotechnology. In addition, these halo bacterial biosurfactants producers isolated from this Chott will help to develop more valuable eco-friendly products to the pharmacological and food industries and will be usefulness for bioremediation in marine environment and petroleum industry.ACKNOWLEDGMENTSOur thanks to Professor Abdelhamid Zoubir for proofreading the English composition of the present paper.CONFLICT OF INTERESTThe authors declare that they have no conflict of interest.Akbari, S., N. H. Abdurahman, R. M. Yunus, F. Fayaz and O. R. Alara, 2018. Biosurfactants—a new frontier for social and environmental safety: A mini review. Biotechnology research innovation, 2(1): 81-90.Association, A. P. H., A. W. W. Association, W. P. C. 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Nitrogen is a paramount important essential element for all living organisms. It has been found to bea crucial structural component of proteins, nucleic acids, enzymes and other cellular constituents which are inevitable for all forms of life. In the atmosphere, the percentage of nitrogen is very high (N2, 78%) compared to other inorganic gases. However, most organisms have practically no direct access to this nitrogen. While plants can not directly uptake nitrogen from atmosphere, they are capable of assimilating other forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-). For agricultural crop production, artificial fixation of nitrogen is heavily utilized and it is an expensive process that requires high temperatures (at least 400 °C) and pressures (around 200 atm). It has been conspicuously demonstrated that indiscriminate use of fertilizer hampers soil physical, chemical and micro biological properties and also a potential risk to environment e.g. water quality. Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching. Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, only 1-2% of the nitrogen fixation in the world occurs through the natural process of lightening. Notably, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae. Against the backdrop, we are accentuated on an environmentally friendlyand themost sustainable approach to increase productivity for legume and non-legume crops. Till today, the term biological nitrogen fixation (BNF) has received much attention as a sustainable alternative; this process facilitates atmospheric nitrogen to convert into ammonia by rhizobia in specialized plan organs termed “root nodules”. This review article seeks to better understand plant mechanisms involved in the development of root nodules in soybean. Soybean (Glycine max) is one of the most important oil crops and a source of animal feed protein in the world. It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Oldroyd, Murray et al. 2011). Biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. A better understanding of how nodules form and function is important for selection or generation of soybean genotypes with better nitrogen fixation capacity. Soybean nodules originate from root cortex via de novo cell differentiation (Oldroyd 2013). Consequently, two major nodule development zones are formed for instance; the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. It is not clear what early signaling pathways driving the conspicuous development of the nodule zones. My research is aimed at filling this knowledge gap by illustrating the molecular signatures that paves the way to cellular differentiation in root nodule development in soybean. Based on initial evidence obtained by the Subramanian lab, we hypothesize that microRNAs (miRNAs) play important regulatory roles in spatio-temporal expression of their target genes during nodule developmental in soybean. For instance, the regulation of auxin sensitivity by miR160 has been found to be crucial for formation of nodule primordia and vasculature in the parenchyma (Marie Turner 2013). Against this backdrop, this review article focused on nuclear and cytoplasmic transcriptome as well as miRNA profiles of parenchyma and primordial tissues and determine the relative abundance and differentially expressed mRNAs and regulatory role of miRNAs in cell differentiation and nodule development. Root nodule a sustainable alternative to fix atmospheric nitrogen Atmospheric nitrogen percentage is very high (N2, 78%) compared to other inorganic gases (Mary Elvira 1932). However, most of the organisms have practically no direct access to this nitrogen. Nevertheless, plants can not directly uptake nitrogen from atmosphere but they are capable of assimilating only very specific forms of nitrogen, for example ammonium (NH4+) and nitrate (NO3-) (Bytnerowicz and Fenn 1996, Peter M. Vitousek 1997) (Sponseller, Gundale et al. 2016). Virtually, nitrogen has been found to be a crucial structural component of proteins, nucleic acids, enzymes, and other cellular constituents which are inevitable for all forms of life (O'Brien, Vega et al. 2016). For agricultural crop production, artificial fixation of nitrogen is heavily utilized. It is an expensive process that requires high temperatures (approx. 400 °C) and pressures (approx. 200 atm) (Witschi 2000). It has been conspicuously demonstrated that indiscriminate use of N fertilizer hampers the diversity of the bacterial community and decreases soil C and N concentrations (Verzeaux, Alahmad et al. 2016). Notably, it has been demonstrated as a potential risk to environment e.g. water quality (Zhao, Sha et al. 2016) (Sponseller, Gundale et al. 2016). Besides, chemically manufactured fertilizers are depleted from soils in various ways, for instance; denitrifying bacteria, volatilization, and leaching (Johnson 1996, Peter M. Vitousek 1997). Consequently, it results relatively poor availability of nitrogen to get into plants. On the flipside, over 90 % of the nitrogen fixation in the world occurs through the natural process of lightening and microorganisms. Furthermore, microbial fixation is well characterized in diazotrophs for example; Rhizobia and Frankia, and blue-green algae (Cheng 2008). It has been demonstrated that Bradyrhizobium strains substantially escalated soybean grain yield, and protein content up to 57% and 26%, respectively (Zimmer, Messmer et al. 2016). Against the backdrop, we are accentuated on an environmentally friendly and a sustainable approach to increase the productivity for legume and non-legume crops. Literature mining depicted that biological nitrogen fixation in soybean nodules reduces the use of chemical nitrogen fertilizers resulting in cost-savings to producers and minimizes environmental damage due to nitrogen run-off. Rhizobia infection leads to the root nodule development In the natural environment, plants are continuously confronted with pathogenic and symbiotic microbes. Symbioses involves mutual exchange of diffusible signal molecules, first endophytic bacteria (rhizobia) are attracted by the plant root exudates flavonoids which are perceived and triggered the bacterial nodulation (nod) genes. Consequently, the bacteria synthesize specific lipochito-oligosaccharides, called nodulation (Nod) factors. This signal is perceived by the LysM receptor like kinase of host plant, it induces the root hair curling, and bacteria get access into the host epidermis through infection threads (ITs) and initiate cell division within the root cortex, leading to the progression of the root nodule meristem. In later stages of the interaction, bacteria are released from the infection threads into the plant cells, surrounded by membrane of plant origin. These bacteria multiply within the host cells and differentiate into the nitrogen fixing bacteroids (Udvardi and Day 1997) (Oldroyd 2013). Till now, integration of genetic and genomic approaches has revealed twenty-six genes to be involved in nodule development of Medicago truncatualaand Lotus japonicum (Kouchi, Imaizumi-Anraku et al. 2010). In addition, deep sequencing of the Medicago truncatularoot transcriptome has uncovered thousands of genes to be induced during Nod factor signaling and its resulting ethylene (ET) biosynthesis throughout the multiple development stages of indeterminate nodule (Larrainzar, Riely et al. 2015). Albeit the molecular mechanism of such regulation is not well understood. There has been a large-scale transcriptome analysis of B. japonicum-inoculated and mock-inoculated soybean root hairs. It has showed that a total of 1,973 soybean genes differentially expressed during root hair infection, particularly NFR5 and NIN genes (Libault, Farmer et al. 2010). Nevertheless, the signaling mechanisms directing the cellular differentiation of nodule are not known. Soybean root nodule organogenesis Soybean (Glycine max) has a genome size of 1.1 to1.5 Gb, it is partially diploidized tetraploid. It is one of the most important oil crops and a source of animal feed protein in the world (soybase.org/sb_about.php). It has a salient feature to fix atmospheric nitrogen through symbioses with compatible rhizobia that yields to determinate type nodule (Udvardi and Day 1997) (Oldroyd, Murray et al. 2011). Notwithstanding of the economic and environmental importance, there has been very few studies about quantitative trait loci (QTL) that controlling BNF traits, for instance nodule number, ration of nodule dry weight with nodule number, and shoot dry weight (SDW). It has been reported via composite interval mapping that approximately six QTLs bears very small effect on BNF traits (Santos, Geraldi et al. 2013). Besides, it has been demonstrated in earlier studies that nodules originate from root cortex via de novo cell differentiation into two different cell types, parenchymal and primordium (Celine Charon 1997) (Oldroyd&Downie 2008; Oldroyd 2013). In addition, early nodulin genes in legume for instance; Enod 40 gene reported to be expressed in root pericycle during the rhizobia infection and later it occupied in the dividing cortical cells (H. Kouchi and S. Hata 1993). Among the two major nodule development zones, the nodule primordium (Npr) in the middle which is encircled by nodule parenchyma (Npa). At later time point, the Npr gives rise to N-fixation zone and the Npa holds vascular bundles. Lately, a β- expansin gene, GmEXPB2 fused with GUS reporter gene which was observed to be preferentially expressed in nodule vascular trace and nodule vascular bundles. It indicated that GmEXPB2 might be crucial for nodule organogenesis. Over expression of GmEXPB2 contrast to suppressed GmEXPB2 transgenic lines found to be escalated nodule number, nodule mass and nitrogenase activity. It further suggested that GmEXPB2 might have influenced over root architecture, nodule formation and development, and profoundly yielding to biological N2 fixation (Li, Zhao et al. 2015). Even though, it is not clear what early signaling pathways driving the conspicuous development of the nodule zones. Against the back drop, to understand the regulation of auxin sensitivity by miR160 which is believed to be crucial for the formation of nodule primordia (Marie Turner 2013). Figure 1 a. Illustrating the progression of root nodule development through Rhizobial bacterial infection in the plant root leading to the determinate nodule (Oldroyd 2013). b. Nodule development zones A. Nodule primordial zone (Enod 40 gene) in the middle B. surrounding parenchyma (Enod 2 gene), differentiated from cortex (collected from Sen Subramanian lab). Regulatory small RNAs biogenesis and its molecular functions Regulatory small RNAs are ranged between 20 to 24 nucleotides which are ubiquitous elements of endogenous plant transcriptomics, a common response to exogenous viral infections and introduced double-stranded RNA (Axtell 2013). Three core enzymes families, for instance; RNAdependent RNA polymerase (RDR), Dicer like (DCL), and Argonaute (AGO) proteins paves the way of small RNA biogenesis and function in plants. Firstly, ribonuclease type III or DICERLIKE1 involves in the yield of a fold-back precursor RNA or primary miRNA (primiRNA) transcripts using an RNA templates in the nuclei. Later, the resulting miRNA-miRNA duplex which is originated in nucleus then translocated into cytoplasm. The guided miRNAmolecule is incorporated into ARGONAUTE (AGO) to form an active RISC complex to specific target RNAs that are complementary to the miRNA, and this process eventually follows up mRNA cleavage, represses the translation of the mRNAs or Chromatin modification. This phenomenon accentuated as an inhibition or silencing of the gene expression, which play a crucial role in the developmental process in plant and animal (Chapman and Carrington 2007) (Axtell 2013). Fig. 2 Regulation of gene expression events via RISC complex (modified from https://www.google.com/?gws_rd=ssl#q=mirna+picture+in+plants accessed on 7th February, 2016) Fig. 3 Gene expression events occurring in typical plant cell (modified https://www.google.com/search?q=transcription+and+translation accessed on 7th February, 2016) It has been found in several studies that most plant miRNAs are non-coding RNA, and small 21-24 nucleotide long (Cuperus, Fahlgren et al. 2011). It requires DCL1-clade DCL for their biogenesis and AGO1-clade AGO for their function (Wu, Zhou et al. 2010, Manavella, Koenig et al. 2012). In rice (Oryza sativa), DCL3 has been reported in the biogenesis of 24nt long miRNA that incorporated in AGO4 to regulate the target gene expression primarily through mRNA cleavage (Wu, Zhou et al. 2010). Argonaute proteins (AGO) form RNA inducing silencing complexes (RISC) with small RNAswhich is known as post-transcriptional gene silencing. It has typically four domains, for instance:N-terminal, PAZ, MID and PIWI domains. The MID-PIWI lobes are belongs to the C-terminus. It has been studied that MID-domains contains the specificity loop to recognize and bind to the 5’-phosphate of smRNAs. The PIWI domains contained the catalytic active site D-E-D-H/D. PAZ domain anchored the 2-nt overhang at the 3’ end of miRNAs. The N-terminal domain involved in the separation of miRNA-miRNA duplex and the slicer activity of the mRNA (Song, Smith et al.2004). There has been an expansion and duplications of AGO family members during plantevolution (Singh, Gase et al. 2015). The functional diversification of AGOs is indicating sRNAdirected regulatory pathways. The binding preference of AGO and sRNA is mainly assigned by the sequence of sRNA. In Arabidopsis, 10 AGO have been extensively studied (Liu et al. 2014). It has been demonstrated that AtAGO10 like AtAGO1, it recognized distinct structural features in miR165/miR166 duplex than involved by AtGO1. AtAGO10 found to regulate shoot apical meristem by decoying miR165/miR166 and subsequent repression of homeodomain-leucinezipper (HD-ZIP) gene expression (Zhu, Hu et al. 2011). Notably, 22 AGO proteins have been reported in Soybean (Glycine max). It has been found that genome duplication in Soybean resulted such a proliferation of AGOs. For example: its genome encodes two copies of AGO1, AGO2, AGO5, AGO4/9, AGO6 and AGO7 (Xiang Liu 2014). However, the molecular function of the plant AGO genes yet not very clear. There are several miRNA families that are conserved across the vast evolutionary distances from flowering plants to mosses (Cuperus, Fahlgren et al. 2011). It has been observed in another study that miRNA, and its target pairing found to be stable for a prolonged periods of plant evolution. On the flip side, another group demonstrated that conserved plant miRNAs and their targets are to somehow flexible. For instance; miR159 is a highly conserved miRNA that targets not only a subset of MYB mRNAs but also observed to target a non MYB mRNA, SGN-U567133 (Buxdorf, Hendelman et al. 2010). A mutant tomato transgenic line (miR159-resistant line) showed higher level of the SGN-U567133 transcript and exhibited defects in leaf and flower development. This result suggests that miR159 involves in a post-transcriptional regulation. Additionally, it is found to be crucial for the normal tomato development. Recently, the identification of miRNAs in the regulation of photoperiodic pathways in soybean have been reported through high throughput sequencing and qRT-PCR. Six libraries were constructed using Illumina Solexa, for instance; 0, 8, and 16 h under short day treatment, similar time points considered for the long the long day treatment. A total of 163 miRNAs families were reported which covered 318 plant miRNAs, and unclassified 81 novel predicted miRNAs. As expected, significant differences in abundance between short day and long day treatment was observed (Wenbin Li 2015). These findings provided evidence of miRNA in the regulation of flowering time that ultimately affects the seed yield and quality of soybean. The complex regulatory network of miRNA-mRNA interactions during viral infection has been revealed via small RNA seq (sRNA), degradome seq, and genome-wide transcriptome analysis. There has been a total of 253 soybean miRNAs found to be two-folds abundance compared with mock-inoculated control demonstrated through sRNA seq analysis. Among them 105 miRNAs were identified as potential targets of 125 transcripts that has been validated by degradome seq analyses. In addition, 2679 genes were detected via genome wide transcriptomic analysis. These genes have been differentially expressed during infection of soybean mosaic virus and among them 71 genes projected to induce in defense response (Hui Chen 2016). These findings suggested the regulatory role miRNA that governed the target gene expression during viral infection. Furthermore, the regulatory role of microRNAs (miRNAs) during Soybean- Bradyrhizobium japonicum mutualistic association was studied first by Subramanian et al. 2008. They sequenced approximately 350000 small RNAs of soybean root sample which were inoculated with B. japonicum. It helps to detect 20 conserved miRNAs loci based on the similarity to miRNAs in another plant species. In addition, 35 novel miRNAs were identified based on potential hairpin forming precursors in Soybean EST as well as shotgun genomic sequences (Subramanian, Fu et al. 2008). These findings advocated the potential role of miRNAs in the regulation of legumerhizobiumsymbiosis. In another study, 120 hairpin-forming precursor genes have been identified in soybean by Turner et al. In addition, they reported three novel miRNAs for instance; miR160, miR164 and miR393 found to be involved in auxin signaling (Turner, Yu et al. 2012). Moreover, the plant hormone auxin is thought to have a pivotal role in nodule organogenesis in determinate and indeterminate type of nodule. It indicates a redundancy and diversity of miRNAs family members that governs the formation of root nodule. It has been illustrated that auxin receptor gene family hushed by over expressed microRNA393. These plant roots found to be hypersensitive to auxin and yielded normal nodule. This observation advocated that only minimal/reduced auxin signaling is required for determinate nodule development. Likewise, overexpressed microRNA160 hushed a set of repressor auxin response transcription factor. These plant roots were hypersensitive to auxin and observed not to be reluctant in epidermal responses to rhizobia. Notably, it yielded to lower sized nodule primordium (Marie Turner 2013). This observation indicated that auxin hypersensitivity inhibits nodule organogenesis Organ specific expression of profile of miRNA and the potential targets were also studied. Two genes (Glyma10g10240 and Glyma17g05920) which were the target of miR169 but detected to be highly expressed in soybean nodule. Likewise, three potential targets of gma-new-miR13587 demonstrated to be highly expressed in the nodules than in the roots. As expected, gma-newmiR13587 found to be poorly expressed in the nodules than in the roots (Turner, Yu et al. 2012). There was an inverse expression pattern observed in between roots and nodules. Li et al., studied the transgene expression of three novel miRNAs namely, miR482, miR1512, and miR1515 in Soybean. They noticed a significant increase of nodule numbers while root length and later root density were normal in all tested miRNA lines. As expected, there were differential expression of these miRNAs in supernodulating and nonnodulating soybean mutants. They reported that 6 novel miRNAs decoyed 22 predicted target genes. And it was estimated via real time polymerase chain reaction and qRT-PCR (Li, Deng et al. 2010). It advocates that miRNAs have the signatory roles in soybean nodule development. Sequencing of small RNAs and Parallel analysis of RNA ends (PARE) libraries revealed to identify 284 nodule miRNAs, more than 500 target genes, and including 178 novel soybean miRNAs. It has been reported that ENOD93 only found to be expressed in nodule tissue not in other plant parts of Soybean. Ectopic expression of miR393j-3p and RNAi silencing approach to ENOD93 expression showed a significant reduction in nodule formation (Zhe Yan 2015). Therefore, this study showed a list of miRNAs and their potential target of nodulation genes. In the model legume (Medicago truncatula), 25 conserved miRNA families and 100 novel miRNA reads were detected by high-throughput sequencing. The expression of MtHAP2-1 (encodes a CCAAT binding transcription factor) to meristematic zones was restricted by miR169a which is found to be critical for the development of indeterminate type of nodule (Combier, Frugier et al. 2006). In another study, HDZIPIII transcripts were inhibited by overexpression of miR166, it dropped the number of symbiotic nodule and lateral root (Boualem, Laporte et al. 2008). To get insights into key genes of nodule zones, transcript profiles of specific cells/tissues were investigated at different time points from indeterminate nodules of M. truncatulausing laser capture micro dissection. It has been demonstrated from the comprehensive gene expression map that selected genes enriched in different cell/tissue types (Limpens, Moling et al. 2013). These findings indicated that organ specific gene expression could be controlled by the presence or absence of miRNAs. Recently, Agrobacterium rhizogenesmediated hairy root transformation has been applied as tool for exploring cell type specific gene expression in tomato. Cell type or tissue specific promoter introduced into INTACT and TRAP constructs via gateway cloning technology to develop binary vectors. INTACT method used to capture biotin tagged nuceli from specific cell types and TRAP method used for profiling of mRNAs or foot printing of individual ribosomes (Ron 2014). TRAP methodology is not required tissue fixation or single cell suspension. It has been successfully used to date in organisms ranging from D. melanogaster to mice and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts (Heiman, Kulicke et al. 2014). In this study, we would perform polysome isolation deploying gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18. Over the last one decade, there has been several microarrays-based studies which characterized transcriptional variations deployed in nodule formation. It has been embedded with couple of shortcomings, for instance; relative late time points study, incomplete representation of plant genes,discrimination of close paralogs, and reduced sensitivity. Lately, next generation sequencingtechnology have widened the horizon of transcription analyses in different legume species to detectsymbiosis induced changes in late nodule developmental stages. Against this backdrop, we areaccentuated to reveal early transcriptional changes induced in determinate type of soybean noduleby Bradyrhizobium japonicum. In determinate type of nodule, two major nodule development zones are formed for instance, the nodule primordium (Npr) in the middle and it is encircled by nodule parenchyma (Npa). At later time point, the Npr converted to N-fixation zone and the Npa contained vascular bundles. Of these facts, it is not clear what early signaling pathways driving the conspicuous development of thenodule zones. In this context, mechanisms regulate the distinct gene expression profiles in Npr andNpa cell types has not understood clearly. The proposed research study is aimed at filling this knowledge gap byillustrating the molecular signatures that paves the way to cellular differentiation in root noduledevelopment in soybean considering four different time points (5 dai, 7 dai, 10 dai& 14 dai). The hypothesisis microRNAs(miRNAs) play important regulatory roles in spatio-temporal expression of their target genesduring nodule developmental in soybean. For example, a gradient of microRNA localizationbetween nodule primordium and parenchyma cells could result in distinct differentiation of thesecell types. To test this hypothesis, one has to obtain both cell type-specific miRNA andtranscriptome (miRNA target) profiles. Since, the majority of miRNA regulation occurs in thecytoplasm, we reasoned that comparison of nuclear and ribosomal transcriptome profiles wouldreveal genes whose expression is potentially regulated by post transcriptional mechanisms such asmiRNA cleavage. Combining this information with cell type-specific miRNA profiles, andto test the above hypothesis and identify key miRNA-target pairs important for nodule celldifferentiation. The use of translating ribosome affinity purification (TRAP) of nodule zonecells, namely from parenchyma and primordial tissues, to obtain cytoplasmic transcriptomes data. Techniques to determine cell type specific expression profiles: TRAP methods TRAP is termed translating ribosome affinity purification, combines cell-type-specific transgene expression with affinity purification of translating ribosomes. It supersedes the need for tissue fixation, and facilitates to study the cell type-specific mRNA profiles of any genetically defined cell type. It has been successfully used to date in organisms ranging from D. melanogaster to mice, and human cultured cells. Multiple ribosomes or Polyribosomes (polysomes) are engaged in translation on a single mRNA. To evaluate the translation state of an mRNA, ribosomal subunits, ribosomes, and polysomes can be isolated from detergent-treated cell extracts. In this study, the polysome isolation using gene cassettes ENOD40p:HF-GFP-RPL18 for primordial tissues, and ENOD2p:HF-GFP-RPL18 for parenchymal tissues in Glycine max root nodules that express an epitope tagged version of ribosomal protein L18 RPL18(Heiman, Kulicke et al. 2014, Ron 2014). Relative abundance and differentially expressed mRNAs profile in two different tissue specific zones would help to understand the effect of regulatory role of miRNAs in cell differentiation and nodule development. References: Axtell, M. J. (2013). "Classification and comparison of small RNAs from plants." Annu Rev PlantBiol 64: 137-159. 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"Arabidopsis Argonaute10 specifically sequesters miR166/165 to regulate shoot apical meristem development." Cell 145(2): 242-256.

All‐polymer photovoltaics (all‐PPVs) that operate under both indoor and outdoor lighting conditions require active layers with appropriately adjusted optical‐absorption ranges. However, the optical absorption of a conventional donor–acceptor binary blend is restricted to the combined absorption bands of its components. Herein, a new conjugated block copolymer (CBC) acceptor, b‐PYT, was designed by integrating polymer acceptor blocks of wide and narrow band gaps in a single structure. Such combination results in the wide absorption range (550–850 nm) of b‐PYT that matches the emission of both artificial and solar light. The b‐PYT CBC acceptor is more crystalline than the corresponding random terpolymer, r‐PYT, owing to improved interactions between its macromolecular acceptor units. Despite exhibiting slightly inferior outdoor performance compared to that of devices using the homopolymer BTTP‐T, the PM6:b‐PYT‐based devices deliver superior power conversion efficiency (PCE) under indoor light emitting diode (LED) light owing to better matched absorption and emission spectra of b‐PYT and a cold white LED, respectively. Additionally, it is worth highlighting that PM6:b‐PYT‐based all‐PPVs can maintain approximately 87% of the initial PCE even after 600 min of thermal aging at 150 °C, which demonstrates the superior thermal stability compared with those of all‐PPVs that use traditional binary active layers.This article is protected by copyright. All rights reserved.

Background: Qinzhi Zhudan Formula (QZZD), optimized from Angong Niuhuang Wan, consists of Radix Scutellariae, Fructus Gardeniae and Pulvis Fellis Suis. We had investigated the neuroprotective effects of QZZD and its active components, and demonstrated that it could treat cerebral ischemia and dementia through multiple pathways and mechanisms. Nevertheless, toxicological data on this formula still remains limited. In the study, we sought to examine the toxicological effects of QZZD during the treatment and recovery periods.Methods: We investigated potential toxicities of QZZD in Sprague-Dawley (SD) rats via 28-day gavage administration. SD rats were randomly divided into control group and treatment groups of A (0.5 g/kg/d QZZD), B (1.5 g/kg/d QZZD), and C (5.0 g/kg/d QZZD). The 56-day course includes treatment period (administration with water or QZZD once a day for 28 consecutive days) and recovery period (28 days). The rats received daily monitoring of general signs of toxicity and mortality, as well as weekly determination of body weight and food consumption. Moreover, the complete blood cell count, biochemistry, coagulation, and urine indicators, organ weights, and histopathological report were analyzed respectively at the end of the treatment and recovery periods.Results: There was no death related to the active pharmaceutical ingredients of QZZD during the treatment period. The maximum no observed adverse effect level (NOAEL) was 0.5 g/kg/d, which is approximately 16.7 times of the equivalent dose of clinical dose in rats. In group TB (1.5 g/kg/d QZZD) and TC (5.0 g/kg/d QZZD), there were adverse effects of blue coloring of tail skin, weight loss, a significant increase of total bilirubin (TBIL), blackening of liver and kidney in gross examination, hyperplasia of bile duct and karyomegaly of hepatocytes in histopathological examination. Besides, in females rats, the food consumption was reduced, while in male rats, there was decrease in triglycerides (TG) and slight increase in white blood cell (WBC) count and neutrophils. In group TC (5.0 g/kg/d QZZD), the indicators of red blood cell (RBC) count, hemoglobin (HGB) and hematocrit (HCT) were decreased slightly, while the platelet count (PLT) was increased. However, these changes were not considered to be toxicologically significant because they resolved during the recovery period.Conclusion: Overall, QZZD exhibited a good safety profile. The maximum no observed adverse effect level was 0.5 g/kg/d, and no target organs toxicity were identified. The present findings might confirm the safety of QZZD in clinical practices.

Objective: The primary objective of the study was to assess the immunogenicity of an HIV-1 Gag conserved element DNA vaccine (p24CE DNA) in people with HIV (PWH) receiving suppressive antiretroviral therapy (ART). Design: AIDS Clinical Trials Group A5369 was a phase I/IIa, randomized, double-blind, placebo-controlled study of PWH receiving ART with plasma HIV-1 RNA less than 50 copies/ml, current CD4+ T-cell counts greater than 500 cells/μl, and nadir CD4+ T-cell counts greater than 350 cells/μl. Methods: The study enrolled 45 participants randomized 2 : 1 : 1 to receive p24CE DNA vaccine at weeks 0 and 4, followed by p24CE DNA admixed with full-length p55Gag DNA vaccine at weeks 12 and 24 (arm A); full-length p55Gag DNA vaccine at weeks 0, 4, 12, and 24 (arm B); or placebo at weeks 0, 4, 12, and 24 (arm c). The active and placebo vaccines were administered by intramuscular electroporation. Results: There was a modest, but significantly greater increase in the number of conserved elements recognized by CD4+ and/or CD8+ T cells in arm A compared with arm C (P = 0.014). The percentage of participants with an increased number of conserved elements recognized by T cells was also highest in arm A (8/18, 44.4%) vs. arm C (0/10, 0.0%) (P = 0.025). There were no significant differences between treatment groups in the change in magnitude of responses to total conserved elements. Conclusion: A DNA-delivered HIV-1 Gag conserved element vaccine boosted by a combination of this vaccine with a full-length p55Gag DNA vaccine induced a new conserved element-directed cellular immune response in approximately half the treated PWH on ART.

The Trans-Mexican Volcanic Belt (TMVB) is an active volcanic chain being deformed by an intra-arc extensional fault network. Although several crustal earthquakes with magnitude>7 have originated in the TMVB since the 16th century, the background seismicity of this geological structure is very low and the region is usually considered of low seismic hazard. In this study, we present an updated probabilistic seismic hazard model of the TMVB. The seismicity catalog used here includes forty-three historically and instrumentally recorded earthquakes, from 1858 to 2014; five of these are large earthquakes that occurred in the TMVB during the XIXth century. Due to the lack of a statically representative sample, we propose, in a qualitative manner, the seismicity catalog is complete for M≥4 since 1964 and for M≥6 since 1858. Moreover, we introduce three different earthquake frequency-magnitude relations. The first one is a conventional Gutenberg Richter fit of the distribution of the instrumentally recorded earthquakes data. The other two are non-conventional, semi-parametric approaches that integrate the historical and the instrumental data to determine seismicity rates in the region. Our preferred model (seismicity model B) fits separately the instrumental and the historical data and merge the two fits into one curve. A uniform seismic hazard (USH) of the TMVB for a return period of 500 years was calculated considering three major sources of earthquakes: 1) Subduction thrust-faulting events in the Middle American Trench (MAT); 2) Earthquakes within the subducted Cocos plate and, 3) Shallow crustal earthquakes in the TMVB. According to the seismicity model B, the average recurrence time of a M≥7 earthqua-ke on the TMVB is approximately 150 years. In contrast, the recurrence time estimated from the instrumental catalog is 12,000 years. The results of this seismicity model, which is based on historical and instrumental data, agrees also with the return periods of prehistoric earthquakes, estimated for short segments of the fault system in the TMVB in paleoseismological studies. When comparing the results of our preferred seismicity model, the PGA estimated using only the instrumental seismicity are 18 to 56% smaller than those predicted by the model using the historical catalog.

Abstract Disclosure: L. El Musa Penna: None. W. Medina-Torres: None. L.R. Sepulveda-Garcia: None. L.N. Madera Marin: None. A. Rosado-Burgos: None. M.A. Ortiz-Rivera: None. B. Torres Rivera: None. M. Alvarado: None. M. Ramirez: None. L.A. Gonzalez-Rodriguez: None. M. Marcos Martínez: None. M. Correa Rivas: None. N. Bracero, MD: None. Hyperandrogenism in premenopausal women is most commonly associated to polycystic ovarian syndrome (PCOS). Approximately 10% of females in reproductive age present with clinical and/or biochemical findings of androgen excess, such as hirsutism, acne, alopecia, oligo-amenorrhea or if hyperandrogenism is severe it can lead to extreme virilization. Androgen excess can also contribute to insulin resistance. In this case we discuss a patient with severe hyperandrogenism, extreme insulin resistance and a rare cause of androgen excess in a woman of childbearing age. 34-year-old female patient G0P0 with type 2 diabetes mellitus (T2DM) on continuous insulin infusion system (CIIS), familial partial lipodystrophy, PCOS and severe hyperandrogenism, who was referred to our clinics for management of uncontrolled T2DM. Patient was on CIIS with regular insulin U-500 using a total daily dose of 95 units. She referred amenorrhea for the past 12 years and significant progression of hirsutism, alopecia and acanthosis nigricans in the past two years. Patient had clinical findings of hyperandrogenism such as hirsutism evaluated with modified Ferriman-Gallwey scale with a score of 32, alopecia Ludwig class 3 and marked acanthosis nigricans in neck and abdomen.Pre-operative laboratories: hemoglobin (Hgb) level 15.13 g/dL, hematocrit (Ht) 44.59 %, total testosterone level 525 ng/dL (13-53 ng/dL) and DHEAS 113 ug/dL (95.8-511.7 ug/dL), suggesting an ovarian source of androgen excess. Transvaginal ovarian ultrasound showed at the posteromedial edge of right ovary a hyperechoic structure measuring 1.5 cm long x 1.0 cm AP representing a lesion of unknown etiology. After discussion with patient a decision for oophorectomy was made. Pathology report described the ovary negative for neoplasia with findings consistent with hyperthecosis. Laboratories two weeks post-operative showed significant decrease in total testosterone to 81 ng/dL and in Hgb/ Ht (12.50 g/dL and 37.8% respectively). Insulin requirement decreased and she was able to be transitioned to U-100 insulin lispro with a total daily dose of 100. Four weeks after surgery patient had her menstrual period. Ovarian hyperthecosis is a disorder where there is an increased tissue with luteinized theca cells in the ovarian stroma; these cells are ovarian interstitial cells that differentiate into steroidogenically active cells. It is most commonly observed in post-menopausal women, but it has been described in women of childbearing age presenting with worsening hirsutism, virilization and insulin resistance. Monitoring patterns and progression of androgen excess is important in premenopausal women with a diagnosis of PCOS. Severe biochemical or clinical presentation, as well as progression of hyperandrogenism should increase suspicion of additional pathological entities as it will improve patient’s quality of life with the appropriate management. Presentation Date: Saturday, June 17, 2023

Forsythia suspensa (Thunb.) Vahl (Oleaceae) is a well-known traditional Chinese medicine. It exhibits antioxidant activity and exerts antibacterial, antiviral, and antiemetic effects (Li and Chen 2005). From May 2020 to October 2021, a disease was observed on field-grown forsythia plants in Lingbao City, Henan Province, China (110°33′25.74″E, 34°30′19.34″W). The diseased plants were characterized by stem rot, retarded growth, a declined fruit quality, and in extreme cases, death of F. suspensa. Approximately 3.0% to 5.0% individuals exhibited stem rotten in the main branches. On average, 60% of the branches of infected individual trees were affected by this disease. During the initial infection stage, the bark of the plants was raised and curled, and the xylem and phloem of the stems turned brown color, whereas in the late stage of the infection, the outer bark had dried and become detached, and the inner xylem and phloem had blackened. Upon infection, the growth of plants was reduced, and the main branches became desiccated as the disease progressed. We randomly selected five diseased branches from five plant fields, the bark tissues (about 25 mm²) of which were surface-sterilized in 75% ethanol for 30 s, treated with 1% NaClO for 5 min, rinsed five times with sterile water, and placed on potato dextrose agar (PDA). After incubating 3 days, 20 clones were observed, and two representative strains (FSJF11 and FSJF13, three replicates for each) was selected for intensive study. Samples of these strains have been deposited in Institutes of Traditional Chinese Medicine, Henan University. On PDA, the colonies of FSJF11 were initially white and fluffy in appearance, later turning gray, and finally black. The vigorously growing hyphae were branched and septate. However, no spores was observed during culture. FSJF13 colonies were rapidly growing, initially white in color and later turning gray. After culturing for 20 days, black conidia appeared and yellow conidial horns were released. The alpha conidia were elliptical, slightly pointed at both ends, and each end possessed an oil ball (6.40±0.60 × 1.86±0.25 μm). The beta conidia were slender, linear, and hook shaped with a slightly curved end (28.92±2.81 × 0.96±0.14 μm). DNA of the isolates was extracted using a Fungal Genome DNA Extraction Kit (Sangon Biotech, Shanghai), and selected genes were amplified using the primer pairs ITS1/ITS4 (Tian et al. 2018), LROR/ LR5, and NS1/NS4 (Aiello et al. 2020). Sequences have been deposited in GenBank (ITS: MW834579 and MW834580; LSU: MW829566 and MW829567; SSU: MW834582 and MW834583). The lengths of the amplified ITS, LSU and SSU sequences were 491, 759, and 1013 bp for FSJF11, respectively, and these in FSJF13 were 543, 927, and 901 bp, respectively. The ITS, LSU, and SSU sequences of FSJF11 were found to have sequence identities of 99.19%, 100%, and 100% with those of Botryosphaeria dothidea stains AY259092, EU673243, and Eu673174, respectively, and a phylogenetic tree was constructed based on the concatenated sequences (ITS, LSU, and SSU) revealed that FSJF11 and B. dothidea formed a clade with 96% support. A BLAST search of the Genbank database revealed that the ITS sequence of FSJF13 showed 99.81% identity with that of Phomopsis velata (MN183778). Given that no LSU or SSU sequences of this species are currently available, we constructed a phylogenetic tree based solely on ITS sequences, which revealed that FSJF13 and P. velata formed a clade with 99% support. Based on the morphological and molecular characteristics(Qi et al. 2007), the isolates of FSJF11 and FSJF13 were identified as B. dothidea and P. velata, respectively. Healthy branches of F. suspensa were wounded in vitro after inoculating active fungal cake of B. dothidea or P. velata (diameter = 5 mm) on the bark, and control branches were treated with PDA. In total, each branch was inoculated via four holes were inoculated on each branch, and three branches were used for each treatment. The inoculation sites were covered with a piece of wet absorbent cotton and then wrapped with plastic film, and the branches were incubated at 26 °C. The branches continued to grow after removal of the cotton and the film on the fourth day. All inoculated points on the branches showed lesions similar to those observed in the field, whereas the control branches were asymptomatic. The pathogenicity rates of FSJF11 and FSJF13 (three replicates for each) were 66.67% and 83.33%, respectively. Both species were re-isolated from the symptomatic branches respectively, thereby fulfilling Koch’s postulates. To the best of our knowledge, this is the first report of B. dothidea and P. velata causing branches rot in F. suspensa. The findings of this study will contribute to developing effective strategies for the control of this newly emerging plant disease.

Abstract Study question Can key elements of ovarian physiology be accurately modelled, thereby providing a tool to generate novel insights and guide development of new therapeutics? Summary answer A physiological computational model was developed to describe follicle growth and follicle size distributions observed in agonist and antagonist ovarian stimulation (OS) protocols. What is known already The outcome of OS remains unpredictable despite identification of several prognostic factors. The average first cycle pregnancy rate is 30-40% across fertility centres, highlighting the need for improved stimulation protocols. This is an active area of clinical research; however, clinical trials are costly, time-consuming, and frequently have outcomes that are unexpected or difficult to interpret. We integrated available physiological knowledge of human follicle growth and development and clinical trial data into a computational model to enable in silico simulations with the aim to optimise clinical trial design. Study design, size, duration Computational model building was performed using a mechanistic approach aimed at capturing well-understood physiology that is relevant in OS protocols. Model inputs included patient characteristics (such as anti-Müllerian hormone (AMH) level), OS regimen and pituitary downregulation with gonadotrophin releasing hormone (GnRH) agonist or antagonist. Key outputs of the model included follicle number and mean size, duration of stimulation and serum hormone concentrations (oestradiol, testosterone, inhibin B and androstenedione). Participants/materials, setting, methods Published physiological data on regulation of follicle development and hormone production informed model design. Following implementation of the model in the Python platform, data from two OS clinical trials were used to calibrate (partially censored) and test the model. The two trials provided data on the effects of the recombinant follicle stimulating hormone preparation follitropin delta over a range of fixed doses and compared the effects of follitropin delta in agonist and antagonist OS protocols. Main results and the role of chance The computational model describes the interplay between pituitary gonadotrophins ovarian steroid hormones and follicle growth and development. Processes spanning the cellular level (such as steroidogenesis and receptor dynamics in theca and granulosa cells), the ovaries (follicle number and size) and the organism (pharmacokinetics and hypothalamic-pituitary-ovarian axis) are included as a system of coupled differential equations. Approximately 70 compartments, 500+ parameters and 1700+ reactions constitute the model framework. Follicles are recruited in the model at variable times, starting from 7 days prior to the start of stimulation. Follicles are initialised with individual sensitivity to gonadotrophins and growth rates. The fate of each follicle (dominance or atresia) is governed by these properties, which determine whether the balance shifts towards proliferation or atresia under the influence of gonadotrophins. Model-simulated numbers of follicles, their average size and mean serum hormone concentrations match closely with observed values in the two clinical trials. For each of these variables, their dependence on time, the dose of follitropin delta, AMH level and downregulation protocol were described with good precision. The inter-patient variability in the simulated number of follicles and in serum hormone levels matched closely with the observed variability based on comparison of the interquartile range. Limitations, reasons for caution The computational model currently describes follicle development during treatment with follitropin delta. We envision extending the model to encompass other treatment outcomes and additional preparations of exogenous gonadotrophins. While the model incorporates our current understanding of mechanisms important for follicle development, ongoing research in the field will likely drive improvements. Wider implications of the findings The computational model is hypothesis-generating and can improve the design of future clinical trials through in silico trial simulation. For instance, the effects of changing subject inclusion/exclusion criteria or of novel dosing regimens can be investigated. Trial registration number NCT01426386, NCT03809429