Journal articles on the topic 'Roche saline'

The taxonomic structure of soil microbial community was studied in six samples taken from a salt marsh along the salinity gradient and in two samples of non-saline soils using pyrosequencing method (454 Roche). The analysis allowed to identify three main ecological groups of microorganisms depending on the degree of the soil salinity. Halophylic microorganisms were mainly represented by bacteria of three phyla Firmicutes, Proteobacteria and Bacteroidetes and included much less of archaea (the Halobacteriaceae family). Within the distance of 150–200 m from the point with the highest levels of salinity, the microbial community tends to have a considerable similarity with control samples of non-saline soils.

Objective Over the past several years, only approximately 50% of HIV-exposed infants received an early infant diagnosis test within the first two months of life. While high attrition and mortality account for some of the shortcomings in identifying HIV-infected infants early and putting them on life-saving treatment, fragmented and challenging laboratory systems are an added barrier. We sought to determine the accuracy of using HIV viral load assays for infant diagnosis of HIV. Methods We enrolled 866 Ugandan infants between March–April 2018 for this study after initial laboratory diagnosis. The median age was seven months, while 33% of infants were less than three months of age. Study testing was done using either the Roche or Abbott molecular technologies at the Central Public Health Laboratory. Dried blood spot samples were prepared according to manufacturer-recommended protocols for both the qualitative and quantitative assays. Viral load test samples for the Roche assay were processed using two different buffers: phosphate-buffered saline (PBS: free virus elution viral load protocol [FVE]) and Sample Pre-Extraction Reagent (SPEX: qualitative buffer). Dried blood spot samples were processed for both assays on the Abbott using the manufacturer’s standard infant diagnosis protocol. All infants received a qualitative test for clinical management and additional paired quantitative tests. Results 858 infants were included in the analysis, of which 50% were female. Over 75% of mothers received antiretroviral therapy, while approximately 65% of infants received infant prophylaxis. The Roche SPEX and Abbott technologies had high sensitivity (>95%) and specificity (>98%). The Roche FVE had lower sensitivity (85%) and viral load values. Conclusions To simplify and streamline laboratory practices, HIV viral load may be used to diagnose HIV infection in infants, particularly using the Roche SPEX and Abbott technologies.

ABSTRACT Although Roche COBAS Ampliprep/COBAS TaqMan (CAP/CTM) systems are widely used in sub-Saharan Africa for early infant diagnosis of HIV from dried blood spots (DBS), viral load monitoring with this system is not practical due to nonspecific extraction of both cell-free and cell-associated viral nucleic acids. A simplified DBS extraction technique for cell-free virus elution using phosphate-buffered saline (PBS) may provide an alternative analyte for lower-cost quantitative HIV virus load (VL) testing to monitor antiretroviral therapy (ART). We evaluated the CAP/CTM v2.0 assay in 272 paired plasma and DBS specimens using the cell-free virus elution method and determined the level of agreement, sensitivity, and specificity at thresholds of target not detected (TND), target below the limit of quantification (BLQ) (<20 copies/ml in plasma or <400 copies/ml in DBS), and VL of <1,000 copies/ml, and VL of <5,000 copies/ml. Reported plasma VL ranged from TND, or <20, to 5,781,592 copies/ml, and DBS VL ranged from TND, or <400, to 467,600 copies/ml. At <1000 copies/ml, agreement between DBS and plasma was 96.7% (kappa coefficient, 0.93; P < 0.0001). The mean difference between DBS and plasma VL values was −1.06 log 10 copies/ml (95% confidence interval [CI], −1.17, −0.97; P < 0.0001). At a treatment failure threshold of >1,000 copies/ml, the sensitivities, specificities, positive predictive values (PPV), and negative predictive values (NPV) were 92.7%, 100%, 100%, and 94.3%, respectively. PBS elution of DBS offers a sensitive and specific method for monitoring plasma viremia among adults and children on ART at the WHO-recommended threshold of >1,000 copies/ml on the Roche CAP/CTM system.

Introduction: Trauma-induced coagulopathy (TIC) is a multifactorial condition secondary to severe trauma. In TIC, early fibrinogen (FI) replacement and low dose of recombinant activated factor VII (rFVIIa) may positively impact outcome. Factor XIII (FXIII), on the other hand, may stimulate in vitro clot formation and clot stability. We hypothesized that combination of FI, rFVIIa and FXIII might normalize clot formation more effectively than the isolated use of each concentrate in a model of TIC. Aim: Evaluation of the procoagulant effect of isolated or combined use of FI, rFVIIa and FXIII in a model of TIC. Methods: TIC in vitro model was obtained by dilution of whole blood from seven healthy controls with isotonic saline (NaCl 0.9%) (2:3 whole blood:saline ratio). FI, rFVIIa and FXIII were spiked in combination or alone until obtaining final levels of 2 g/L, 1 μg/mL and 100 IU/dL respectively. Procoagulant effects of the different concentrates or their mixtures were evaluated by Rotational Thromboelastometry (ROTEM®, Werfen) triggered using starTEM® (calcium chloride 0,2 M) and exTEM® reagent (source of tissue factor) diluted with saline up to 1:100.000 (final dilution) for a better evaluation of both the extrinsic and intrinsic pathways of coagulation. The values of clotting time (CT: time until 2 mm of amplitude, in seconds), amplitude (parameter proportional to the clot strength) at 5 minutes (A5, in mm) and clot formation time (CFT: time from CT to 20 mm of amplitude, in seconds) were evaluated. Statistical analysis of differences was performed by One-Way ANOVA test assuming no paring of data and using the Holm-Sidak's correction for multiple comparisons with a family-wise significance and confidence level of 0.01. Statistical significance was set at p&lt; 0.05. Results/Discussion: Data are summarized in Table I and Figure 1. CT needed the combination of two of more concentrates to reach the normal range suggesting that the administration of FI alone in TIC may not be enough to restore the patients' hemostatic potential. In regard to the clot strength evaluated by A5, the addition of FXIII or rFVIIa alone or in combination did not improve the value of A5 that was only normalized by the addition of FI. This effect of FI was increased in the presence of FXIII or rFVIIa which indicated that normal levels of FI might be required for rFVIIa or FXIII to be effective emphasising the possible benefit of the combinatory therapy. Like observed in A5, the velocity of clot formation evaluated by the CFT was normalised only by the addition of FI. However, the combination of FI plus FXIII + rFVIIa had a stronger effect on CFT compared with the combination of FI + FXIII or FI + rFVIIa, indicating that the improvement of thrombin generation due to rFVIIa plus an increment of fibrin formation and net stabilization through the contribution of higher levels of FI and FXIII respectively, might provide a beneficial synergistic procoagulant effect in TIC. Conclusion: The use of FI in TIC may contribute to increase the patient's hemostatic potential but might not be enough. Combinatory therapies based on the administration of FI, rFVIIa and FXIII might be of better benefit in this setting. Ex-vivo studies using blood of patients with stablished TIC might bring new insights on the possible advantages of this combinatory therapy to design more effective protocols to treat this frequent and life-threatening acquired condition. Disclosures Canales: Sandoz: Honoraria; iQone: Honoraria; Janssen: Speakers Bureau; Janssen: Honoraria; Roche: Speakers Bureau; Karyopharm: Honoraria; Sandoz: Speakers Bureau; Novartis: Honoraria; Takeda: Speakers Bureau; Roche: Honoraria; Sandoz: Honoraria; Janssen: Speakers Bureau; Roche: Speakers Bureau; Sandoz: Speakers Bureau; Takeda: Speakers Bureau; Janssen: Honoraria; Karyopharm: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Gilead: Honoraria. Butta:NovoNordisk: Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI: Speakers Bureau; Pfizer: Speakers Bureau; ROCHE: Research Funding, Speakers Bureau; Novartis: Speakers Bureau; Grifols: Research Funding. Alvarez Román:NovoNordisk,: Research Funding, Speakers Bureau; Takeda: Research Funding, Speakers Bureau; SOBI,: Consultancy, Research Funding, Speakers Bureau; Pfizer,: Research Funding, Speakers Bureau; Roche: Speakers Bureau; Novartis: Speakers Bureau; Bayer: Consultancy; Grifols: Research Funding. Jiménez-Yuste:F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer: Consultancy; Grifols, Novo Nordisk, Takeda, Sobi, Pfizer: Research Funding; F. Hoffman-La Roche Ltd, Novo Nordisk, Takeda, Sobi, Pfizer, Grifols, Octapharma, CSL Behring, Bayer: Honoraria.

Abstract Abstract 4920 Within a prospective phase II study (HOVON 80) of patients with recurrent diffuse large B-cel lymphoma in the CNS, three of 13 patients treated with intrathecal rituximab developed an acute, transient, extremely painful lumbosacral radiculopathy. All were treated with systemic R-DHAP every 4 weeks with i.v. HD-MTX on day 15 for three cycles. In addition intrathecal Rituximab was administered twice weekly via lumbar puncture starting on day -1. According to protocol the first administration consisted of 10 mg of rituximab after premedication with acetaminophen, thereafter the dose was increased to 25 mg. No patient experienced side effects of the first intrathecal administration of rituximab. However, after the first administration of 25 mg rituximab three of 13 treated patients reported extremely painful tingling sensations in the buttocks, legs and feet immediately after administration and lasting 30–60 minutes. Concomitantly a temporarily increased bloodpressure was documented. Premedication with an antihistaminic in the third patient was ineffective. No neurologic deficits occurred and the pain resolved completely. The patients refused further treatment with intrathecal rituximab, and therapy was changed to intrathecal methotrexate, without any side effects. After these events the rituximab was diluted in saline to 5 mg/ml, the dose reduced to 10 mg per administration, and 4 mg dexamethasone was administered concomitantly in all subsequent patients. Twelve additional patients were thus treated and no further incidents of painful radiculopathy were observed. This serious, though completely transient, adverse effect of intrathecal rituximab precludes intrathecal administration of higher doses of rituximab via lumbar route. It has never been described after intraventricular administration. Disclosures: Bromberg: Roche: Research Funding. Off Label Use: rituximab administration intrathecally. Doorduyn: Roche: Research Funding. van den Bent: Roche: Consultancy, Research Funding.

Abstract Background Serum free light chain (FLC) assays are used clinically to measure the concentration of κ and λ FLC in patients with suspected or diagnosed plasma cell proliferative disorders. Previous studies have demonstrated a loss of linearity in low concentration ranges of these assays. We hypothesized that this result could be caused by a matrix effect. Methods Recovery studies were performed for κ and λ FLC in both serum and saline using the Freelite assay (Binding Site) on a Cobas c502 system (Roche). Samples were analyzed either at the recommended dilution or undiluted. Follow-up studies were performed in varying matrices ranging from 0% to 100% saline. Retrospective patient data were analyzed to assess the impact on reported κ FLC, λ FLC, and κ/λ ratio. Results FLC in a serum matrix demonstrated underrecovery relative to samples diluted in saline for both κ and λ FLC. Of 255 patient samples with λ FLC measured undiluted (λ FLC &lt;6.0 mg/L), an unexpected gap was observed in patient results between 2.0 and 6.0 mg/L. In addition, 23 patients measured serially with λ FLC between 2.0 and 6.0 mg/L demonstrated dramatic changes in κ/λ ratio, with no changes in κ FLC, likely because of the matrix effect. Conclusions The κ and λ Freelite assays exhibit a matrix effect when samples are tested undiluted, which has the potential to affect the κ/λ ratio. Consequently, our laboratory has stopped reporting λ FLC &lt;6.0 mg/L.

The aim of our study was to compare the diagnostic performance of cardiac troponin T (cTnT) and cardiac troponin I (cTnI) in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) daunorubicin (3 mg/kg i.v.); 3) daunorubicin (3 mg/kg i.v.) + dexrazoxane (60 mg/kg i.p.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination (R2 = 0.79) was acceptable only in the control group. In the remaining cases (i.e. in the daunorubicin group and in the daunorubicin + dexrazoxane treated group) R2 was too small (0.53, and 0.06). We may conclude that in rabbits after repeated administration of cardiotoxic or cardioprotective drugs meaningful dependence between cTnT and cTnI was not found. The choice of the most suitable cardiomarker in laboratory animals deserves further studies.

Abstract Background Widespread testing of SARS-CoV-2 has resulted in shortages of collection devices and transport media. We evaluated the stability of flocked swabs inoculated with SARS-CoV-2-containing specimen incubated dry (i.e., without transport medium) at room temperature. Methods A pool of SARS-CoV-2 positive specimen was used to inoculate flocked swabs. Five swabs were placed immediately into universal transport media (UTM) following inoculation, and tested immediately (day 0). Fifteen of the swabs were placed into sterile 15-mL conical tubes and incubated at room temperature for 1, 2, or 7 days. Following incubation, swabs were hydrated in separate vials of UTM and tested. This protocol was repeated for viral transport media (VTM) and saline. As a comparison, a series of swabs was prepared and tested in parallel, but stored in the corresponding liquid transport media (UTM, VTM, or saline) and incubated at room temperature. Testing was performed at 1, 2, and 7 days postinoculation in duplicate. All molecular testing was performed using the Roche cobas SARS-CoV-2 assay. Results All dry swabs tested on days 1, 2, and 7 provided results that were within 2 cycle thresholds (CTs) of the average CT values for swabs hydrated in the same media and tested on day 0. There was no statistical difference in CT values between swabs incubated in liquid media versus dry swabs incubated at room temperature prior to hydration in liquid media. Conclusions The utilization of “dry swabs” may simplify specimen collection, negate the need for liquid transport media, and mitigate safety risks while preserving the accuracy of testing.

Both cardiac troponin T (cTnT) and cardiac troponin I (cTnI) are considered to be reliable biomarkers with sufficient sensitivity and specificity for cardiac injury in the majority of laboratory animals. The aim of our study was to compare the diagnostic performance of cTnT and cTnI in three groups of rabbits: 1) control (saline 1 ml/kg i.v.); 2) Salicylaldehyd Isonicotinoyl Hydrazone – SIH (50 mg/kg, once weekly, i.p.; partially dissolved in 10 % Cremophor solution); 3) 10 % Cremophor solution in water (2 ml/kg i.v.). The drugs were given once a week, 10 administrations. The concentration of cTnT was measured using Elecsys Troponin T STAT Immunoassay (Roche). The concentration of cTnI was measured using AxSYM Troponin I (Abbott). The linear regression model was applied to see if there is a dependence between cTnT and cTnI. The coefficient of determination was not acceptable in all groups. The highest value of R2was found in the control group (R2= 0.424). We may conclude that in rabbits meaningful dependence between cTnT and cTnI was not found. According to our long-term experiences cTnT seems to be more suitable cardiomarker in rabbits in comparison with cTnI where the data are characterized by the large scatter.

The authors examined the effect of selective endothelin (ET) receptor type A (ETA) antagonism on histological and functional recovery in cat at 24 hours after reversible middle cerebral artery occlusion (MCAO). A novel and specific ETA antagonist, Ro 61-1790 [5-methylpyridine-2-sulfonic acid-6-(2-hydroxyethoxy)-5-(2-methoxyphenoxy)-2-(2-1 H-tetrazol-5-yl-pyridin-4-yl)-pyrimidin-4-ylamide sodium salt (1:2)] (Roche, Basel, Switzerland), was used at doses that produced steady-state plasma concentrations and abolished ET-induced pial arteriolar vasoconstriction. In a cranial window preparation, 8 nmol/L ET constricted pial arterioles by 33 ± 18% (mean ± SD), but this response was ablated by intravenous Ro 61-1790 treatment (10-mg/kg bolus, 4-mg/kg/h infusion). In additional animal cohorts, halothane-anesthetized cats were treated with 90 minutes of MCAO and 24 hours of reperfusion. Animals received Ro 61-1790 infusion beginning at the onset of reperfusion and continuing for 6 or 24 hours (n = 41). Control cats were treated with 0.9% saline by intravenous infusion throughout reperfusion. There was no difference in injury volume or neurologic evaluation score in saline-treated cats (n = 11; caudate 24 ± 28%, cortical injury 7.5 ± 5% of ipsilateral structure; score 52 ± 8) versus the results in cats treated with Ro 61-1790 for either 24 hours (n = 6; caudate 22 ± 23%, cortex 6 ± 5%, injury volume of ipsilateral structure; score 55 ± 3) or 6 hours (n = 11; caudate 33 ± 30%, cortex 12 ± 14%, injury volume of ipsilateral structure; score 50 ± 10). Mortality was greatest in the 24-hour drug treatment group. These data suggest that blockade of ETA receptor activity is not beneficial to tissue or functional outcomes from experimental stroke in cat.

N-Chlorotaurine (NCT) is a promising endogenous agent for topical treatment of infections. We tested the tolerability and pharmakokinetics of NCT in the bovine mammary glands in a phase 1 study. Three concentrations of NCT in water (0·1%, 1·0%, 2·0%) were administered intramammarily in each of two cows. Into two quarters of the udder 100 ml NCT was injected into each twice daily for 5 d, while 0·9% NaCl was injected into the other two quarters in a randomized and blinded manner. Samples of milk were taken to determine the number of leucocytes and the activity of NCT, and samples of urine and blood to determine the taurine and chloride concentration. Chloride concentrations in serum samples were determined by an ISE-Unit of a Modular-System of the Roche Diagnostics company. The udder was monitored clinically for signs of inflammation. Oxidative activity could be detected in the milk after single irrigations for 15 min (0·1% NCT) and for maximally 5 h (1% and 2% NCT), respectively. On day 2, leucocytes increased to 4×106/ml in the NCT group, while they remained ⩽1×106/ml in the saline group. However, on days 3–5 they increased to (5–7)×106 in both the NCT and control group without any statistical difference. One day after the end of dosing the number decreased significantly and reached the baseline (<1×106/ml) on day 10. The decrease was similar in both groups. Except for sporadic slight induration of single quarters in both groups and slight reduction of milk performance no disorders occurred. Taurine levels in blood and urine did not change. Irrigation of the bovine mammary gland with both NCT and saline caused a transient increase of leucocytes in the milk, but no severe side effects. The absence of residues and decay products may be a great advantage of NCT over other antimicrobial agents.

Background:Intra-articular therapies (IAT) are routinely used in rheumatic and musculoskeletal diseases (RMDs); however large variability exists regarding current practice of delivery amongst health professionals.Objectives:To inquire about common practice aspects to inform the EULAR Taskforce for the IAT of arthropathies.Methods:A steering committee prepared a 160-item questionnaire based on the information needs of the Taskforce. The survey was disseminated via EULAR professional associations and social media and it was open to any health professional treating persons with RMDs, regardless of using IAT personally.Results:The survey was answered by 186 health professionals from 26 countries, the large majority of whom (77%) were rheumatologists, followed by nurses (12%), general practitioners (2%) and orthopaedic surgeons (2%). The two collectives that perform IAT routinely are rheumatologists (97%) and orthopaedic surgeons (89%), with other professionals <50%. Specific training was compulsory for 32%. The most frequent indication for IAT is inflammatory arthritis (76%), followed by osteoarthritis (74%), crystal arthritis (71%) and bursitis (70%); and all joints are injected, with knee (78%) and shoulder (70%) being the most frequent. When questioned about specific contexts, such as pre-surgical, diabetic or hypertensive patients, variability among respondents was evident, with around 30 to 69% of professionals considering it acceptable to inject glucocorticoids (GC), while in others there was less variability (prosthetic or septic joints, <1%). GCs are the most used compounds, followed by hyaluronic acid and saline/dry puncture. Only 66 (36%) use ultrasound to guide IAT. In their opinion, to be accurately in the joint is moderately to largely important for large joints (80%) and very important in small joints. The maximum number of injections to perform safely in the same joint within one year was “2 to 3” for 65% (2% thought there is “No limit”). The majority reported that they informed patients about side-effects (73%), benefits (72%), and the nature of the procedure (72%), and less frequently about other aspects; with 10% obtaining written consent and 56% oral consent (mandatory only for 32%). Other questions help to understand the setting and procedures followed, including use of local anaesthetics and care after injection.Conclusion:Although often performed in clinical practice for RMDs, there is apparent variability in several elements related to delivery of this treatment. This information, together with patient input, will help design current recommendations where research evidence is not available.Acknowledgments:Eular Taskforce grant CL109Disclosure of Interests:Jenny de la Torre-Aboki: None declared, IRENE Pitsillidou: None declared, Jacqueline Uson Jaeger: None declared, Esperanza Naredo: None declared, Lene Terslev: None declared, Mikael Boesen Consultant of: AbbVie, AstraZeneca, Eli Lilly, Esaote, Glenmark, Novartis, Pfizer, UCB, Paid instructor for: IAG, Image Analysis Group, AbbVie, Eli Lilly, AstraZeneca, esaote, Glenmark, Novartis, Pfizer, UCB (scientific advisor)., Speakers bureau: Eli Lilly, Esaote, Novartis, Pfizer, UCB, Hemant Pandit Grant/research support from: Glaxo Smith Kline (GSK) for work on Diclofenac Gel, Speakers bureau: Bristol Myers Squibb for teaching their employees about hip and knee replacement, Ingrid Möller: None declared, Maria Antonietta D’Agostino Consultant of: AbbVie, BMS, Novartis, and Roche, Speakers bureau: AbbVie, BMS, Novartis, and Roche, Willm Uwe Kampen: None declared, Terence O’Neill: None declared, Michael Doherty Grant/research support from: AstraZeneca funded the Nottingham Sons of Gout study, Consultant of: Advisory borads on gout for Grunenthal and Mallinckrodt, Francis Berenbaum Grant/research support from: TRB Chemedica (through institution), MSD (through institution), Pfizer (through institution), Consultant of: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Bone Therapeutics, Regulaxis, Peptinov, 4P Pharma, Paid instructor for: Sandoz, Speakers bureau: Novartis, MSD, Pfizer, Lilly, UCB, Abbvie, Roche, Servier, Sanofi-Aventis, Flexion Therapeutics, Expanscience, GSK, Biogen, Nordic, Sandoz, Regeneron, Gilead, Sandoz, Valentina Vardanyan: None declared, Elena Nikiphorou: None declared, Sebastian C Rodriguez-García Speakers bureau: Novartis Farmaceutica, S.A., Merck Sharp & Dohme España, S.A., Sanofi Aventis, UCB Pharma, Raul Castellanos-Moreira: None declared, Loreto Carmona Grant/research support from: Novartis Farmaceutica, SA, Pfizer, S.L.U., Merck Sharp & Dohme España, S.A., Roche Farma, S.A, Sanofi Aventis, AbbVie Spain, S.L.U., and Laboratorios Gebro Pharma, SA (All trhough institution)

Many studies have reported ischemia protection using various preconditioning techniques, including single dose 3-nitropropionic acid (3-NPA), a mitochondrial toxin. However, the cellular signal transduction cascades resulting in ischemic tolerance and the mechanisms involved in neuronal survival in the tolerant state still remain unclear. The current study investigated the mRNA and protein expression of the antiapoptotic bcl-2 and the proapoptotic bax, two antagonistic members of the bcl-2 gene family, in response to a single dose of 3-NPA, to global cerebral ischemia–reperfusion, and to the combination of both 3-NPA-pretreatment and subsequent global cerebral ischemia–reperfusion. Brain homogenates of adult Wistar rats (n = 25) were analyzed for bcl-2 and bax mRNA expression using a new highly sensitive and quantitative polymerase chain reaction (PCR) technique that allows real-time fluorescence measurements of the PCR product (LightCycler; Roche Diagnostics, Mannheim, Germany). Animals for mRNA analysis received 3-NPA (20 mg/kg, intraperitoneal; “chemical preconditioning”) or vehicle (normal saline), and were either observed for 24 plus 3 hours or were subjected to 15 minutes of global cerebral ischemia 24 hours after the pretreatment and observed for 3 hours of reperfusion. Immunohistochemistry was applied to serial brain sections of additional rats (n = 68) to determine amount and localization of the respective Bcl-2 and Bax protein expression in various brain areas. One set of animals was injected with 3-NPA and observed for 3, 12, 24, and 96 hours; a second set was exposed to 15 minutes global cerebral ischemia, 3, 12, and 24 hours reperfusion; and a third set was pretreated with 3-NPA or saline 24 hours before the ischemic brain insult and observed for 96 hours of reperfusion. The authors found single dose 3-NPA treatment to be associated with an elevated bcl-2:bax ratio (increased bcl-2 expression, decreased bax expression), both on the transcriptional (mRNA) and the translational (protein) level. The differential influence of 3-NPA was maintained during early recovery from global cerebral ischemia (3 hours), when 3-NPA pretreated animals showed higher bcl-2 and lower bax mRNA levels compared with rats with saline treatment. Respective changes in protein expression were localized predominately in neurons vulnerable to ischemic damage. Compared with baseline, Bcl-2 protein was significantly higher in surviving neurons at 96 hours after the insult, whereas Bax protein remained unchanged. However, at this late time of postischemic recovery (96 hours), the protein expression pattern of surviving neurons was not different between animals with and without 3-NPA pretreatment. To the authors' knowledge, the current study is the first report on the differential expression of pro-and antiapoptotic genes after a single, nonlethal dose of 3-NPA. The current results suggest alterations in the balance between pro-and antiapoptotic proteins as a potential explanation for the reported protection provided by chemical preconditioning using 3-NPA in rats.

BackgroundSystemic autoimmunity is a complex and multifactorial process for which underlying mechanism are unclear. Several theories have been formulated to explain the development of autoimmunity, including excessive self-immunosurveillance, danger, and antigen discontinuity. The antigen discontinuity and the self-criticality theories presume that repetitive antigen exposure leads to a dysregulated immune response. Supporting these theories, Tsumiyama and Shiozawa have shown that repetitive immunization of BALB/c mice with ovalbumin induces auto-antibodies production and a glomerulonephritis with immune deposit resembling lupus nephritis [1].These data prompted us to consider the potential impact of repetitive SARS-CoV-2 infection in the onset of autoimmune diseases in human. Indeed, since 2020, a large majority of the population has encountered SARS-CoV-2 antigens multiples times, either through vaccination or through infection/reinfection. Hence, we hypothesized that repetitive exposure to the SARS-CoV-2 spike antigen may induce autoimmunity in healthy individuals.ObjectivesEvaluate whether repetitive exposure to SARS-CoV-2 spike protein induces autoimmunity in mice.MethodsWe immunized intraperitoneally C57Bl/6 mice (n = 5/group) with recombinant SARS-CoV-2 spike protein (1µg per injection) or vehicle (phosphate buffered saline [PBS]);Figure 1A). The first injection was mixed with Alum as an adjuvant, and the other injections were diluted in PBS, administered intraperitoneally every 5 days, 16 times in total. Five days after the final injection, the mice were sacrificed to retrieve serum. We measured anti-spike and anti-dsDNA IgG using ELISA. The serum from 5 34-week-old C57Bl/6.lpr autoimmune mice were used as positive control. Briefly, 96-well plate were coated overnight with either spike protein (0.2µg/mL) or calf thymus DNA (0.1mg/mL; after precoating with poly-L-lysin 0.05mg/mL). After blocking with PBS-BSA, the diluted serum samples were incubated on the plate for 2 hours at room temperature. Plate-bound IgG were revealed using an alkaline phosphatase-conjugated anti-mouse IgG (1:5000). For anti-dsDNA IgG measurement, the serum of an autoimmune mice was used as a reference standard and the result expressed in unit per mL.ResultsAfter immunization, the phenotype of control and spike-immunized mice was clinically similar. All the immunized mice were exempt of proteinuria (Figure 1B). Only the mice which were injected with spike protein developed measurable amounts of anti-spike IgG as determined by ELISA, while those injected with PBS and the C57Bl/6.lprmice did not (Figure 1C). None of the mice in the immunization group developed measurable anti-dsDNA IgG (Figure 1D).ConclusionIn our study, the repetitive exposure of C57Bl/6 mice to the SARS-CoV-2 spike antigen did not lead to autoimmunity. Although these results are reassuring, large scale epidemiological studies are needed to evaluate the incidence of autoimmune diseases in individuals with multiple exposure to SARS-CoV-2 antigens.Reference[1]K. Tsumiyama, Y. Miyazaki, S. Shiozawa, Self-Organized Criticality Theory of Autoimmunity, PLOS ONE. 4 (2009) e8382.Figure 1.Acknowledgements:NIL.Disclosure of InterestsMarc SCHERLINGER Speakers bureau: Amgen, Sandoz, Nordic Pharma, Jean Sibilia Speakers bureau: Roche, Chugai, Bristol-Myers Squibb, UCB, GSK, LFB, Actelion, Pfizer, MSD, Novartis, Amgen, Abbvie, Sandoz, Gilead, Lilly, Sanofi Genzyme, Janssen, Mylan, Galapagos, Sobi., Grant/research support from: Pfizer, BMS, Roche, MSD, George Tsokos: None declared, Jacques-Eric Gottenberg Speakers bureau: AbbVie, BMS, CSL Behring, Galapagos, Gilead, Lilly, Roche-Chugai, Pfizer, Sanofi, and UCB., Grant/research support from: BMS.

Abstract Background The Roche Cobas chemistry analyzer’s hemolysis index (HI) check function can directly report hemoglobin (Hb) concentrations. We aimed to validate the HI check function for the measurement of plasma cell-free Hb. Methods Plasma samples (6 μl) were taken by the analyzer and diluted in normal saline to measure the absorbance for Hb at 570 and 600 nm. Hb concentrations were calculated based on the molar extinction coefficient. Imprecision, lower limit of quantification (LLOQ), and analytical measurement range (AMR) of the assay were evaluated. The accuracy was determined by comparing the results between the new method and an existing spectrophotometric method. We further studied interference of icterus and lipemia and carryover. The performance of the assay in proficiency testing was also evaluated. The reference range was transferred from the existing method. Results Within-run and total CVs were 1.7%–4.2% and 2.1%–7.0%, respectively (n = 20). The LLOQ was 11 mg/dL (CV = 8.1%) with the upper limit of AMR of 506 mg/dL. The results of the new method correlated well with the existing reference assay: Y (new method) = 0.974 x (reference method) + 4.9, r = 0.9990, n = 52. Bilirubin with a concentration up to 60 mg/dL and lipemic index up to 389 did not show significant interference. No significant carryover was detected. The average standard deviation index in proficiency testing was 0.03 ± 0.29. The reference range was &lt;22 mg/dL. Conclusions Plasma cell-free Hb measurement using the HI check function meets the analytical requirements of the plasma cell-free Hb assays. It is simple and cost-effective.

This work describes the treatment with total parenteral nutrition (TPN) of a 37-day cloned calf after suffering ruminal acidosis by ingestion of milk. Cells for SCNT were obtained by using a bicistronic vector for human lysozyme and lactoferrin. We obtained 7 embryos and 2 pregnancies. Only one fetus was born alive weighing 47 kg and it was presented to the neonatal unit of INTA showing a deep depression, diarrhoea, dehydration (10%), hyperthermia and inability to stand (Table 1). A 20-cm-length 20-Ga-diameter catheter (Arrow) was placed in the jugular vein. Blood samples from catheter and brachial artery showed leukocytosis, hypoglycemia and metabolic acidosis (Table 1). Esophageal tube was placed to remove 5 L of ruminal content and for the administration of 2 L of a solution of sodium bicarbonate (40 g L–1 of water). Saline (NaCl, 9 g L–1), sodium bicarbonate (8.4 g/100 mL) and 10% dextrose were administered IV until dehydration; blood pH and glucose were corrected. Ceftriaxone 1 g IM/12 h (Acantex, Roche) to prevent bacterial translocation, 1.175 mg of flunixin meglumine (PharmaVet) as anti-endotoxic dose and 80 mg of ranitidine IV/12h (Vetanco) to prevent laminitis were also administered. Two litres of bovine plasma were administered during the first 2 days and, after this, we began with a TPN regimen due to lack of sucking reflex and animal anorexia. Kabiven (Fresenius Kabi AV) was administered at 1 g of lipids/kg/24 h by a regimen of 18 drops min–1 to prevent hyperlipidemia at the recommended dose for humans. For this reason, we also administered dextrose 25% 12.5 drops min–1 and amino acids 11.5% 504 mL (Rivero), to reach a dose of 10 g/kg/24 h and 2 g/kg/24 h, respectively. Saline (NaCl, 9 g L–1) and vitamin complex (Rivial Paediatrics, Rivero) was also administered to cover water and vitamin requirements. The TPN therapy lasted 24 days during which the animal regularized its metabolic functions, reversed signs of ruminal acidosis and learned to eat a balanced ration and hay. To our knowledge, no information is available on such a long period of TPN in bovine neonates. This work shows that TPN can increase the survival chances of high risk animals and thus the final efficiency of cloning and transgenesis. Table 1.Clinical and haematological responses to 24-day total parenteral nutrition in a 37-day cloned female calf The authors thank Fresenius Kavi and Rivero Laboratories for their support.

L’exploitation intensive des ressources en eaux souterraines du bassin Chougafiya a largement influencé le fonctionnement hydrodynamique de la nappe phréatique. Elle a provoqué une inversion du gradient hydraulique et une invasion des eaux minéralisées en provenance de la nappe du Sabkhas El Batten. Afin de mettre en évidence l’effet de cette intrusion salée sur l’hydrodynamisme de la nappe phréatique, une étude multidisciplinaire basée sur les méthodes de l’hydrogéologie et la géochimie a été menée. L’étude hydrochimique a montré que le faciès des eaux évolue entre un pôle chloruré sulfaté calcique et chloruré sodique. Les principaux phénomènes géochimiques intervenant dans l’acquisition de la charge saline sont liés à l'interaction eau-roche (dissolution des minéraux évaporitiques) et à l'échange cationique. Cependant, la composition chimique des eaux minéralisées situées dans les parties centrale et méridionale du bassin d'étude est nettement influencée par un processus de mélange avec l'eau de la nappe du Sabkhas. Le traçage naturel des eaux au moyen des isotopes stables (18O, 2H) a permis d’identifier des eaux d’origine météorique et à caractère non évaporé, tandis que les eaux à caractère évaporé s’alignent selon une droite de mélange avec l’eau souterraine de la Sabkhas. L’évolution des teneurs en 3H et 14C témoigne d’une forte contribution des eaux récentes à la recharge de la nappe phréatique dans les secteurs nord et ouest, alors que les faibles activités enregistrées au centre et à l’est de la plaine reflètent la présence d’une lithologie peu perméable. L’étude des isotopes du carbone a permis de comprendre le schéma du fonctionnement de la nappe étudiée et d’estimer le temps de séjour des eaux. D’ailleurs, les âges calculés varient de l’actuel jusqu’à 7 814 ans BP. L’enrichissement isotopique en carbone-13 qui apparaît au fil de l’écoulement est révélateur de phénomènes d’échanges isotopiques avec la matrice carbonatée de l’aquifère.

Abstract Background Body fluid samples (BFs) with low pH have demonstrated underrecovery of enzyme activity. The aims of this study were (1) measure the frequency of abnormal pH in BFs received for clinical chemistry testing, (2) calculate recovery of analytes in BFs undergoing pH titration, and (3) investigate the mechanism of pH interference. Methods Residual BFs submitted for physician-ordered testing to the Central Clinical Laboratory (Mayo Clinic) were utilized for this study. BFs were centrifuged 10 minutes (3,200 rpm) prior to pH measurement (pH meter, Accumet X15; Fisher Scientific) and analysis. A Roche Cobas c501 (Roche Diagnostics) analyzer was used to measure amylase, lactate dehydrogenase (LDH), blood urea nitrogen (BUN), creatinine, glucose, albumin, total protein, bilirubin (all Roche Diagnostics reagents), and lipase (Sekisui Diagnostics). pH was measured in 122 BFs received between 10/23/2018 and 10/26/2018 within 24 hours of receipt. pH titration was performed using pooled BFs of similar source/type. Each pool was titrated over a range of pH (≤2 to ≥10) in 1-unit increments by addition of a fixed volume of acid (6M HCL) or base (6M NaOH). Analytes were measured before and after acid/base addition. Average percent recovery was calculated (measured/expected × 100%) from n = 3 to 9 pH measurements. A low pH BF pool was spiked (<5% by volume) with a high-concentration BF to investigate mechanism of enzyme underrecovery. Additionally, low pH BF pool was neutralized by addition of base and % recovery calculated. For all titration/spiking experiments, a control sample having the same volume of diluent (7% bovine serum albumin or saline) was used to account for the effects of dilution. Results BFs received (n = 122) had mean (SD) pH = 8.0 (0.6) with 6.6% (n = 8) having pH <7.0 and 2.5% (n = 3) having pH <6.0. All recovery (%) and pH data are expressed as mean (range) values. Amylase recovery (initial pH = 8.4-8.9) was 1.3 (0.9-2.2)% at pH = 1.6 (1.0-2.2) (n = 6), 55.6 (37.4-67.0)% at pH = 4.4 (3.5-5.4) (n = 6), 81.4 (74.0-85.2)% at pH = 6.2 (6.1-6.5) (n = 3), 90.1 (84.4-95.4)% at pH = 7.5 (7.3-7.7) (n = 3), 98.8 (96.2-101.7)% at pH = 10.0 (9.8-10.2) (n = 3), and 14.5 (1.4-38.9)% at pH = 11.9 (11.5-12.1) (n = 3). LDH recovery (initial pH = 8.2-8.5) was 1.4 (0.1-3.7)% at pH = 1.7 (1.0-2.4) (n = 6), 18.3 (0.5-39.7)% at pH = 4.5 (3.5-5.4) (n = 6), 63.6 (54.9-68.4)% at pH = 6.2 (6.1-6.5) (n = 3), 85.9 (80.2-90.7)% at pH = 7.2 (6.8-7.4) (n = 3), 46.1 (36.4-61.0)% at pH = 10.0 (9.8-10.2) (n = 3), and 9.8 (0.0-28.9)% at pH = 11.3 (10.4-12.1) (n = 3). Lipase recovery (initial pH = 8.2-8.9) was 10.4 (<1-18)% at pH = 1.8 (1.0-2.4) (n = 6), 73.9 (62.6-85.5)% at pH = 4.2 (3.4-5.2) (n = 3), 92.4 (90.8-93.5)% at pH = 6.2 (6.0-6.5) (n = 3), 96.3 (95.6-96.8)% at pH = 7.3 (6.8-7.7) (n = 3), and 92.9 (89.3-96.8)% at pH = 9.9 (9.8-10.1) (n = 3) and 80.9 (0.0-78.6)% at pH = 11.3 (10.4-12.1) (n = 3). Creatinine, BUN, albumin, glucose, total protein, and bilirubin recovery were 97.9 (92.2-102.9)%, 100 (96.6-103.1)%, 99.9 (97.6-102.4)%, 100.2 (97.8-103)%, 100.2 (98.7-101.9)%, and 100.1 (92.-105.9)%, respectively, between pH 1.3-11.7 (n = 9). Recovery of amylase, LDH, or lipase was <1% after spiking enzymes into BF pool with pH <3. pH adjustment to normal (pH = 8.6-9.0) also resulted in recovery of <1%. Conclusion Enzyme activity in BFs was not affected (90%-110% recovery) when pH = 7.4-8.5 for LDH, pH = 7.3-10.2 for amylase, pH = 6.0-9.9 for lipase, and pH = 1.3-11.7 for all other analytes. An irreversible loss of enzyme activity occurs at low pH. Few clinical BFs have pH <7.0, but laboratories should have awareness.

Abstract Introduction/Objective Histologic frozen section analysis is typically used for parathyroid tissue identification during parathyroidectomy when needed, in conjunction with a rapid intraoperative plasma parathyroid hormone (PTH) assay to confirm falling levels of circulating PTH after parathyroid excision. As an alternative to frozen section consults, we hypothesize that automated analysis of intraoperative fine needle aspiration (ioFNA) tissue samples using a rapid PTH immunoassay can accurately identify parathyroid tissue and reduce the need for frozen section consults. Methods A rapid PTH immunoassay (Elecsys PTH STAT; Roche Diagnostics, Indianapolis, IN), currently used for intraoperative plasma samples, was validated for FNA samples on a Cobas® e411 using tissue aspirates of ex vivo parathyroid and control specimens rinsed in 1mL saline. ioFNA PTH results during parathyroidectomy were then prospectively assessed for accuracy over a 4-month period by comparing values to final histopathologic diagnoses. The number of frozen section consults requested was compared to a 5-month period prior to the availability of the ioFNA PTH assay. Results Ninety patients underwent parathyroidectomy (128 excised parathyroids) during the study period, performed by a single experienced endocrine surgeon. Indications included primary (81/90), tertiary (5/90), and recurrent (4/90) hyperparathyroidism. Thirty-nine cases (55.5 excised parathyroids) were performed after the availability of the ioFNA PTH assay. ioFNA samples were sent for PTH analysis in 7/39 cases (18%; 12 samples total) and had a sensitivity/specificity of 100% (parathyroid [n=7] PTH values 1968 - &gt;5000pg/mL; non-parathyroid [n=5] PTH values &lt;2 - 16pg/mL). Parathyroidectomies requiring frozen section consult significantly decreased from 41% (21/51 cases; 40 specimens) to 10% (4/39 cases; 9 specimens) with the availability of the ioFNA PTH assay (p&lt; 0.05, Fisher exact test). Conclusion Analysis of ioFNA tissue samples using an automated rapid PTH immunoassay can accurately identify parathyroid tissue and can be used as an alternative to frozen section consult when needed.

Background:In recent years, diverse compounds for intra-articular administration were brought into the market with a subsequent significant and heterogeneous literature production. Understanding the efficacy of intra-articular therapies (IAT) on pain implies bearing in mind the related placebo (PBO) effect. To date, most studies analyzing it were focused on the compound being administered rather than the route of administration.Objectives:We aimed at evaluating the size of the PBO effect after intra-articular injections.Methods:We conducted an overview of systematic reviews (SRs) including randomized-controlled trials (RCTs) of frequently used IAT. SRs with a saline solution PBO arm and high-confidence results according to the AMSTAR-2 tool were selected for analysis.Data on the change in pain in the PBO arms from baseline to 3-6 and 12-16 weeks after the IA procedure was extracted. The standardized mean differences (SMD) from baseline were calculated as the ratio between the size of the intervention effect in each study and the variability observed in that study. A meta-analysis was then performed using an inverse-variance random-effects model in Review Manager 5.3. The overall effect sizes obtained refer to versions of the SMD, which corresponds to the Hedges’ (adjusted) g. e.g. a “g” of 1 indicates the two groups being compared differ by 1 standard deviation and so on.Results:Two SR were included comprising 50 RCTs, 44 not meeting inclusion criteria were excluded so pain, measured by visual analogue scale (VAS) and Lequesne index, was retrieved from 6 RCT.At 3-6 weeks, an SMD [95%CI] = 0.74 [0.47-1.00] was found. One study showing too large an effect was excluded after conducting sensitivity analysis resulting in a significant reduction of heterogeneity with an SMD = 0.62 [0.45-0.79] (Fig.1). At 12-16 weeks, we found a SMD = 0.33 [0.13-0.52] (Fig.2)Figure 1.Forest plot for intra-articular PBO effect at 3-6 weeks after injection.Figure 2.Forest plot for intra-articular PBO effect at 12-16 weeks after injection.Using the interpretation suggested by Cohen1, our results would confirm a moderate to large effect of IA saline (PBO) at 3-6 weeks with a subsequent reduction to a small but persistent effect at 12-16 weeks.Conclusion:Our results showed a moderate to large short-term effect of intra-articular PBO that persisted on the mid-term although reduced. Based on these findings we suggest this effect should be considered when assessing the efficacy of IAT in RCTs and also in clinical practice where it could be maximized as well.References:[1]Cohen J. Statistical Power Analysis in the Behavioural Sciences (2nd edition). Hillsdale (NJ): Lawrence Erlbaum Associates, Inc., 1988.Disclosure of Interests: :Sebastian C Rodriguez-García Speakers bureau: Novartis Farmaceutica, S.A., Merck Sharp & Dohme España, S.A., Sanofi Aventis, UCB Pharma, Raul Castellanos-Moreira Speakers bureau: Lilly, MSD, Sanofi, UCB, Jacqueline Uson Jaeger: None declared, Esperanza Naredo: None declared, Loreto Carmona Grant/research support from: Novartis Farmaceutica, SA, Pfizer, S.L.U., Merck Sharp & Dohme España, S.A., Roche Farma, S.A, Sanofi Aventis, AbbVie Spain, S.L.U., and Laboratorios Gebro Pharma, SA (All trhough institution)

Maternal oocyte Cyclin B1 mRNA is known to be stored in the cytoplasm with a short poly(A) tail and be translationally dormant at GV stage. During maturation, Cyclin B1 poly(A) tail is elongated by a process called cytoplasmic polyadenylation and driven by A/U-rich cis-acting elements in its 3′ untranslated region (UTR) known as cytoplasmic polyadenylation elements (CPEs). The objective of this study was to elucidate whether GV-stage bovine oocytes possess a stockpile of Cyclin B1 mRNA stored with a short a poly(A) tail that is elongated during maturation by CPE regulation. The mRNA poly(A) tail length was measured by Rapid Amplification of cDNA Ends Polyadenylation test (Race-PAT) on oocytes (n=100) at the GV stage and 3, 5, 8, 10, 15, 20, and 25h of in vitro maturation. The mRNA poly(A) tail length was also measured in triplicate (n=20) on cold oocytes in GV (all manipulations on ice), warm oocytes in GV (ovaries transported in warm saline and manipulations on ice) and warm+2h 30min oocytes in GV (oocytes left for an additional 2h and 30min at room temperature). To assess for variation in mRNA quantity, Cyclin B1 mRNA level was quantified by real-time PCR (Lightcycler, Roche, Indianapolis, IN, USA) in cold, warm or warm+2h 30min GV oocytes (n=20). The data were treated as factorial design, using treatment and type of RT as factors, and analysed by ANOVA (SAS Inst., Cary, NC, USA). Differences between means were checked using Tukey’s test. Oocyte Cyclin B1 transcript show two different 3′ UTRs. These transcripts had the same ORF but different 3′ UTR lengths because of an alternative nuclear polyadenylation element AAUAAA (NPE). The longest form (Cyclin B1L) that possessed a putative CPE (UUUUAAUAAA) fused to the last NPE was studied. In warm GV oocytes, Cyclin B1L had a long poly(A) tail of 100 adenosine residues, and this length did not change during in vitro maturation. Interestingly, we found that Cyclin B1L showed an expected short poly(A) tail when the ovaries and the oocytes were transported and manipulated on ice. We showed that Cyclin B1L mRNA is cytoplasmically polyadenylated (addition of 75 adenosine residues) between the time of collection and the end of manipulation. This lengthening is most probably sufficient to promote translation. There was no significant difference between the Cyclin B1 mRNA quantity of cold oocytes or warm oocytes when the oligo used for the reverse transcription was either dt or decamers. Therefore, we believe that the increase in poly(A) tail length is not the result of Cyclin B1L mRNA degradation in cold oocytes or de novo transcription in warm oocytes. We report for the first time that Cyclin B1L cytoplasmic polyadenylation is carried out well before the beginning of in vitro maturation in bovine oocytes when ovaries are transported from the slaughterhouse in warm saline. Studying the real early mechanisms leading to resumption of meiosis in bovine oocytes is complicated by Cyclin B1 polyadenylation occurring prior to in vitro maturation. (Supported by NSERC.)

Abstract Background The current susceptibility breakpoint for MIN against STM is 4mg/L, yielding &gt;99% of isolates susceptible. Unfortunately, there are limited pre-clinical and clinical data to support this breakpoint for STM. The purpose of this study was to evaluate the efficacy of a MIN human simulated regimen (HSR) against STM across a wide range of MICs in the murine neutropenic thigh model. Methods Clinical STM with modal MIN MICS of 0.25-8mg/L were included. Confirmatory pharmacokinetic (PK) studies were performed in infected neutropenic mice to develop a MIN HSR providing an area under the curve (AUC) and maximum concentration (Cmax) exposure similar to MIN 100mg intravenous (IV) q12h at steady-state based on PK parameters from critically ill adult patients. The murine neutropenic thigh infection model was utilized to examine the antibacterial effects of the confirmed MIN HSR against 17 STM. Both thighs of neutropenic ICR mice were inoculated with bacterial suspensions of 107 colony forming units (CFU)/mL. Two hours after inoculation, the MIN HSR was administered subcutaneously (SC) over 24h. Control mice received normal saline. Efficacy was measured as the change in log10CFU/thigh at 24h compared with 0h controls. Results MIN 22, 10, 14, and 10mg/kg dosed SC at 0, 6, 12, and 18h best recapitulated the human Cmax and AUC profile. Mean ± standard deviation bacterial burden at 0h across all isolates was 6.03±0.32 log10CFU/thigh. Bacterial growth was 1.35±0.68 log10CFU/thigh in 24h controls. Six of 7 isolates (86%) with MIC ≤ 0.5mg/L achieved 1-log kill with MIN HSR (-1.44±1.37 log10CFU/thigh). All STM with MIC ≥ 1mg/L experienced bacterial growth (1.18±0.79 log10CFU/thigh) (Figure). Figure. Efficacy of a minocycline human simulated exposure of 100mg intravenous Q12h in the murine neutropenic thigh model against 17 clinical Stenotrophomonas maltophilia isolates Conclusion These data describe the in vivo efficacy of a MIN HSR with exposures similar to MIN 100mg IV q12h in critically ill adults. Lack of antibacterial activity against STM with MICs ≥ 1mg/L justifies a reassessment of the current susceptibility breakpoint. The study was funded under FDA Contract 75F40120C00171. Disclosures David P. Nicolau, PharmD, Abbvie, Cepheid, Merck, Paratek, Pfizer, Wockhardt, Shionogi, Tetraphase (Other Financial or Material Support, I have been a consultant, speakers bureau member, or have received research funding from the above listed companies.) Joseph L. Kuti, PharmD, Allergan (Speaker’s Bureau)BioMérieux (Consultant, Research Grant or Support, Speaker’s Bureau)Contrafect (Scientific Research Study Investigator)GSK (Consultant)Merck (Research Grant or Support)Paratek (Speaker’s Bureau)Roche Diagnostics (Research Grant or Support)Shionogi (Research Grant or Support)Summit (Scientific Research Study Investigator)

Abstract Introduction: Venetoclax (Ven), an oral BCL2 inhibitor, is approved for the treatment (tx) of relapsed/refractory (R/R) chronic lymphocytic leukemia (CLL). Ven is generally well tolerated, and side effects observed in clinical trials have been consistent with other CLL tx. Clinical trials using the approved dose escalation schedule report negligible rates of clinical tumor lysis syndrome (TLS). We aimed to understand rates of select adverse events (AEs) including cytopenias, infections, and TLS in CLL patients (pts) treated with Ven in community and academic settings. To do so, we examined 297 pts with CLL who received Ven, either alone or paired, in this multicenter, international study. Methods: We conducted a retrospective cohort study of Ven treated pts with CLL across at 15 academic (n=169) and 51 community (n=128) centers outside of the clinical trial setting. This study represents a collaboration between US centers, CLL Collaborative Study of Real World Evidence (CORE), and UK CLL Forum. Demographics, baseline disease characteristics, Ven dosing, TLS risk (per FDA Ven label) and prophylaxis, and AEs were collected. Lab vs. clinical TLS was defined by Howard criteria. PFS was estimated by Kaplan Meier methodology. All comparisons were descriptive. Results: Of the 297 pts examined, median age at Ven initiation was 67 (range 37-91). The group was 69% male, 96% had R/R CLL, and 45% had del17p. Baseline characteristics stratified by practice setting are included in Table 1. 80% received Ven as monotherapy while 20% received it paired with another agent (anti-CD20 mAb (75%), ibrutinib (8.5%), other (16.5%)). All pts were treated outside of clinical trials. During dose escalation, 81% achieved a 400 mg dose and 65% maintained 400 mg following escalation (cyp3A4 use unknown). TLS risk was low in 40%, intermediate (int) in 32%, and high risk in 28%. CT scan prior to Ven initiation was performed in 62%. At least one hospitalization occurred for 56% of low, 80% of int, and 88% of high risk pts (63% of the total cohort). Table 1 describes the distribution of TLS risk and frequency of hospitalizations in academic, community centers. TLS prophylactic measures were available for a subset of pts. Allopurinol was used for 91% (n=68/75) of low, 93% (n=52/56) of int, and 94% (n=29/31) of pts at high risk for TLS. Rasburicase was used for 27% (n=28/102) of low, 42% (n=34/81) of int, and 72% (n=57/79) of high risk pts. Normal saline was used in 85% (n=62/73) of low, 88% (n=49/56) of int, and 97% (n=30/31) of high risk pts. TLS occurred in 8.4% of pts (n=25/297). Three lab and 2 clinical events occurred in low risk pts, 7 lab and 3 clinical events in int risk pts, and 7 lab and 3 clinical events in high risk pts. Of pts with TLS, 1 has discontinued Ven. Of pts with clinical TLS, all were hospitalized, received allopurinol and normal saline, and 28% received rasburicase. 72% with TLS had creatinine clearance <80 mg/mL vs. 44% who did not have TLS (p=0.02). One int risk pt received hemodialysis. One death from TLS (previously reported) was observed in a pt hospitalized with rapid disease progression and tx-related neutropenia re-challenged with 400 mg Ven after dose interruption without dose escalation. Select AEs were neutropenia (ANC<1000) 39.6%, thrombocytopenia (plt <100) 29.2%, infection 25%, neutropenic fever 7.9%, and diarrhea (>7 stools/day) 6.9%. For the subset who received Ven paired, AEs were not increased: 35% neutropenia, 29% thrombocytopenia, 22% infection, 6.3% neutropenic fever, and 6.4% diarrhea. TLS was observed in 3.4% of pts who received Ven paired vs. 9.3% who received Ven monotherapy. 29% pts required ≥1 dose reduction and 32% had ≥1 dose interruption. Median length of dose interruption was 7 days (range 1 - 132). 22 pts (7.4%) discontinued Ven due to an AE. PFS was similar in pts with ≥1 dose interruption vs. 0, pts who required dose interruption ≥8 days vs. <8 days, and pts who achieved a stable Ven dose of <400 mg vs. 400 mg (Figure 1). Conclusions: Ven was well tolerated in this cohort; AE rates were similar to those reported in clinical trials. Both academic and community sites employed TLS prophylaxis consistent with FDA/EMA recommendations resulting in a small proportion of clinical TLS events (<3%). Of note, Ven paired with another agent did not appear to result in increased rates of AEs and TLS events. Dose reduction and interruptions were consistent with clinical experience with other novel agents though these did not appear to impact PFS. Disclosures Fox: Sunesis: Consultancy; Celgene: Consultancy, Other: Travel support, Speakers Bureau; Roche: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Gilead: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; AbbVie: Consultancy, Other: Travel support, Research Funding, Speakers Bureau; Janssen: Consultancy, Other: Personal fees and non-financial support, Speakers Bureau. Eyre:Gilead: Consultancy, Other: travel support; Abbvie: Consultancy, Other: travel support; Roche: Consultancy; Janssen: Consultancy, Other: travel support; Celgene: Other: travel support. Allan:Verastem: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; Acerta: Consultancy; Genentech: Membership on an entity's Board of Directors or advisory committees; Sunesis: Membership on an entity's Board of Directors or advisory committees. Schuster:OncLive: Honoraria; Physician's Education Source, LLC: Honoraria; Dava Oncology: Consultancy, Honoraria; Genentech: Honoraria, Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Research Funding; Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Nordic Nanovector: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Nabhan:Cardinal Health: Employment, Equity Ownership. Hill:Amgen: Research Funding; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees. Shah:Lentigen Technology: Research Funding; Miltenyi: Other: Travel funding, Research Funding; Juno Pharmaceuticals: Honoraria; Exelexis: Equity Ownership; Oncosec: Equity Ownership; Geron: Equity Ownership. Lamanna:Jannsen: Consultancy, Membership on an entity's Board of Directors or advisory committees; Abbvie: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Verastem: Research Funding; AstraZeneca: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; TG Therapeutics: Research Funding; Gilead: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Consultancy, Membership on an entity's Board of Directors or advisory committees; Acerta: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Cheson:AbbVie, Roche/Genentech, Pharmacyclics, Acerta, TG Therapeutics: Consultancy. Coombs:AROG: Other: Travel fees; H3 Biomedicine: Honoraria; Abbvie: Consultancy; DAVA Oncology: Honoraria; Incyte: Other: Travel fees. Barr:AbbVie, Gilead: Consultancy. Skarbnik:Gilead Sciences: Honoraria, Speakers Bureau; Pharmacyclics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Genentech: Honoraria, Speakers Bureau; Seattle Genetics: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Jazz Pharmaceuticals: Honoraria, Speakers Bureau; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Shadman:Pharmacyclics: Research Funding; Acerta Pharma: Research Funding; AstraZeneca: Consultancy; Genentech: Consultancy; Celgene: Research Funding; Verastem: Consultancy; Gilead Sciences: Research Funding; TG Therapeutics: Research Funding; AbbVie: Consultancy; Genentech: Research Funding; Mustang Biopharma: Research Funding; Beigene: Research Funding; Qilu Puget Sound Biotherapeutics: Consultancy. Ujjani:AbbVie: Consultancy, Speakers Bureau. Pagel:Pharmacyclics, an AbbVie Company: Consultancy; Gilead: Consultancy. Jacobs:Genentech: Honoraria. Schuh:Giles, Roche, Janssen, AbbVie: Honoraria. Brander:Genentech: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Novartis: Consultancy, Other: DSMB; BeiGene: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; DTRM: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; TG Therapeutics: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Acerta: Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; AbbVie: Consultancy, Honoraria, Other: Institutional research funding for non investigator initiated clinical trial, Research Funding; Pharmacyclics, an AbbVie Company: Consultancy, Honoraria, Research Funding; Teva: Consultancy, Honoraria. Mato:Pharmacyclics: Consultancy, Honoraria, Research Funding; TG Therapeutics: Research Funding; Regeneron: Research Funding; Sunesis: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Acerta: Research Funding; Abbvie: Consultancy; AstraZeneca: Consultancy; Celgene: Consultancy; Prime Oncology: Speakers Bureau.

Abstract Background: PTHrP-secreting pancreatic neuroendocrine tumors (PNET) are a recognized cause of malignancy associated hypercalcemia. Herein, we report a case of severe hypercalcemia due to an extreme elevation of PTHrP from a PNET, where after treatment of the hypercalcemia, symptomatic hypocalcemia occurred. Clinical Case: A 59 year-old-woman with a recurrent PNET with liver metastases undergoing an evaluation for multi-visceral transplant presented with acute confusion, nausea and vomiting. Diagnostic testing identified an extreme elevation of total calcium (Ca) [&gt;20.1 mg/dL (8.5 - 10.2)] from two different samples [serum albumin 4.1 g/dL (3.9 - 4.9)]. The total Ca level one month earlier was 8.3 mg/dL with a serum albumin of 3.1 g/dL. Total Ca measurements were performed with the Ca Gen.2 assay on a cobas c702 chemistry analyzer (Roche Diagnostics). Results greater than the analytical measurement range (0.8 – 20.1 mg/dL) were diluted with saline and confirmed (22.6 mg/dL). A Radiometer ABL 800 Flex blood gas analyzer was used to determine the ionized Ca concentration [2.94 mmol/L (1.08 - 1.30)]. Upon presentation the serum creatinine (Cr) was 2.07 mg/dL (0.58 - 0.96); eGFR utilizing the MDRD equation 24 mL/min/1.73m2; baseline serum Cr 0.78 mg/dL. Her serum 25-OH vitamin D was 31 ng/mL (31.0 - 80.0), PTH 12 pg/mL (15 - 65), phosphate 4.3 mg/dL (2.7 - 4.8) and 1, 25-OH vitamin D 39.1 (15.0 - 60.0). PTHrP measurements were performed by ARUP Laboratories via liquid chromatography tandem mass spectrometry (LC-MS/MS) and resulted in a reported value of &gt;2500 pmol/L (0.0 - 3.4). Her symptoms resolved and the corrected Ca gradually decreased to 8 mg/dL after treatment with IV fluids, calcitonin 200 units sc every 12 hours for 48 hours, 60 mg IV pamidronate, and five sessions of hemodialysis. Within thirteen days of receiving pamidronate, her corrected Ca slowly increased to 12mg/dL; thus, she received a single dose of 120 mg sc denosumab. Nine days later, the patient developed symptomatic hypocalcemia (7.3 mg/dL) manifested by paresthesia in the hands and feet and perioral numbness. She then received multiple doses of oral and intravenous Ca along with 50,000 units of oral ergocalciferol twice weekly. The corrected Ca normalized (8.1 mg/dL) and symptoms resolved. The patient was discharged with plans for future treatment of her underlying malignancy. Conclusion: This is the first report of a PNET producing an extreme elevation of PTHrP of higher than 2500 pmol/L, resulting in a concordant extreme elevation of total calcium within a month of documented normocalcemia. Treatment of hypercalcemia with denosumab may result in the development of hypocalcemia requiring treatment.

Abstract Coronavirus disease (COVID-19) caused by the SARS-CoV-2 virus has exposed clinical laboratories to unprecedented challenges. With surging case numbers, clinical laboratories were forced to acquiesce and integrate multiple testing platforms with varying workflows and analytical sensitivities in order to meet testing volumes. Now a new challenge has emerged with the evolution of viral variants, both globally and locally, raising concerns for uncontrolled spread, increased disease severity, and weakened responses to vaccinations. Preliminary data suggests that these variants may be associated with higher viral titers and prolonged infections. While primarily leveraged for epidemiologic surveillance, the clinical utility of variant detection may quickly become paramount. Furthermore, laboratories must remain vigilant and nimble enough to pivot should variant identification play a role in the patient care. To prepare for the validation of clinical assays that identify important viral variants, we designed a novel method, termed VariantDirect, to screen SARS-CoV-2 positive samples for the presence of variants, focusing initially on the increasingly prevalent UK and South African (SA) variants. The detection strategy is based on primers designed to specifically target the viral receptor-binding domain mutation, N501Y, shared by the UK and SA strains. Screening for variants will be limited to nasopharyngeal swab samples of high viral titers (Ct values &lt;25 by RT-qPCR assay, Roche Diagnostics). Pools of 9 different samples, 50 µl each, are mixed and stored at -80°C along with aliquots of the 9 original samples. These pools will then be tested, and if positive for the N501Y variant, the pooled 9 samples will be thawed and tested separately to identify the affected specimen. Most of these specimens are also being independently sequenced via a comprehensive but more resource-intensive NGS approach. Advantages of our pooled workflow are primarily in time and cost, with the capacity of screening up to 837 specimens on a single run. In addition, our collection strategy establishes a “time capsule” to document the evolution of viral strains within our geographical region. Finally, these studies serve to optimize technical parameters for the development of clinical assays. A validated nucleic acid (NA) extraction-free RT-qPCR method will be utilized for this assay. Our internal validation data showed comparable analytical sensitivities to NA extraction-based methods. Pooled samples in transport medium are diluted in normal saline at a ratio of 1:1, and then heat-inactivated in the presence of proteinase-K and ultimately analyzed on the Applied Biosystems™ 7500 Fast Dx instrument. As new variants of interest emerge, primers and probes can be quickly redesigned and validated on clinical samples within our NGS-confirmed “time capsule”. This study will provide important information needed for current or future genomic and epidemiologic studies.

PPARα and c-Fos are involved in regulation of gene expression and are known to be dependent on retinoic acid (RA), which in turn influences oocyte growth and developmental competence (Duque et al., 2002 Hum. Reprod. 17, 2706–2714; Hidalgo et al., 2003. Reproduction 125, 409–416), probably acting in part through granulosa cells. Peroxisome proliferator-activated receptor-α (PPARα) heterodimerizes with the retinoid receptor X (RXR), while c-Jun/c-Fos heterodimerizes with liganded retinoic acid receptors (RARs), then preventing formation of transcription factor activator protein 1 (AP-1) complexes capable of DNA binding. Cellular retinoic acid binding protein (CRABP) limits RA excess and regulates the transcriptional potential of RA;; CRABPII has been detected in rat granulosa cells from mature follicles and luteal cells. The aim of this study was to investigate PPARα, c-Fos and CRABPII mRNA expression in bovine granulosa cells. In parallel, other genes whose expression can be influenced by RA were analyzed: luteinizing hormone receptor (LHr), follicle stimulating hormone receptor (FSHr), aromatase and growth hormone (GH). Ovaries were collected at a local abattoir and kept in saline at 30–35°C. Granulosa cells were obtained by aspirating 2- to 7-mm antral follicle contents, pelleted at 700g for 4min and resuspended in RNA-later (Ambion®). Total RNA was isolated with a NucleoSpin® RNAII kit (Macherey-Nagel), and mRNA was reverse transcribed into single-stranded cDNA using a 1st Strand cDNA Synthesis Kit for RT-PCR (AMV) (Roche). A PCR standard method was made using 1μL of the cDNA as a template. All PCR primer couples were designed on the basis of the bovine sequence, but c-Fos and CRABPII primers were designed based on the human-murine sequences. Primers within the couple were located in different exons to distinguish DNA from RNA amplification. CRABPII was further investigated in bovine whole ovary, corpus luteum (CL) and liver, in a search for positive controls. Bovine β-actin, 18S and 28S were examined in each sample as positive controls for RNA isolation and cDNA synthesis efficiency. TenμL of product were loaded into an agarose 2% gel in TBE buffer containing ethidium bromide, and were separated by horizontal electrophoresis. Gels were visualized with ultraviolet light and photographed using a digital camera. Gene expression in granulosa was demonstrated for PPARα, c-Fos, LHr, FSHr, aromatase, GH and controls (β-actin, 18S and 28S) but CRABPII gene did not express in granulosa cells, whole ovary, CL or liver under our experimental conditions. While lacking CRABPII expression remains intriguing, the expressed genes support a role of retinoid pathway within granulosa cells under both in vivo and in vitro conditions, because granulosa cells used in the present experiments were derived from follicles providing oocytes for IVM-IVF. Grant support: Spanish Ministry of Science and Technology (AGL-2002-01175).

Abstract Pre-analytical error may occur when collecting blood samples from intravenous (IV) lines. Blood samples can be inadvertently diluted by exogenous solutions if an insufficient amount of blood is discarded prior to collection. Rules built in the laboratory information system or middleware can be used to alert laboratory staff to specimens that require co-investigation with the clinical team to determine if sample dilution is possible or likely. Currently, however, there is a lack of literature to guide laboratorians on how to define quality rules of this nature, particularly for detecting subtle, rather than gross contamination by commonly administered IV-fluids. Accordingly, the primary objective of this study is to derive sensitive and specific, multivariate delta checks to detect more subtle blood sample contamination by commonly used crystalloid solutions. To derive the rules, we began with in vitro experiments by spiking increasing volumes of major IV-fluids (normal saline (NS), lactated ringers (LR), and 5% dextrose (D5W)) into clinical blood samples that were collected from healthy donors (n=3). Crystalloid solutions were serially spiked into blood samples at 10% (solution:sample) increments for NS and LR from 10% up to 90%, and in 5% increments for D5W, up to 50%. Basic metabolic panel (BMP) analytes were measured and compared between neat and contrived samples. All testing was performed on Roche Cobas 8000 chemistry auto-analyzers. Based on in vitro data, we derived multivariate delta checks using analytes in a BMP that would reflect 10-40% contamination by a given fluid. We then performed a retrospective data analysis on more than 28 thousand, serially collected BMP results to identify samples that were flagged by these rules. Chart review was then performed to identify EHR-based evidence of temporally associated administration of crystalloid solutions through a peripheral IV access line. Increasing sample dilution from the in vitro study showed significant changes in several BMP analytes across all fluid types. With respect to NS, the most significant changes relative to baseline were observed with potassium, calcium, and chloride, while for LR, the most significant changes relative to baseline were observed with glucose, bicarbonate, and calcium. For D5W, significant changes were observed with glucose, calcium, chloride and sodium. Accordingly, delta check rules were implemented using relative changes of the aforementioned analytes. On chart review, we found 80-100% of flagged samples came from patients with administration of that given fluid in the last 12-24 hours. The results from this study support delta checks that consider multiple analytes, and fluid-specific bias profiles, as this may provide a more sensitive and specific approach to detecting contamination of clinical blood samples by crystalloid solutions. Further evaluation using retrospective data analysis will be used to validate these rules sensitivity and specificity followed by prospective testing before implementation.

Expression of some X-chromosome linked genes has recently been shown to be altered in bovine somatic cell nuclear transfer (SCNT) derived embryos (Wrenzycki et al. 2002 Biol. Reprod. 66, 127), implying that the regulatory mechanisms of X-linked transcription are affected by embryo in vitro production (IVP) methods. We analyzed the transcriptional pattern of X-linked genes (BIRC4, GAB3, HPRT1, MECP2, RPS4X, SLC25A6, and XIST) in bovine in vitro fertilized (IVF) and SCNT male and female blastocysts to determine X-inactivation status and changes resulting from IVP. We collected pools of male (n = 5 pools) and female (n = 3 pools) IVF-derived blastocysts (Bousquet et al. 1999 Theriogenology 51, 59) and male (n = 5 pools) and female (n = 3 pools) SCNT-derived blastocysts (Mastromonaco et al. 2004 Reprod. Domest. Anim. 39, 462). Each pool consisted of five blastocysts. Embryos were washed in phosphate buffered saline (PBS) + 0.1% polyvinyl alcohol (PVA), collected, and stored at -80�C. Total RNA was extracted with an Absolutely RNA Microprep kit (Stratagene, La Jolla, CA, USA), DNase I treated, and precipitated with isopropanol and linear acrylamide (Ambion, Inc., Austin, TX, USA) as a carrier. Reverse transcription was performed with Oligo-dT (Invitrogen, Burlington, Ontario, Canada) and Superscript II RT (Invitrogen). Transcript quantification was performed by quantitative real-time PCR using SYBR Green I (LightCycler system, Roche, Diagnostics, Laval, Quebec, Canada). Data analysis was performed with SAS (SAS Institute, Inc., Cary, SC, USA) using a mixed-model factorial ANOVA and with results presented as estimates of the median, ratios of estimates, and 95% confidence intervals with � = 0.05. IVF-derived male and female blastocysts possessed similar levels of the transcripts analyzed, suggesting successful dosage compensation at this developmental stage for embryos fertilized in vitro. XIST was not detected in male IVF embryos. GAB3 was not detected in any of the female groups and, in addition, HPRT1 transcripts were not detected in SCNT derived female embryos. Male and female SCNT-derived blastocysts possessed marked differences in their transcript levels, with males showing statistically significantly higher levels of BIRC4 and RPS4X and females possessing higher levels of MECP2 and SLC25A6 transcripts although differences between the latter two were not statistically significant. XIST was detected in both male and female SCNT blastocysts. We conclude that dosage compensation between male and female IVF blastocysts is achieved at this developmental stage for the transcripts examined. However, this pattern was markedly changed in the SCNT group, affecting especially female SCNT blastocysts, suggesting that the regulatory mechanisms of X-inactivation and X-linked gene expression are substantially altered in SCNT embryos probably due to aberrant epigenetic patterns and faulty genome reprogramming. We are currently analyzing X-linked transcription in male and female in vivo-derived blastocysts in order to compare this group with IVP-derived embryos. This work was funded by NSERC, CIHR, and CRC.

The aim of this study was to compare the differential effects of the addition of IGF-LongR3 or insulin-like growth factor-1 (IGF-1), during in vitro maturation (IVM) of bovine oocytes on embryonic development, by analysing embryonic viability and apoptosis. Ovaries collected at a slaughterhouse were transported in saline solution (NaCl 0.9%) at 38°C. Follicles of 2–8 mm of diameter were aspirated. Only cumulus-oocyte complexes (COC) with homogeneous cytoplasm, surrounded by at least 3 layers of compact cumulus cells were selected in Dulbecco’ modified PBS 0.3% polyvinyl alcohol (PVA) and transferred to TCM HEPES at 38°C. Maturation medium was composed by TCM199, 0.2 mM pyruvate, 1 μg mL–1 FSH, 50 μg mL–1 LH, 100 μg mL–1 streptomycin sulfate, 100 UI mL–1 penicillin, and 85 μg mL–1 amikacin. Four experimental groups were determined using basic medium supplemented with fetal bovine serum (FBS; 10%), PVA (3 mg mL–1 Polyvinylpyrrolidone), IGF-1 (3.100 ng mL–1 insulin growth factor), LongR3-IGF (14.100 ng mL–1 long-chain human insulin growth factor 1-r3) respectively. The IVM was performed in Petri dishes (35 × 15 mm) with 90-μL droplets of the respective media, covered with sterile mineral oil, in 5% CO2 and 38.5°C temperature atmosphere for 22–24 h. The matured oocytes were fertilised with 2 × 106 spermatozoa mL–1 and incubated for 18 h. Embryos were denuded and cultivated in SOFaa medium (2.7 mM myoinositol, 0.2 mM pyruvate, 5 mg mL–1 BSA, 100 μg mL–1 streptomycin, 100 UI mL–1 penicillin, 85 μg mL–1 amikacin, and 25 μL mL–1 of FBS). Six repetitions were performed. After 7 days, blastocyst formation was analysed and all blastocysts were submitted to the TUNEL reaction. (In Situ Cell Death Detection Kit with Fluorescein, Roche®, Mannheim, BW, Germany), according to the technique adapted by Paula-Lopes and Hansen (2002).Green nuclei fluorescence (fluorescein isothiocyanate; FITC) was considered with fragmented DNA. Data were analysed by ANOVA), and least-squares means was used to verify differences of means using the PROC GLIMMIX model of SAS statistical software package (SAS Institute, Cary, NC, USA). Cleavage rate was similar for all groups. There was no statistical difference (P > 0.05) between groups regarding to embryo production. Most of the blastocysts obtained at Day 7 of culture had good quality (grade I). However, a difference (P = 0.03) on the number of expanded blastocyst was found between FBS (20.83 ± 3.22) and PVA (10.18 ± 3.22) groups. Furthermore, IGF-1 (12.03 ± 1.60) group showed a higher (P = 0.04, P = 0.02) apoptosis rates compared to groups FBS (7.96 ± 1.29) and PVA (6.97 ± 1.48). In the present study, it was observed that the addition of IGF-1 or LongR3-IGF during IVM provided a similar embryo production compared to FBS. On the other hand, embryos obtained from oocytes maturated on medium with the addition of IGF-1 had a high number of cells with fragmented DNA, indicating a more apoptosis.

Background:Cystatins are cysteine proteases-inhibitors secreted byFasciola hepaticain order to modulate the host immune response to promote survival of the parasite. These molecules are able to inhibit different mammal cathepsins, to regulate the immune balance via Th2 and T regulatory responses, to downregulate antigen presentation and the release of pro-inflammatory cytokines (1,2) – mechanisms that are important in the development and maintenance of several immunopathology, as rheumatoid arthritis (RA) (3).Objectives:To evaluate the therapeutic effect of recombinants cystatin 1 and cystatin 3 fromFasciola hepaticain a mice model of collagen-induced arthritis (CIA).Methods:Twenty-seven DBA/1J mice were induced with CIA by an injection of collagen type-II and Freund’s adjuvant at days 0 and 18. Animals were randomly divided into three groups: vehicle (n=9, treated with phosphate-buffered saline), cystatin 1 (n=9, treated with 100 µg/dose of recombinant cystatin 1) and cystatin 3 (n=9, treated with 100 µg/dose of recombinant cystatin 3). Treatment started after day 18 by intraperitoneal injection once a day until the end of the experiment, at day 45 after CIA induction. Clinical arthritis score, nociception, paw edema, body and spleen weight were evaluated. Lymphocytes were isolated from lymph nodes and CD4+CD25+Foxp3+ T regulatory subset was assessed by flow cytometry. Data are expressed as mean ± SEM and were evaluated by one-way or two-way ANOVA followed by Bonferroni post-test.Results:Treatment with cystatin 1 did not alter any of the analyzed parameters. On the other hand, cystatin 3 was able to reduce clinical arthritis score from day 38 with 32% of reduction at day 45 (9.22±1.22) compared to vehicle (13.56±0.73) (p<0.05). In addition, treatment with cystatin 3 diminished nociception (cystatin 3: 4.0±0.36g, vehicle: 2.7±0.32g) (p<0.05) and paw edema (cystatin 3: 0.051±0,012ml, vehicle: 0.093±0.007ml) (p<0.05). Moreover, the treatment did not alter body weight (cystatin 3: 21.67±0.31g, vehicle: 21.05±0.38g) and spleen weight (cystatin 3: 7.04±0.31, vehicle: 7.16±0.38), as well as the T regulatory population (cystatin 3: 63.38±3.66%; vehicle: 58.31±6.77%).Conclusion:Treatment with cystatin 3 improved collagen-induced arthritis by attenuating the disease score, nociception and paw edema. Moreover, the treatment did not induce body weight loss or spleen weight alteration. These results suggest that recombinant cystatin 3 fromFasciola hepaticahas the potential as a treatment for inflammatory and autoimmune diseases such as RA.References:[1]Klotz C, Ziegler T, Daniłowicz-Luebert E, Hartmann S. Cystatins of parasitic organisms. Adv Exp Med Biol. 2011;712:208-21.[2]Vray B, Hartmann S, Hoebeke J. Immunomodulatory properties of cystatins. Cell Mol Life Sci. 2002 Sep;59(9):1503-12.[3]Smolen JS, Aletaha D, McInnes IB. Rheumatoid arthritis. Lancet. 2016 Oct 22;388(10055):2023-2038.Disclosure of Interests:Mirian Farinon: None declared, Renata Ternus Pedo: None declared, Thales Hein da Rosa: None declared, Barbara Jonson Bartikoski: None declared, Thaís Karnopp: None declared, Martin Cancela: None declared, Henrique Bunselmeyer Ferreira: None declared, Ricardo Xavier Consultant of: AbbVie, Pfizer, Novartis, Janssen, Eli Lilly, Roche

Abstract Background: IFN-α2 is indicated for the therapy of hairy cell leukemia, chronic myelogenous leukemia, follicular lymphoma, and malignant melanoma. As is the case for most cytokines, the pharmacokinetic (PK) properties of IFN-α2 are critical for dosing and efficacy. In vivo, the protein is quickly degraded, diffuses widely throughout the body, and has a rapid rate of renal clearance. Pegylation of IFN-α2 significantly increases the serum half-life and reduces renal clearance, thus enhancing its efficacy. However, established pegylation of IFN-α2 results in a mixture of positional isomers and reduced in vitro activity, as known for PEGASYS (Roche) and PEG-INTRON (Schering-Plough). Methods: To improve the PK properties and the potency, the DNL method (Rossi et al. Proc. Natl. Acad. Sci. USA, 2006,103:6841) was used to generate novel agents having two copies of IFN-α2b conjugated to polyethylene glycol (PEG). Results: A fusion protein (DDD2-IFN-α2b) composed of IFN-α2b with a dimerization-and-docking domain (DDD2) and a six-His tag was expressed both in myeloma cells and in E. coli. Two PEG-based modules, each composed of a fluorescent molecule, an anchor domain (AD) and either a 20-kDa PEG (IMP362) or a 30-kDa PEG (IMP413), were synthesized. Combining DDD2-IFN-α2b and IMP362 or IMP413 under redox conditions resulted in the desirable DNL conjugates consisting of two copies of IFN-α2b and one PEG linked site-specifically via the DDD and AD interaction. The cytotoxic activity of DDD2-IFN-α2b on Daudi lymphoma cells was similar to that of commercially available recombinant IFN-α2 (rhIFN-α2). The purity and identity of the two DNL conjugates (α2b-413 and α2b-362) were demonstrated by SDS-PAGE, immunoblotting, and fluorescence imaging. Both also showed potent cytotoxic activity on Daudi cells in vitro and superior PK properties to PEG-INTRON. For example, the mean blood residence times for α2b-362 (10.3 h) and α2b-413 (21.7 h) were significantly longer than those of rhIFN-α2 (0.7 h) and PEG-INTRON (5.1 h). Initial studies in mice bearing Daudi xenografts showed a significant therapeutic advantage over PEG-INTRON for both α2b-362 and α2b-413. Animals given 14,000 IU of PEG-INTRON had a median survival (MS) of 32 days compared to 21 days for saline control, whereas those receiving α2b-362 at 14,000 IU, 7,000 IU and 3,500 IU resulted in MS of 45, 41 and 32 days, respectively. More remarkably, α2b-413 was the most effective, achieving MS of 46, >53, and >53 days with 3,500 IU, 7,000 IU and 14,000 IU, respectively, all statistically significant improvements (P< 0.0028) compared to PEG-INTRON given at each equivalent activity. Conclusions: The DNL method provides a novel pegylation strategy for generating a dimeric IFN-α2b that is linked site-specifically to a single PEG at the predetermined location. Since the resulting conjugates exhibit improved PK and efficacy in a Burkitt lymphoma model, they may represent a new class of interferons for use in cancer and infectious disease therapy.

Background Primary biliary cirrhosis (PBC) is an autoimmune liver disease, and 50% of patients with this disease experience fatigue. This is a debilitating symptom affecting quality of life and resulting in social isolation, which is highlighted by patients as a research priority. PBC is characterised immunologically by the presence of high-titre autoantibodies that are directed at the pyruvate dehydrogenase complex (PDC) and are highly effective at blocking its energy generation function. We hypothesised that if anti-PDC antibodies were a driver of fatigue through bioenergetic dysfunction, then the B-cell-targeting biological agent rituximab (MabThera®, Roche Products Ltd, Welwyn Garden City, UK) might be a therapeutic option. Objective To assess whether or not rituximab safely improved moderate or severe fatigue in PBC patients. Design A Phase II, double-blind, randomised controlled trial comparing rituximab with placebo in fatigued PBC patients. Randomisation was conducted using a web-based system. Participants received two infusions on days 1 and 15 and were followed up at 3, 6, 9 and 12 months. Setting A single-centre UK study in Newcastle upon Tyne Hospitals NHS Foundation Trust. Participants Seventy-one participants aged ≥ 18 years with PBC and moderate or severe fatigue (score of > 33 on the PBC-40 fatigue domain) were screened. The PBC-40 questionnaire is a fully validated disease-specific health-related quality-of-life measure for use in patients with PBC. Fatigue, with a maximum score of 55, is one of its six domains. Fifty-seven participants were randomised to the trial, 55 of whom reached the primary end-point assessment. Intervention Participants were randomised in a 1 : 1 ratio to receive either rituximab (1000 mg) or a saline intravenous infusion (placebo) on days 1 and 15. The infusions were delivered in a double-blind manner using the same protocol. Main outcome measures The primary outcome measure was the PBC-40 fatigue domain at 3 months, assessed on an intention-to-treat basis. Secondary outcome measures included markers of bioenergetics function (anaerobic threshold and post-exercise muscle pH assessed using magnetic resonance imaging) and physical activity levels. Impact on biochemical markers of liver disease severity was assessed as an experimental outcome. Results Rituximab therapy was safe, with no serious adverse events linked to the drug. There was no statistically significant difference in fatigue score at 3 months between the rituximab and placebo arms [adjusted mean difference –0.9, 95% confidence interval (CI) –4.6 to 3.1]. However, improvement in fatigue was observed in both arms {mean score decreasing from 41.2 [standard deviation (SD) 5.5] to 36.2 (SD 8.4) in the rituximab arm and from 43.0 (SD 5.9) to 38.1 (SD 8.7) in the placebo arm}. There was little difference in any of the secondary outcomes between arms. However, anaerobic threshold improved significantly in the rituximab arm (adjusted mean difference at 3 months 1.41, 95% CI 0.03 to 2.80). No change in muscle bioenergetics characteristics was seen. A suggestive improvement in liver biochemistry was observed. Limitations Recruitment was lower than the original target, leading to a reduction in study power. A clinically significant placebo effect on PBC-40 fatigue scores was seen. Conclusions Rituximab is ineffective for the treatment of fatigue in unselected PBC patients despite metabolic modulation through improvement of anaerobic threshold. Future work Results from the trial demonstrate that metabolic effect of rituximab is not translated into clinical benefit. This will help to guide us to design future trials and when looking at completely different targets. Trial registration Current Controlled Trials ISRCTN03978701, ClinicalTrials.gov identifier NCT02376335 and EudraCT number 2012-000145-12. Funding This project was funded by the National Institute for Health Research (NIHR) Efficacy and Mechanism Evaluation programme and will be published in full in Efficacy and Mechanism Evaluation; Vol. 5, No. 2. See the NIHR Journals Library website for further project information. Additional funding was received from the Medical Research Council and a Department of Health and Social Care subvention.

Abstract Abstract 4801 Redox metabolism plays an important role in self-renewal and differentiation of hematopoietic and leukemic cells. Reactive oxygen species (ROS) level is highly regulated. This regulation involves antioxydative enzymes and it has been recently described that leukemic stem cells (LSC) overexpress glutathione peroxydase 3 (Herault O et al, J. Exp. Med, 2012). This overexpression is associated with a decrease in ROS level and p38MAPK inactivation. ROS level in leukemic cells could be also regulated by the activity of ROS producers, such as NADPH oxidase, known to catalyze an electron transfer from NADPH to oxygen producing superoxides which could generate other downstream ROS. The expression of this enzymatic complex (NOX family, 6 isoforms) has been established in the plasma cell membrane of normal CD34+ hematopoietic progenitors (Piccoli C et al, Biochem. Biophys. Res. Commun., 2007). The aim of this study was to decipher the expression of NADPH oxydase components in various human acute myeloid leukemia (AML) Different leukemic cell lines were used according FAB classification: KG1a (MO/M1), KG1 (M1), HL60 (M2), Kasumi 1 (M2), NB4 (M3), ML2 (M4), THP1 (M5), U937 (M5), MV4–11 (M5), K562 (M6). The cells were cultured (2.105 cells/mL, 37°C in 95% humidified air and 5% CO2) in RPMI 1640 with 20mmoL/L L-glutamine supplemented with 10% FCS, 100 units/mL penicillin G, and 100mg/mL streptomycin. The expression of NOX1, NOX2, NOX3, NOX4, NOX5, DUOX1, DUOX2, P22phox and P40phox, P47phox, P67phox, NOXO1, NOXA1 was quantified by RT-qPCR (Universal Probe Library, Roche). NOX2 and its regulatory subunits expression was quantified by SDS-PAGE and western-blot experiments. The effects of diphenylene iodonium (DPI), a specific NOX inhibitor, were evaluated by ROS quantification using dichlorodihydrofluorescein diacetate (DCF-DA) staining followed by fluorimetry and flow cytometry analyses. The cells were washed twice in the physiological saline buffer (PBS) without calcium and magnesium, then incubated in PBS complemented with 0.5M MgCl2, 0.9M CaCl2, 20mM glucose (Picciocchi A et al, J. Biol. Chem., 2011) with or without 20μM DPI for 1 hour. The cells were distributed at 106cells per 200μL well in 96 wells plates. DCF-DA (10μM) was added to quantify the ROS level (flow cytometry) and to monitor ROS production kinetic (fluorimetry). NOX family genes expression showed that phagocyte oxidase NOX2 is expressed in all leukemic cell lines. Conversely the NOX2 isoforms were not expressed, or very weakly expressed in leukemic cell lines (NOX3 in KG1a; NOX4 in K562; DUOX1 in KG1a, KG1; DUOX2 in KG1a, KG1, HL60). P22phox, the second cytochrome b558 component was also expressed in all cell lines, this expression being higher than NOX2. The cytochrome b558 components were more expressed in differentiated leukemic cells (granulocytic and monocytic) than in undifferentiated cells (KG1a, KG1). NOX2 regulatory subunits were expressed in all leukemic cell lines, the lower level (especially P40phox, P47phox) being observed in KG1a. Proteins quantification confirmed RNA results. Cytochrome b558 components and regulatory subunits were expressed in all cell lines with a higher level in differentiated leukemias. Interestingly, the regulatory subunits were not observed in KG1a cells. Functional flow cytometry and fluorimetry studies revealed a decrease in ROS production in DPI exposed leukemic cell lines. This effect was higher in monocytic cell lines than in granulocytic and undifferentiated leukemias. In conclusion, NADPH oxidases are present in the AML cell membrane, and NOX contribution to the ROS level is higher in differentiated cells than in immature leukemias. Altogether these results suggest that NADPH oxidase is constitutively active in leukemic cells and influences the ROS level, suggesting a role in the pathophysiology of AML. Disclosures: No relevant conflicts of interest to declare.

Abstract Background We showed earlier that prolonged treatment with a combination of azacitidine (DNMT1 inhibitor) and panobinostat (HDAC inhibitor) induced complete remission in a disseminated xenograft model of KMT2A rearranged AML (Gopalakrishnapillai et al., 2017, Leuk Res). Pretreatment with epigenetic drugs has been shown to induce chemo-sensitivity in some malignancies. However, studies on the use of epigenetic drugs in overcoming chemoresistance mediated by the bone marrow microenvironment are limited. The aim of this study was to determine the merit of incorporating epigenetic therapy with chemotherapy in AML treatment regimen and to identify the mechanism by which epigenetic drugs can chemosensitize AML. Methods AML cell lines and PDX lines were co-cultured with HS5 bone marrow stromal cells. Cell viability following drug treatment was assessed by flow cytometry. For determination of cell adhesion, violet proliferation dye stained AML cells were co-cultured with HS5 cells. Following treatment, non-adherent cells were removed by washes with phosphate buffered saline. The number of adherent AML cells was determined by flow cytometry. NSG-B2m mice were engrafted with MV4;11 cells and bled regularly to evaluate disease progression. Once engraftment was confirmed mice were assigned to treatment groups. Epigenetic therapy constituted azacitidine and panobinostat (2.5 mg/Kg each; Qd5). Chemotherapy constituted cytarabine (50 mg/Kg; Qd5) and daunorubicin (1.5 mg/Kg; Qd3). Mice were euthanized when they reached predetermined experimental endpoints. All animal studies were approved by the Institutional Animal Care and Use Committee. Results AML cells in co-culture with HS5 bone marrow stromal cells were less sensitive to chemotherapeutics cytarabine and daunorubicin compared to AML cells in monoculture. This chemoprotection was not achieved when AML cells were cultured in HS5 conditioned media or in Transwell inserts suspended over HS5 monolayers, suggesting that direct cell-to-cell contact was required. MV4;11 cells exposed to epigenetic drugs or cytarabine alone retained 70% viability. Pre-treatment with the epigenetic drug combination of azacitidine and panobinostat prior to cytarabine exposure greatly reduced cell viability in MV4;11 cells harboring KMT2A fusion (Fig. 1A). Similar chemo-sensitization was observed when four distinct KMT2A rearranged PDX lines were used ex vivo. This sensitization was accompanied by reduced binding of AML cells to HS5 cells following treatment with epigenetic drugs (Fig. 1B). We evaluated the efficacy of the epigenetic therapy and chemotherapy combination in disseminated xenograft models of pediatric AML. NSG-B2m mice transplanted with MV4;11 cells via the tail vein were randomly assigned to four groups - 1) vehicle, 2) epigenetic therapy 3) chemotherapy (cytarabine + daunorubicin), and 4) epigenetic therapy followed by chemotherapy. The mice receiving the epigenetic therapy and chemotherapy combination survived significantly longer than mice treated with any other condition (Fig. 1C). To assess the leukemic cell distribution in peripheral blood, bone marrow and spleen following treatment, a cohort of mice receiving epigenetic therapy alone or chemotherapy alone were euthanized a day after treatment concluded. The percentage of leukemic cells in bone marrow and spleen was lower in mice treated with epigenetic therapy than those treated with chemotherapy, consistent with the survival data. Surprisingly, the peripheral blood counts were significantly higher in mice receiving epigenetic drug combination. These results together with our in vitro data indicate that epigenetic therapy induces mobilization of AML cells to the blood stream. This increased availability of AML cells may promote enhanced sensitivity to chemotherapy. Conclusion Our data suggest that direct cell-to-cell contact plays a major role in mediating chemoprotective effects of the bone marrow microenvironment. These chemoprotective effects can be overcome by pretreatment with epigenetic drug combination azacitidine and panobinostat. Epigenetic drugs interfere with AML cell interactions with the microenvironment and dislodge AML cells from their protective niche. Thus, mobilization of AML cells to peripheral blood is a potential mechanism of chemo-sensitization mediated by epigenetic drugs. Disclosures Kolb: Servier: Membership on an entity's Board of Directors or advisory committees; Roche- Genentech: Membership on an entity's Board of Directors or advisory committees.

Strategi komunikasi dapat diartikan keseluruhan perencanaan, taktik, cara yang akan digunakan agar proses komunikasi dapat berjalan dengan lancar dengan memperhatikan seluruh aspek yang ada pada proses komunikasi untuk mencapai tujuan yang diinginkan. Sedangkan ukhuwa Islamiyah adalah kekuatan iman dan spiritual yang dikaruniakan Allah kepada hamba-Nya yang beriman dan bertakwa, sehingga menumbuhkan perasaan cinta dan saling percaya terhadap sesame muslim atau saudara seiman. Dalam hal ini penulis mengambil peran strategi komunikasi anggota zona tahfidz Universitas Darussalam Gontor dalam meningkatkan Ukhuwwah Islamiyah.Penelitian deskriptif dengan mengunakan pendekatan kualitatif. Kualitatif adalah penelitian yang digunakan untuk meneliti kondisi atau objek yang bersifat alamiah Jenis penelitian yang digunakan dalam penelitian ini adalah penelitian lapangan (Field Reaserch). Teknik pengumpulan data yang digunakan peneliti adalah observasi, wawancara dan dokumentasi dengan beberapa anggota dan pengurus zona tahfidz. Hasil penelitian menunjukkan bahwa strategi komunikasi yang digunakan zona tahfidz Program ON-Roce merupakan strategi komunikasi yang di terapkan oleh anggota Zona Tahfidz Universitas Darussalam Gontor Kampus Putri. Adapun program tersebut antara lain: Happy family Zona Tafidz, Sharing personal, Mix senjo competition, Penerapan SCE.Key word: Stategi komunikasia, ukhuwah islamyah, ON-Roce

CONCENTRAÇÃO DE SAIS DA SOLUÇÃO AVALIADA PELA CONDUTIVIDADE ELÉTRICA NA ZONA RADICULAR DO CRISÂNTEMO SOB IRRIGAÇÃO POR GOTEJAMENTO Poliana Rocha D’Almeida Mota1; Roberto Lyra Villas Bôas1; Valdemício Ferreira de Sousa21Departamento de Recursos Naturais/Ciência do Solo, Faculdade de Ciências Agronômicas, Universidade Estadual Paulista, Botucatu, SP, polimota@fca.unesp.br2Embrapa Meio-Norte, Teresina, PI 1 RESUMO Avaliou-se o efeito de concentrações salinas a partir da condutividade elétrica (CE) no ambiente radicular de plantas de crisântemo (Dendranthema grandiflora Tzvelev.) cultivadas em substrato sob duas metodologias de amostragem em ambiente protegido. O experimento foi desenvolvido em Paranapanema, São Paulo usando o delineamento experimental em blocos casualizados com quatro repetições. Os tratamentos referem-se a oito épocas de amostragem da solução no substrato dos vasos: 7, 14, 21, 28, 35, 42, 49 e 56 dias após enraizamento (dae) e cinco níveis de concentração salina da solução aplicada: 1,42; 1,65; 1,89; 2,13 e 2,36 dS m-1 na fase vegetativa e 1,71; 1,97; 2,28; 2,57 e 2,85 dS m-1 durante a fase reprodutiva de emissão de botões florais. O monitoramento da CE da solução do substrato foi realizado por meio das metodologias: extrator de solução e solução diluída em água 1:2. As metodologias pelo extrator de solução e pela solução diluída 1:2 permitem o monitoramento da concentração de sais na solução do substrato ao longo do ciclo da cultura; a elevação da concentração salina da água aplicada no substrato promove o aumento da salinidade do substrato; a metodologia com o uso de extrator de solução apresenta maiores valores de condutividade elétrica do substrato. UNITERMOS: Dendranthema grandiflora, extrator de solução, salinidade MOTA, P. R. D’A.; VILLAS BÔAS, R. L.; SOUSA, V. F. de. SALT CONCENTRATION IN SOLUTION EVALUATED BY THE ELECTRICAL CONDUCTIVITY IN CHRYSANTHEMUM ROOT ZONE UNDER TRICKLE IRRIGATION 2 ABSTRACT The effect of salt concentration levels in electrical conductivity (EC) were evaluated in chrysanthemum root, cultivated in substrate using two sampling methods, under greenhouse conditions. The experiment was carried out in Paranapanema, São Paulousing the experimental design in randomized blocks and four replications. The treatments consisted of eight sampling periods of substrate solutions in pots: 7, 14, 21, 28, 35, 42, 49 and 56 days after strike root and five salt concentration levels of applied saline solution: 1.42;1.65; 1.89; 2.13and 2.36dS m-1 in the vegetative period and during the reproduction period of flower budding: 1.71; 1.97; 2.28; 2.57 and 2.85 dS m-1. The substrate solution EC monitoring was done using two methods: solution extractors and 1:2 water diluted solution. The use of solution extractors and 1:2 water diluted solution allowed substrate solution EC monitoring along the culture cycle; the amount of salt concentration applied in the substrate caused the substrate salinity increase; the method using solution extractors presented higher EC values in the substrate. KEYWORDS: Dendranthema grandiflora, solution extractor, salinity

Abstract Hematopoietic stem cells (HSC) reside in specific peri-vascular niches in the bone marrow (BM). We have shown interactions with the inflammatory vascular adhesion molecule E-(endothelial)-selectin awakens HSC (Nat Med 2012). We now report that BM vascular cell-surface E-selectin expression is strongly upregulated during HSC mobilization regimens raising the question of a role for endothelial E-selectin in HSC transplant outcome. G-CSF was administered to cohorts of E gene-deleted or wildtype C57BL/6 mice together with E-selectin antagonist Uproleselan (Upro, GMI-1271). We found absence (Sele-/- mice) or therapeutic blockade (Upro) of E-selectin alone in steady-state hosts did not alter levels of circulating peripheral blood (PB) HSC. In contrast absence or therapeutic blockade of E-selectin strongly synergized with mobilizing regimens such as G-CSF or cyclophosphamide+G-CSF by boosting long-term engraftment and reconstitution potential of mobilized blood. The effect was pronounced boosting reconstitution potential over G-CSF-alone-mobilized blood 24-fold (<p=0.0001; analyzed by Poisson Statistics following serial-dilution long-term competitive-reconstitution [LT-CR] transplantation assays of 4 PB dilutions). Despite the increased reconstitution potential no change in mobilized phenotypic HSC nor in colony forming cell numbers were observed. Together these results suggest the significant 24-fold boost in peripheral blood reconstitution potential induced by Upro + G-CSF is due to increases in HSC potency alone. Two hypotheses may explain how therapeutic E-selectin-blockade promotes potency of mobilized HSC;Dampening of inflammatory activation at the BM niche induced by G-CSF that drive HSC exhaustion,Directly preventing adhesion-mediated HSC commitment during trans-endothelial transmigration from BM into blood. To investigate inflammatory mediator profiles in the BM, cohorts of mice were administered saline control or G-CSF ± Upro (125ug/kg G-CSF, 40mg/kg Upro BiD for 3 days) then femoral BM fluids flushed for cytokine profiling (LegendPlex). As anticipated, G-CSF administration significantly increased concentration of classic pro-inflammatory cytokines (TNF-α, IL-1β, IL-23, IFN-β) in BM associated with HSC activation and loss of quiescence in the BM. No similar boost in inflammatory mediators was observed in BM from mobilized mice co-administered Upro. Similarly pronounced metabolic changes could be observed in BM HSC following in vivo G-CSF administration (such as a significant doubling in HSC Mitochondrial Membrane potential [MOMP] suggesting increased energy demands with HSC activation) was similarly reversed in vivo by Upro co-administration. Together these results suggest blockade of E-selectin on activated vasculature significantly dampens BM HSC activation following G-CSF administration - potentially shielding HSC self-renewal potential. Next we investigated whether the transient interactions with endothelial E-selectin during trans-endothelial transmigration from BM into blood also directly affects HSC potency. BM HSPC harvested from G-CSF plus Upro-treated mice were loaded in transwells pre-coated with recombinant adhesion molecules (P-selectin, E-selectin and CD14-Fc as control) and HSC induced to transmigrate through coated pores towards CXCL12 gradient. After 3hr cell numbers were enumerated and exactly 100 transmigrated HSC from each well transplanted/recipient in a LT-CR transplant assay. Analysis of % CD45.2+ donor HSC reconstitution confirmed a pronounced 10-fold drop in reconstitution potential of HSC transmigrated through E-selectin compared to P-selectin or control coated transwells (p=0.0027) confirming that transient adhesive interactions with E-selectin, such as would occur on activated vasculature during HSC transmigration from BM into the blood, strongly impact subsequent HSC reconstitution upon transplantation. In summary, transient interactions between intravasating HSC with E-selectin on BM vasculature inadvertently compromises reconstitution potential of up to 96% of conventionally harvested HSC, indicating an unexpected disadvantage with current HSC harvesting procedures. These studies also point the way forward to a simple remedy, administration of E-selectin antagonist (Upro) together with G-CSF during HSC mobilization, to improve HSC transplant outcomes. Disclosures Winkler: GlycoMimetics: Patents & Royalties. Magnani:GlycoMimetics: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Marlton:Celgene: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; AbbVie: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlycoMimetics: Research Funding; Pfizer: Membership on an entity's Board of Directors or advisory committees; Astellas: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Perkins:Novartis Oncology: Honoraria. Levesque:GlycoMimetics: Equity Ownership, Patents & Royalties.

Maracujá-do-mato (Passiflora cincinnata) is a species adapted to the climatic conditions of the Brazilian semi-arid region and widely used as rootstock, however, studies related to the production of seedlings and their resistance to abiotic stresses are scarce in literature. The objective was to study the production of maracujá-do-mato seedlings under the effect of the electrical conductivity on the irrigation water as a function of the application of organic fertilizers. The experiment was developed at the State University of Paraíba, Catolé do Rocha-PB, in a completely randomized experimental design, in a 5 x 3 factorial scheme, with 6 replicates. The factors evaluated were five electrical conductivities of irrigation water (ECw: 1; 2; 3; 4 and 5 dS m-1) and application of three organic fertilizers (bovine urine, bovine biofertilizer and liquid earthworm humus). It was verified that the increase of ECw affected the morphology and the quality of the seedlings negatively, while the bovine biofertilizer presented better efficiency in comparison to the others. It is inferred that the use of organic fertilizers as mitigating effects of salinity may be a strategy for production of maracujá-do-mato seedlings in saline conditions.

Introduction Breast Implant Associated Anaplastic Large Cell Lymphoma (BIA-ALCL) is a rare subtype of ALCL that arises as a seroma or a mass in the space surrounding textured breast implants. In July 2019 the FDA had reported 573 cases worldwide and 33 deaths connected to the lymphoma. However, collections of cases usually come from large groups of institutions or countries, with different approaches regarding surgery and treatment. Here we describe a cohort of 18 cases undergoing implant removal and capsulectomy and followed at Memorial Sloan Kettering Cancer Center (MSKCC) Methods We retrospectively analyzed all the cases of women with breast implants undergoing implant removal and capsulectomy for BIA-ALCL at MSKCC from January 2011 to June 2020. Results are expressed in absolute numbers and percentages, survival is calculated with the Kaplan Maier method. Results Of 27 total BIA-ALCL cases seen at MSKCC, 18 women had their primary surgery for implant removal and capsulectomy by our surgery division, analyzed in our central pathology and treated and/or expectantly monitored in our T cell lymphoma clinic. Sixteen patients (89%) had implants as reconstruction after breast cancer (of which 12/16, 75% had received adjuvant/neoadjuvant chemotherapy (CT), 3, 19% radiotherapy (RT). Two subjects (11%) had implants for cosmetic reasons). Four patients (25%) had undergone implants exchange after initial reconstruction. BRCA mutational status was known in 9 patients (56%), with 3/9 women (33%) having BRCA mutations (2 BRCA2, 1 BRCA1) and one additional woman initially thought to have a mutation of BRCA2, subsequently re-classified as polymorphism. After a median exposure of 11 years (STDEV 6 y), 10 years from last implant change, median age at BIA-ALCL diagnosis was 57 years (STDEV 10 y). Implant characteristics were as follows: 18/18 (100%) silicone surface, 17/18 (94%) had a biocell textured surface, 1/18 (6%) unknown surface, 9/18 (50%) silicone filled, and 9/18 (50%) saline filled. BIA-ALCL presented as effusion in 14/18 (78%) of cases, mass in 3/18 (17%) of cases, and PET+ intramammary lymphadenopathy in 1/18 (6%). Pre-surgery, cases were assessed with PET/CT in 15/18 (83%), US in 15/18 (83%) and MR in 7/18 (39%). Initial treatment was implant removal and capsulectomy for all women, with removal of all the implants in place. One early case received implant removal and replacement with a smooth textured implant with partial capsulectomy prior to confirmed diagnosis. Three of 18 (16%) who presented with higher stage disease received additional treatment: one received chemotherapy (Brentuximab Vedotin - cyclophosphamide - doxorubicin - prednisone, BV-CHP) followed by RT consolidation, and 2 received RT consolidation only. Patients were followed for a median of 26 months (STDEV 2.22 months) after diagnosis, with clinical exam every 3-6 months (100%), PET/CT in 16/18 (89%), and MR in 4/18 (22%). No patient died of lymphoma progression or recurrence. One patient died from progression of breast cancer. One woman (described above) recurred 2 years after receiving incomplete capsulectomy and smooth surface implant re-placement. She underwent repeat implant removal and bilateral capsulectomy and remains disease free at 6.5 years from the second surgery. Another patient was likely diagnosed at recurrence: she underwent unilateral implant removal and partial unilateral capsulectomy for recurrent delayed seroma (at the time of this implant removal BIA-ALCL could not be confirmed). 1.5 years later she developed a recurrent seroma on the contralateral side where a textured device remained. BIA-ALCL diagnosis was confirmed at this time. Overall survival (Figure 1), was 94% at 2 years and progression-free survival was 89% at 2 years. Conclusions Our data on this cohort of patietns with BIA-ALCL surgically treated and followed at a single institution, confirm the importance of adequate surgery (bilateral implant removal and complete capsulectomy) in patients presenting with seroma-confined disease. This dataset reinforces the high rates of progression free and overall survivalwhen diagnosis is identified and treatment performed in those with limited stage disease. Studies are ongoing to determine the role of somatic mutations like BRCA1-2. Figure: overall and progression-free survival of BIA-ALCL women operated and followed at MSKCC Figure 1 Disclosures Cordeiro: Inamed: Consultancy, Research Funding; Acelity: Consultancy; Allergan: Consultancy, Research Funding. Moskowitz:Bristol-Myers Squibb: Research Funding; Merck: Research Funding; Merck: Consultancy; Miragen Therapeutics: Consultancy; Seattle Genetics: Consultancy; Seattle Genetics: Research Funding; Incyte: Research Funding; Imbrium Therapeutics, L.P.: Consultancy. Dogan:Takeda: Consultancy; AbbVie: Consultancy; National Cancer Institute: Research Funding; Seattle Genetics: Consultancy; EUSA Pharma: Consultancy; Corvus Pharmaceuticals: Consultancy; Roche: Consultancy, Research Funding; Physicians Education Resource: Consultancy. Horwitz:Vividion Therapeutics: Consultancy; Affirmed: Consultancy; Daiichi Sankyo: Research Funding; GlaxoSmithKline: Consultancy; Janssen: Consultancy; Kura Oncology: Consultancy; ASTEX: Consultancy; C4 Therapeutics: Consultancy; Beigene: Consultancy; Portola: Consultancy, Research Funding; Mundipharma: Consultancy; Innate Pharma: Consultancy; Corvus: Consultancy; Trillium: Consultancy, Research Funding; Seattle Genetics: Consultancy, Research Funding; Millenium/Takeda: Consultancy, Research Funding; Kyowa Hakka Kirin: Consultancy, Research Funding; Infinity/Verastem: Research Funding; Forty Seven: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Aileron: Consultancy, Research Funding; ADCT Therapeutics: Consultancy, Research Funding; Verastem: Consultancy, Research Funding; Myeloid Therapeutics: Consultancy; Miragen: Consultancy.

ARRANJO ESPACIAL E PODA NA PRODUÇÃO E QUALIDADE QUÍMICA DE MARACUJÁ IRRIGADO COM ÁGUA SALINA RAFAEL RAMOS MORAIS1; JOÃO PAULO SILVA MACÊDO2; LOURIVAL FERREIRA CAVALCANTE3; JACKSON TEIXEIRA LOBO1; ANTÔNIO GUSTAVO LUNA SOUTO1 E EVANDRO FRANKLIN MESQUITA4 1 Programa de pós-graduação em Agronomia, Departamento de Fitotecnia e Ciências Ambientais do Centro de Ciências Agrárias, Universidade Federal da Paraíba, Rodovia PB 079, SN, Km 12, 58.397-000, Areia, Paraíba, Brasil, agro.rafaelmorais@gmail.com; jacksonteixeira78@gmail.com, gusluso@hotmail.com. 2 Empresa Paraibana de Pesquisa, Extensão Rural e Regularização Fundiária, Rodovia BR 230, S/N, Km 13, Morada Nova, 58.108-502, Cabedelo, Paraíba, Brasil, jp_agro@hotmail.com. 3 Professor do Departamento de Solos e Engenharia Rural, Universidade Federal da Paraíba, Rodovia PB 079, S/N, Km 12, 58.397-000, Areia, Paraíba, Brasil, lofeca@cca.ufpb.br. 4 Professor do Centro de Ciências Humanas e Agrárias, Universidade Estadual da Paraíba, Sitio Cajueiro, S/N, Zona Rural, 58.884-000, Catolé do Rocha, Paraíba, Brasil, elmesquita4@uepb.edu.br. 1 RESUMO Um experimento foi desenvolvido no município de Coronel Ezequiel, Estado do Rio Grande do Norte, Brasil, para avaliar os efeitos da densidade de plantio e poda da haste principal em plantas de maracujazeiro amarelo, acesso Guinezinho, sob irrigação com água salina de 3,4 dS m-1. Os tratamentos, com distância de 2,0 m entre linhas, foram distribuídos em blocos ao acaso, com quatro repetições de 12 plantas por parcela, em esquema fatorial 4 × 2, correspondente as distâncias de 3, 6, 9 e 12 m entre plantas nas linhas com e sem poda da haste principal, ao atingir o sistema de sustentação. Os componentes avaliados foram número de frutos colhidos, produção por planta, produtividade e na polpa dos frutos, os valores de sólidos solúveis, acidez titulável, pH e a relação sólidos solúveis/ acidez titulável. A interação entre os fatores estudados exerceu efeitos significativos no número de frutos colhidos, produção por planta, teor de sólidos solúveis e acidez titulável. A produtividade foi influenciada pelos dois fatores de forma isolada. O pH da polpa respondeu apenas ao espaçamento entre plantas nas linhas e a relação SS/AT não foi influenciada por nenhuma das fontes de variação estudadas. O aumento das distâncias de plantio nas linhas promove ganho de produção por planta, mas reduz a produtividade. Plantas podadas na haste principal apresentam maior produtividade. A irrigação com água de qualidade restritiva à agricultura não inibiu a capacidade produtiva do maracujazeiro amarelo acesso Guinezinho e não prejudicou a qualidade química dos frutos. Palavras-chave: acesso Guinezinho, densidade de plantio, Passiflora edulis Sims. MORAIS, R. R.; MACÊDO, J. P. S.; CAVALCANTE, L. F.; LOBO, J. T.; SOUTO, A. G. L.; MESQUITA, E. F. SPATIAL ARRANGEMENT AND PRUNING IN THE PRODUCTION AND CHEMICAL QUALITY OF YELLOW PASSIONFRUIT IRRIGATED WITH SALINE WATER 2 ABSTRACT An experiment was carried out in Coronel Ezequiel municipality, Rio Grande do Norte State, Brazil, to evaluate the effects of planting density and pruning of the main stem on yellow passion fruit plants access Guinezinho under irrigation with saline water (3.4 dS m-1). The treatments, with inter-row distance of 2 m, were distributed in randomized blocks, with four replications of 12 plants per plot, in a 4 × 2 factorial scheme, corresponding to intra-row distances of 3, 6, 9 and 12 m for plants with and without pruned main stem, upon reaching the support system. The evaluated components were number of fruit harvested, production per plant, fruit yield and in the fruit pulp, the analyzed components were soluble solid content, titratable acidity, pH and soluble solid content/ titratable acidity ratio. The interaction between the factors studied significantly affected the number of fruit harvested, production per plant, solids content and titratable acidity. The fruit yield was influenced by the two isolated factors, but the pH of the pulp responded only to the intra-row plant spacing, and the solids content/ titratable acidity ratio was not influenced by any of the sources of variation studied. Increasing the distances intra-row raises the production per plant, but reduces fruit yield. Plants pruned on the main stem have higher fruit yield. Irrigation with restrictive water quality to agriculture did not inhibit the productive capacity of yellow passion fruit access Guinezinho and did not impair the chemical quality of the fruits. Keywords: Guinezinho access, planting density, Passiflora edulis Sims.

As atividades de projeto e instalação de colunas de revestimento em poços de petróleo correspondem a elementos fundamentais para garantir a segurança e a operacionalização de campos exploratórios. Com o desafio da perfuração de poços em áreas geologicamente mais complexas, a exemplo das chamadas reservas do pré-sal e em reservatórios sujeitos à alta pressão e alta temperatura (High Pressure High Temperature - HPHT), novas variáveis de projeto devem ser consideradas. Este trabalho propõe-se a avaliar o comportamento de modelos constitutivos de rochas salinas por meios de curvas de fluências objetivando avaliar o impacto do efeito de fluência da rocha salina na estabilidade do poço aberto na fase de perfuração. Também são avaliados os incrementos de esforços advindos do comportamento viscoso das rochas salinas nas colunas de revestimento de poços de petróleo e a respectiva influência no tratamento de projetos para dimensionamento de revestimento de poços. Emprega-se uma estratégia baseada no método dos elementos finitos para modelar o comportamento de fluência em rochas salinas através de modelos viscoelásticos em função da série de Pronny. O processo de dimensionamento das colunas de revestimento é efetuado seguindo as recomendações normativas da API5C3 e ISO10400.

Obesitas terus meningkat di seluruh dunia, khususnya di Indonesia. Kondisi ini ditandai oleh peningkatan lemak tubuh yang berlebihan berdampak negatif pada kesehatan. Pengukuran obesitas menggunakan perhitungan IMT yaitu berat badan dalam kilogram dibagi dengan kuadrat tinggi badan dalam meter. Dikatakan obesitas jika IMT > 25kg/m2. Jaringan lemak pada orang dengan obesitas memiliki respons inflamasi rendah. Sel lemak yang meradang melepaskan sitokin pemicu rasa lapar seperti interleukin-6 (IL-6) dan tumor necrosis factor-alpha. Kadar IL-6 yang meningkat secara terus menerus dapat menyebabkan berbagai macam penyakit. Hubungan antara IL-6 dan obesitas adalah kompleks atau saling terkait dengan faktor lain. Obesitas merupakan kondisi multifaktorial yang dipengaruhi oleh genetik, gaya hidup, pola makan, dan lingkungan. Tujuan dalam penelitian ini adalah untuk mengetahui hubungan antara obesitas dengan kadar Interleukin-6 dan mengetahui apa penyebab peningkatan kadar Interleukin-6 pada populasi anak laki-laki di Kota Makassar. Penelitian ini bersifat deskriptif analitik dengan studi observasional yang memakai desain kohort untuk melihat hubungan dari kadar IL-6 terhadap kondisi obesitas. Pengukuran kadar IL-6 pada plasma menggunakan metode Roche- cobas 6000. Hasil analisis didapatkan bahwa kadar Interleukin 6 pada kondisi obesitas merupakan kriteria rendah dengan nllai kolerasi p= 0.372; p < 0.05. Sementara hubungan usia dengan kadar interleukin 6 diperoleh nilai kolerasi 0.096; p >0.05 artinya tidak terdapat hubungan diantara kedua variabel atau termasuk kriteria sangat rendah. Hubungan kadar interleukin-6 pada populasi anak laki-laki dengan kondisi besitas terbilang cukup rendah dimana tidak terjadi peningkatan signifikan diantara keduanya. Tetapi, semakin tinggi Indeks massa tubuh seseorang kadar interleukin 6 terus meningkat walaupun tidak secara drastis.

Today’s Economy is rapidly facing changes in globalization and Knowledge-based products and services. The survival and performance of an organization are influenced by its ability and speed in Developing knowledge-based competencies among the employees (Salina and Fadzilah,2008) According to Goh, SC. (2002), One of the major challenges an organization faces are managing its knowledge assets. Increasingly, knowledge transfer is seen as a basis for competitive advantage. Organizations always face issues with the kind of information, competencies, and expertise that are essential for it to take widespread advantage of available opportunities (Husnain et al., 2021). In addition to these competencies, an organization must possess unique features that competitors find difficult to imitate while operating in the same market and industry (Sousa and Rocha 2019; Sohail et al., 2020). This paper explores, in-depth, the role that key strategic organizational capabilities play in facilitating or preventing knowledge transfer. The paper converses knowledge transfer in an organizational context, then discusses significant organizational capabilities that trigger knowledge transfer and further discuss how these organizational capabilities influence the ability to transfer knowledge, an important area of knowledge management. Each of these key capabilities and their influence on Knowledge transfer is discussed separately and then integrated into a framework to explain how effective knowledge transfer can be managed in an organization with such capabilities. Conclusions are drawn about the complexity of knowledge transfer and the need to take a balanced approach to the process.

<p>Dalam rangka mendeskripsikan status pemanfaatan dan pengelolaan sumberdaya tuna neritik di perairan Samudera Hindia (WPP 572 dan 573) telah dilakukan analisis terhadap informasi tentang jenis dan produksi tuna neritik yang disajikan dalam Statistik Perikanan Tangkap di Laut Menurut WPP tahun 2005-2012 (DJPT, 2013) serta data hasil penelitian berbasis di PPS Cilacap dan PPN Sibolga tahun 2011. Rekomendasi ‘working party’ tentang tuna neritik dari IOTC dikaji sebagai langkah pengelolaan perikanan tuna neritik di Indonesia. Hasil analisis dan kajian menunjukkan bahwa sumberdaya ikan tuna neritik yang tertangkap nelayan Indonesia di perairan WPP 572 dan 573 meliputi tongkol lisong (<em>Auxis rochei</em>), tongkol krai (<em>Auxis thazard</em>), tongkol komo atau kawakawa (<em>Euthynnus affinis</em>) dan tongkol abu-abu (<em>Thunnus tonggol</em>). Tuna neritik tertangkap sebagai by-product dari pukat cincin, jaring insang hanyut, pancing tonda, pancing ulur dan bagan. Tahun 2011 produksi neritik tuna di Samudera Hindia khususnya WPP 572 dan 573 mencapai 121.818 ton atau 29,4% dari total produksi tuna neritik nasional. Tuna neritik jenis tongkol lisong dan krai yang tertangkap jaring insang hanyut yang berbasis di Cilacap &gt; 70% merupakan ikan yang telah dewasa. Adapun tongkol komo yang tertangkap pukat cincin yang berbasis di Sibolga sekitar 55,5% merupakan ikan dewasa. Belum ada langkah-langkah pengelolaan secara spesifik terhadap sumberdaya tuna neritik di Indonesia. Merujuk hasil Working Party on Neritic Tuna pertama dan kedua tahun 2011 dan 2012, Indian Ocean Tuna Commission (IOTC) merekomendasikan adanya kerjasama antar negara anggota IOTC yang saling berdekatan didalam melakukan pengelolaan sumberdaya neritik tuna. Langkah pertama adalah dilakukan perelitian mengenai populasi melalui studi mtDNA untuk memastikan status stok dan populasinya.</p><p> </p><p>The species of neritic tuna caught by fishers in the Indian Ocean particularly FMAs 572 and 573 consisted of frigate tuna (<em>Auxis thazard</em>), bullet tuna (<em>Auxis rochei</em>), longtail tuna (<em>Thunnus tonggol</em>) and kawa-kawa/eastern little tuna (<em>Euthynnus affinis</em>). These species are by-product of purse seine, drifting gillnet, trolling lines, and lift net. In 2011, production of the neritic tuna from FMAs 572 and 573 reached 121,818 mt or about 29.4% of the national production. More than 70% of catch of neritic tuna especially frigate and bullet tuna caught by drifting gillnet based at Cilacap were matured fish, and kawa-kawa caught by purse seine based at Sibolga about 55.5% of total catch was mature. There are no specific management measures for neritic tuna resources in Indonesia. First and Second IOTC Working Parties on Neritic Tuna in 2011 and 2012 recommended among IOTC’s member countries that are geographically close to each other to conduct a management collaboration of neritic tuna which begins with identifying the status of stock and population through a study mtDNA or other proper methodology.</p>

LEAF GAS EXCHANGE AND NUTRIENTS ACCUMULATION IN COWPEA PLANTS UNDER DIFFERENT MANAGEMENT STRATEGIES WITH BRACKISH WATER ANTÔNIA LEILA ROCHA NEVES1; CLAUDIVAN FEITOSA DE LACERDA2; MAILSON PEREIRA ALVES3; CARLOS HENRIQUE CARVALHO DE SOUSA4 E ENÉAS GOMES FILHO5 1Doutora em Engenharia Agrícola, Universidade Federal do Ceará (UFC), Campus do Pici, Bloco 804, Fortaleza, Ceará, CEP: 60.455-760. leilaneves7@hotmail.com2Professor do Departamento de Engenharia Agrícola, Universidade Federal do Ceará (UFC), Campus do Pici, Bloco 804, Fortaleza, Ceará, CEP: 60.455-760. cfeitosa@ufc.br3Agrônomo, Universidade Federal do Ceará (UFC), Campus do Pici, Bloco 804, Fortaleza, Ceará, CEP: 60.455-760. mailsonpa@gmail.com4Doutor em Engenharia Agrícola, Universidade Federal do Ceará (UFC), Campus do Pici, Bloco 804, Fortaleza, Ceará, CEP: 60.455-760. sousaibiapina@yahoo.com.br5Professor do Departamento de Bioquímica e Biologia Molecular, Universidade Federal do Ceará (UFC), Campus do Pici, Bloco 907, Fortaleza, Ceará. egomesf@ufc.br 1 ABSTRACT Many studies have shown that the use of strategies for the management of saline water in irrigation reduces impacts on the soil and crops. We aimed to evaluate the effect of management strategies of irrigation with brackish water on leaf gas exchange and mineral concentrations of cowpea (Vigna unguiculata L.Walp.). The experiment was conducted in randomized blocks with thirteen treatments and five replications. The treatments consisted of: T1 (control), T2, T3 and T4 using water of 0.5 (A1), 2.2 (A2), 3.6 (A3) and 5.0 (A4) dS m-1, respectively, during the entire crop cycle; T5, T6 and T7, use of A2, A3 and A4 water, respectively, only in the flowering and fructification stage of the crop cycle; using different water in a cyclic way, six irrigations with A1 followed by six irrigations with A2 (T8), A3 (T9) and A4, (T10), respectively; T11, T12 and T13, using water A2, A3 and A4, respectively, starting at 11 days after planting (DAP) and continuing until the end of the crop cycle. Continuous application of the high salinity water (above 3.6 dS m-1) over the whole cycle of cowpea inhibits leaf gas exchange, being stomatal conductance the most sensitive variable. The alternate use of brackish water or only in the salt tolerant growth stage reduces the accumulation of potentially toxic ions (especially chloride) and maintains the similar values of leaf gas exchange and total essential nutrients ( K, Ca and Mg) extracted by plants, in relation to plants irrigated with canal water (low salinity). Index terms: Salt stress, irrigation, stomatal conductance, photosynthesis, transpiration. NEVES, A. L. R.; LACERDA, C. F.; ALVES, M. P.; SOUSA, C. H. C.; GOMES FILHO, E.TROCAS GASOSAS FOLIARES E ACUMULAÇÃO DE NUTRIENTES EM PLANTAS DE FEIJÃO-CAUPI SOB DIFERENTES ESTRATÉGIAS DE MANEJO DE IRRIGAÇÃO COM ÁGUAS SALOBRAS 2 RESUMO Muitos estudos têm mostrado que a utilização de estratégias para o manejo de águas salobras na irrigação reduz os impactos sobre o solo e as culturas. Objetivou-se avaliar o efeito de estratégias de manejo de irrigação com água salobra sobre as trocas gasosas foliares e os teores de íons em feijão-caupi (Vigna unguiculata L.Walp.). O experimento foi conduzido em blocos ao acaso com treze tratamentos e cinco repetições. Os tratamentos consistiram de: T1 (controle), T2, T3 e T4, utilizando água de 0,5 (A1), 2,2 (A2), 3,6 (A3) e 5,0 (A4) dS m-1, respectivamente, ao longo de todo o ciclo da cultura; T5, T6 e T7, utilizando águas salinas A2, A3 e A4, respectivamente, apenas na fase de floração e frutificação; uso de diferentes fontes de água de forma cíclica, com seis irrigações com A1 seguido por seis irrigações com A2 (T8), A3 (T9) e A4 (T10), respectivamente; T11, T12 e T13, usando água A2, A3 e A4, respectivamente, a partir de 11 dias após o plantio (DAP) e continuando até ao final do ciclo da cultura. A aplicação contínua de água de elevada salinidade (acima de 3,6 dS m-1) ao longo do ciclo do feijão-caupi inibe as trocas gasosas foliares, sendo a condutância estomática a variável mais sensível. O uso alternado de água salobra ou apenas na fase de maior tolerância da cultura do feijão-caupi reduz o acúmulo de íons potencialmente tóxicos (especialmente o cloreto) e mantém os valores de trocas gasosas foliares e dos totais de nutrientes essenciais (K, Ca e Mg) extraídos pela cultura similares aos das plantas irrigadas apenas com água do canal (baixa salinidade). Palavras-chave: Estresse salino, irrigação, condutância estomática, fotossíntese, transpiração.

Abstract Umbilical cord blood transplantation (UCBT) has been used to treat malignant and non-malignant diseases. UCBT offers the advantages of easy procurement, immediate availability, and acceptable partial HLA mismatches. Still, patients treated with UCBT show delayed hematopoietic and immunological recoveries, and have higher rates of infection. The problem of slower hematopoietic recovery post-UCBT has been addressed using different approaches: infusion of two CB units, ex-vivo expansion of CB, transplantation of a UCB combined with a selected CD34+ peripheral stem cell unit from an haplo-identical donor. Intrabone (IB) injection is a known emergency access route for infusion of fluids and drugs in children. Frassoni et al published a study on the IB injection of a single unit of CB into 32 adult patients (Frassoni F, et al. Lancet Oncol. 2008) and showed a neutrophil (0.5x10^9/L) and platelet (20x10^9/L) recoveries with a median of 23 and 36 days, respectively, with a median nucleated cell dose of only 2.6 x 10^7 cells/kg. These results have been confirmed in a retrospective study comparing IB infusion of CB with infusion of two units of CB in adult patients (Rocha V, et al. Transplantation. 2013). The only data of IB infusion in pediatric patients is a case report of 5 patients (Saglio F, et al. J Pediatr Hematol Oncol. 2012). In order to confirm the role of IB infusion of CB to improve hematopoietic reconstitution after UCBT in pediatric patients, we started a Phase II single arm, exploratory clinical trial (NCT01711788). Our primary hypothesis is that use of IB infusion of CB reduces stem cell trapping seen with IV infusion, maximizing the number of stem cells and providing a faster short- and robust long-term engraftment. Inclusion criteria includes age up to 21 years, diagnosis of hematopoietic disorders (malignant or not), availability of a single CB with at least 3 x 10^7 nucleated cells (NCs)/kg at freezing (use of two CB units is allowed provided a single CB unit fulfilling the above criteria is not available), and use of a myeloablative-conditioning regimen. On the day of UCBT, frozen CB unit is thawed, washed and re-suspended in 20 ml of a saline/albumin solution and aliquoted into four 5-ml syringes. Patient is sedated with propofol and ketamine according to the current protocol for sedation in children. After sedation, a first aliquot of CB stem cell suspension is injected into the bone marrow, followed by a of 0.5-1 ml saline solution rinse. This procedure is repeated for all remaining aliquots at different places and can be performed in one or both iliac crests, depending on patient size. Each injection is performed within a 2-minute timeframe. If patient requires two CB units, one unit is infused IB while the second unit is infused IV after recovery from sedation. G-CSF is given from D+7. A total of 12 patients has been included since Nov/2012. Eight were male, and median age was 7 (2 - 15) years. Diagnosis were AML (n=6), ALL (n=4) and sickle cell anemia (n=2). Most UCB were unrelated (n=10) and 6/6, 5/6 and 4/6 HLA matching comprised four, five and three UCBT. Busulfan-based conditioning was used in eight patients. Ten patients received a single unit UCB. Median NC infused was 3.4 (2.17-8.4) x 10^7/kg. There was no toxicity related to IB infusion. Engraftment occurred in 10 out of 12 patients and median time to engraftment (neutrophil ≥ 0.5x10^9/L) was 14.5 days. Platelet recovery (≥ 50x10^9/L) occurred in just 6/12 patients (due mainly to early relapse) and median time to recovery was 41 (28-77) days. Three patients presented a grade II aGVHD. There were three relapses. Ten out of twelve patients are alive, one patient died from UCBT complication (ARDS) and other from relapse. In conclusion, IB infusion of UCB is feasible in pediatric patients without any particular toxicity and there is a positive impact on neutrophil recovery. Inclusion of more patients are needed to confirm the impact of IB-UCBT on platelet recovery, duration of hospitalisation, and survival. IB-UCBT might be a more affordable alternative to improve hematopoietic recovery comparing to other methods of UCB expansion. Disclosures No relevant conflicts of interest to declare.

In the current study, methanolic and selective extracts from different parts of Gymnocarpos decander were screened for total phenolic, flavonoid, flavonol and condensed tannin contents. The antioxidant activity of extracts was also determined. The highest values of total contents of phenolics (156.097 ± 2.312 mg GAE/g DM), flavonoids (14.878 ± 0.275 mg CE/g DM), condensed tannins (39.388 ± 1.599 mg CE/g DM) and flavonols (6.506 ± 1.021 mg QE/g DM) were found in flowers. The most powerful antioxidant was found in the methanolic extract of flowers (32.27 ± 2.400 mg AAE/g DM). Tannins extracted from flowers showed an interesting antioxidant activity to trap the 1,1-Diphenyl- 2-picrylhydrazyl (DPPH) free radical (0.063 ± 0.000 mg/mL) and to reduce iron absorption (0.083 ± 0.004 mg/mL). The highest activity in the β-carotene test was found in the butanolic fraction of flowers (0.314 ± 0.008 mg/mL). 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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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Abstract Objectives The aims of this study were to (1) establish the maximum allowable interference limits for hemolysis, lipemia, and icterus for chemistry analytes tested in body fluid samples and (2) assess the effectiveness of serial dilution to mitigate spectral interferences. Methods Residual body fluids from clinically ordered testing were mixed (&lt;10% by volume) with stock solutions of interferent (spiked) and compared with a control spiked with an equal volume of 0.9% saline. The analytes were measured on the Roche cobas c501 instrument. Difference and percentage difference were calculated and compared with allowable total error limits. A subset of samples were serially diluted with 0.9% saline. Mean (SD) difference and percentage difference were calculated. Results The interference thresholds were lower than the package insert for lactate dehydrogenase, cholesterol, triglycerides, and total protein for hemolysis; amylase, cholesterol, and total protein for icterus; and albumin for lipemia. Only cholesterol and triglyceride results returned to baseline upon dilution of icteric samples. Conclusions Interference thresholds in body fluids were lower than blood for 6 analytes. Diluting interferences that surpass these limits does not produce reliable results that are comparable to the baseline results before spiking in the interferent.

Background Spuriously high results using the Abbott Architect enzymatic creatinine assay were noted to be particularly associated with very small sample volumes. This led us to query the effect of under-filling lithium heparin tubes on the measured enzymatic creatinine result. Methods Blood was provided by 5 laboratory personnel and then decanted into 5 x1.2 mL Sarstedt S-Monovette tubes, giving final blood volumes of 200, 400, 600, 800 and 1200 μL. Plasma was analysed using Abbott Architect Jaffe, enzymatic creatinine, Beckman Coulter (AU500) enzymatic creatinine and Roche (Cobas c702) enzymatic creatinine assays. Saline was also added to Sarstedt 1.2 mL and Teklab 2 mL tubes and analysed using the Abbott Jaffe and enzymatic creatinine methods. Results Increasing degrees of under-fill were associated with greater over-estimation of creatinine using the Abbott enzymatic assay, but no difference was noted using Jaffe methodology on the same platform or enzymatic assays provided by Roche or Beckman. On average, creatinine was 40.6% (+27.7 μmol/L) higher when only 200 μL of blood was present in the tube. Small volumes of saline added to lithium heparin tubes measured significant creatinine concentrations using the Abbott enzymatic method. Conclusions Lithium heparin directly interferes in the Abbott Architect enzymatic creatinine assay. Under-filling lithium heparin tubes can lead to clinically significant over-estimation of creatinine results by this assay. Users of this assay should be aware of the potential for spurious results in small sample volumes collected into lithium heparin tubes and implement robust procedures for identifying and reporting results on these samples.