Статті в журналах з теми "Magee mechanism"

ABSTRACT Candida albicans is a diploid yeast with a predominantly clonal mode of reproduction, and no complete sexual cycle is known. As a commensal organism, it inhabits a variety of niches in humans. It becomes an opportunistic pathogen in immunocompromised patients and can cause both superficial and disseminated infections. It has been demonstrated that genome rearrangement and genetic variation in isolates of C. albicans are quite common. One possible mechanism for generating genome-level variation among individuals of this primarily clonal fungus is mutation and mitotic recombination leading to loss of heterozygosity (LOH). Taking advantage of a recently published genome-wide single-nucleotide polymorphism (SNP) map (A. Forche, P. T. Magee, B. B. Magee, and G. May, Eukaryot. Cell 3:705-714, 2004), an SNP microarray was developed for 23 SNP loci residing on chromosomes 5, 6, and 7. It was used to examine 21 strains previously shown to have undergone mitotic recombination at the GAL1 locus on chromosome 1 during infection in mice. In addition, karyotypes and morphological properties of these strains were evaluated. Our results show that during in vivo passaging, LOH events occur at observable frequencies, that such mitotic recombination events occur independently in different loci across the genome, and that changes in karyotypes and alterations of phenotypic characteristics can be observed alone, in combination, or together with LOH.

e12555 Background: Recurrence score (RS) from Oncotype Dx (ODx) test predict chemotherapy benefit in estrogen receptor (ER) positive, and HER2-negative patients with breast cancer. ODx is an expensive test in an Indian practice setting - cost of adjuvant chemotherapy is one-sixth of the cost of the test. Magee equations use histopathological variables, and semiquantitative results from ER, PR and Ki-67, to calculate Magee score (MS) and provide a cost-effective surrogate mechanism for predicting RS. We aimed to assess the correlation between MS and RS in an Indian setting. Methods: Pathology reports were obtained from oncologists in India. Immunohistochemistry for ER and PR were reported using the Allred system. Therefore, two methods were identified to estimate H-scores - using a calculated method (A), and using an expert-approximated (B) method. MS were calculated by a single investigator blinded to the RS. MS and RS were then assessed for correlation via the Pearson correlation method. MS were arbitrarily stratified into ≤ 15, 15 - 30, and ≥ 30, and their overall agreement (OA) or concordance with RS risk categories, ≤ 15, 15 - 30, and ≥ 30, were assessed. The OA was assessed by determining the proportion of patients in similar risk categories across low, intermediate and high-risk RS and MS. Results: Among 34 patients included in the study, 38% (13) had an RS ≤ 15, 38% (13) had an RS between 15 and 30, and 23% (8) had an RS ≥ 30. Correlation coefficient between MS and RS was 0.78 for both methods of estimation (p<0.0001). The OA between MS-A and RS, and MS-B and RS were 59% (20/34) and 47% (16/34) respectively. The concordance between low-risk MS-A and RS is 78% (n=9) and between low-risk MS-B and RS is 50% (n=20). The concordance between high-risk MS-A and RS is 100% (n=2) and high-risk MS-B and RS is 100% (n=2). Conclusions: Although there is good correlation between RS and MS, the concordance between the two in similar risk categories is not optimal. Of the two methods to estimate H-score from Allred score, the calculated method seems to be more optimal. Our pilot study shows good correlation between MS and RS. We recommend that pathologists incorporate H-score as part of the reporting of ER and PR. A larger prospective study will be useful to test our hypothesis for the use of MS as a predictive tool for adjuvant chemotherapy in breast cancer.

Abstract Clonal hematopoiesis (CH), the age-related expansion of specific clones in the blood system has been considered as a pre-malignant condition of blood cancers. Approximately 50% of CH manifests from DNMT3A mutations acquired in the hematopoietic stem cells (HSCs), suggesting these mutations may convey a fitness advantage to HSCs during hematopoietic stress. However, a unifying molecular mechanism to illuminate how DNMT3A-mutant HSCs outcompete their counterparts is still lacking. Here, we used interferon-gamma (IFNg) as a model to study the mechanisms by which Dnmt3a mutations enhances HSC fitness under recurrent inflammatory stress. To represent the spectrum of DNMT3A variants found in humans, mouse genetic models were generated by specifically deleting Dnmt3a heterozygously (Vav-Cre;Dnmt3afl/+ = Dnmt3aHET) or homozygously (Vav-Cre;Dnmt3afl/fl = Dnmt3aKO) from the hematopoietic system. A hematopoietic system-specific knockin model analogous to the hotspot point mutation most prevalent in AML (Vav-Cre;Dnmt3aR878H/+ = Dnmt3aR878) was also included in the analysis. Competitive transplantation assays coupled with inflammatory and proliferative challenges suggested that Dnmt3aKO and Dnmt3aR878 HSCs were specifically tolerant of the deleterious effects of IFNg on HSC self-renewal and clonal expansion. When the competition between Dnmt3a-mutant and control HSCs under IFNg exposure was directly quantified in a novel mouse model, we found Dnmt3a-mutant HSCs resisted IFNg-mediated depletion due to an enhanced fitness advantage. Mechanistically, DNA hypomethylation-associated over-expression of Txnip in Dnmt3a-mutant HSCs was identified by coupling single-cell RNA-sequencing and Whole-Genome Bisulfite sequencing. The sustained Txnip levels in Dnmt3aKO HSCs lead to p53 stabilization and upregulation of p21 under IFNg challenge, further correlated with a retained quiescence due to a prolonged G0 exit in response to IFNg exposure. Implementing biochemical studies, we observed Txnip mediated an enrichment of p53 at p21 promoter under IFNg exposure in Dnmt3aKO but not control 32D cells. Knocking down Txnip by shRNA normalized p53 occupancy at p21 promoter and rescued IFNg-associated p21 upregulation in Dnmt3aKO 32D cells. Functionally, knocking down Txnip and p21 sensitized Dnmt3aKO HSCs to IFNg-induced cell cycle activation ex vivo. In vivo, down-regulation of p21 had no effect on control HSCs in response to IFNg exposure, but it completely primed Dnmt3aKO and Dnmt3aR878 HSCs to IFNg-associated exhaustion in a transplantation experiment. In summary, our data suggest an augmented Txnip-p53-p21 axis preserves the functional potential of Dnmt3a-mutant HSCs under inflammatory stress, which highlights a novel mechanism to explain the increased fitness of Dnmt3a-mutant HSCs and supports rationale for developing interventions to mitigate expansion of pre-malignant clones as a method of blood cancer prevention. Citation Format: Christine R. Zhang, Elizabeth Ostrander, Won Kyun Koh, Ostap kukhar, Hamza Celik, Jeffrey Magee, Grant Challen. Augmented Txnip-p53-p21 axis preserves Dnmt3a mutant hematopoietic stem cells during inflammatory stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB555.

Abstract ICB therapy has transformed cancer treatment; durable responses in difficult-to-treat cancers have been observed. Despite this most patients (pts) don’t respond to ICB (primary resistance), and many pts who initially respond eventually relapse (acquired resistance). Primary resistance is associated with tumor cell extrinsic factors, e.g., the immunosuppressive nature of the tumor microenvironment (TME), while acquired resistance is associated with tumor intrinsic factors, e.g., downregulation of MHC1, preventing T cell recognition. Treatments to overcome ICB resistance are necessary. WU-NK-101 is a PBMC-derived, cytokine-reprogrammed, expanded, and cryopreserved off-the-shelf memory NK cell product. WU-NK-101 cells capture the memory-like NK cell biology of cytokine-induced memory-like (CIML) NK cells, exhibiting enhanced cytotoxicity, metabolic fitness/flexibility, and resistance to TME immunosuppression (Muth et al. EHA 2022; Rutella et al. ESMO 2022). Bone marrow biopsies, collected from R/R AML pts who received CIML-NK cells (NCT01898793), were interrogated using immune gene expression (GE) profiling and spatially resolved proteomics (IO360® panel, n = 740 genes, and GeoMx® DSP; NanoString Technologies). Higher T-cell infiltration was noted post CIML-NK. CIBERSORT deconvoluted GE data inferred higher abundance of macrophages, γδT cells and activated dendritic cells on day 28 post-treatment. GE signatures showed downregulation of NFIL3 and FAM30A (log2 fold-change &lt;1.0; p&lt;0.05) post CIML-NK. TIDE algorithm modelling indicated that lower expression of NFIL3 and FAM30A correlated with high CTL infiltration and improved outcomes in many TCGA tumors (Jiang et al. Nat. Med. 2018). Hence, CIML-NK treatment led to modifications in the TME towards a more T-cell amenable environment. Deleting MHC1 on NALM6 cells significantly improved WU-NK-101 killing compared to WT (p&lt;0.0001), highlighting one mechanism whereby WU-NK-101 overcomes acquired ICB resistance. In transwell assays, co-incubation of tumor cells with WU-NK-101 in the lower chamber led to dose dependent killing; in the upper chamber (tumor cells alone), viability was preserved but a dose-dependent increase of MHC1/PDL1 expression was noted. Similar results were observed using cell-free conditioned media from WU-NK-101 cytotoxicity assays, suggesting that soluble factors released from WU-NK-101 augment MHC1/PDL1 expression. Treatment of tumor cells with IFNγ alone led to similar increases in MHC1/PDL1 expression. Importantly, in vivo experiments revealed that residual tumor cells post WU-NK-101 treatment exhibited higher levels of MHC1/PDL1. Overall, these data highlight a further mechanism through which WU-NK-101 overcomes acquired ICB resistance. In summary, WU-NK-101 has the potential to reverse primary and acquired mechanisms of ICB resistance. A Phase 1b clinical trial of WU-NK-101 as salvage therapy post-ICB is in development. Citation Format: Tom A. Leedom, Barbara Muz, Jaykumar Vadakekolathu, John J. Muth, Xiao-Hua Li, Gregory Watson, Kristaan Magee, Ryan P. Sullivan, Melissa Berrien-Elliott, Todd Fenigher, Sergio Rutella, Matthew L. Cooper, Ayman Kabakibi, Jan K. Davidson-Moncada. xWU-NK-101 as salvage therapy post immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6418.

Alcohol consumption and associated harms are an issue among emerging adults, and protective behavioral strategies (PBS) are actions with potential to minimize these harms. We conducted two studies aimed at determining whether the associations of at-risk personality traits (sensation-seeking [SS], impulsivity [IMP], hopelessness [HOP], and anxiety-sensitivity [AS]) with increased problematic alcohol use could be explained through these variables’ associations with decreased PBS use. We tested two mediation models in which the relationship between at-risk personality traits and increased problematic alcohol use outcomes (Study 1: Alcohol volume; Study 2: Heavy episodic drinking and alcohol-related harms) was partially mediated through decreased PBS use. Two samples of college students participated (N1 = 922, Mage1 = 20.11, 70.3% female; N2 = 1625, Mage2 = 18.78, 70.3% female). Results partially supported our hypotheses, providing new data on a mechanism that helps to explain the relationships between certain at-risk personality traits and problematic alcohol use, as these personalities are less likely to use PBS. In contrast, results showed that AS was positively related to alcohol-related harms and positively related to PBS, with the mediational path through PBS use being protective against problematic alcohol use. This pattern suggests that there are other factors/mediators working against the protective PBS pathway such that, overall, AS still presents risks for alcohol-related harms.

MAGnetic Expansion Control (MAGEC) rods are used in the surgical treatment of children with early onset scoliosis. The magnetically controlled lengthening mechanism enables rod distractions without the need for repeated invasive surgery. The CE certification of these devices was suspended in March 2021 due, primarily, to performance evidence gaps in the documents provided by the manufacturer to regulators and notified bodies. MAGEC rods are therefore not permitted for use in countries requiring CE marking. This was a survey of 18 MAGEC rod surgeons in the UK about their perception of the impact of the CE suspension on the clinical management of their patients. Unsurprisingly, virtually all perceived a negative impact, reflecting the complexity of this patient group. Reassuringly, these surgeons are highly experienced in alternative treatment methods. Cite this article: Bone Jt Open 2022;3(2):155–157.

Pregnant women who smoke are at greater risk of delivering a growth-restricted infant than nonsmoking mothers. We wanted to see if apoptosis could be involved in the mechanisms behind smoke-induced growth restriction, and our aim was to compare apoptosis in the placenta of smoking mothers giving birth to growth-restricted infants and nonsmoking mothers with infants of appropriate weight. The project was conducted at the Magee—Womens Hospital and Magee—Womens Research Institute, University of Pittsburgh, PA. Histological sections from 20 placentas were selected from smoking mothers who had given birth to small-for-gestational-age infants (birth weight ≤ 2 SD). The controls were gestational-age matched nonsmoking mothers with infants having appropriate-for-gestational-age weight. The TUNEL method was used to demonstrate DNA fragmentation in nuclei, and a monoclonal antibody M30, specific for a neo-epitope on cytokeratin 18, was used to identify apoptotic epithelial cells. The positive nuclei (TUNEL) and positive cells (M30-positive cytoplasm) were counted blindly both in villous tissue and in decidual/basal plate tissue. M30-positive cells in villous tissues were significantly increased in placentas from smoking mothers compared to nonsmoking mothers. When evaluated by the TUNEL method, the difference between the two groups of women was not significant. Our study shows that apoptosis was increased in the placentas of smoking mothers with growth-restricted infants. The difference between the two groups was mainly in the syncytiotrophoblast layer and in connection with perivillous fibrin deposition. Cigarette smoke with reduction in blood flow has previously been shown to increase apoptosis, and it is possible that this could be one of the mechanisms playing a role in the growth restriction.

Abstract Introduction: Glioblastoma is resistant to standard treatment, leading to tumor recurrence and early death of patients. Using organoid models, we aim to explore the mechanisms of resistance to cancer therapies and identify novel therapeutic vulnerabilities, including stress protecting melanoma-associated antigens (MAGEs). MAGEs are normally restricted to expression in testis but are abnormally activated in cancer and are associated with therapy resistance. Methods: Organoids have been established from fresh tumor biopsies and are stored in Gliobank, a Slovenian collection of patient tumor samples with corresponding clinical data. Organoids were first characterized and compared with the corresponding parental tumor tissue by immunofluorescence and qPCR of selected markers. Organoids were then treated with a combination of irradiation, the chemotherapeutic agent temozolomide and/or a chemokine receptor antagonist, and response to therapy was assessed by immunofluorescence, qPCR, cell viability and invasion assays, multiplex ELISA and proximity ligation assay. Results: Organoids recapitulated the gene expression profile of parental glioblastoma tissues, including expression of genes related to cancer stem cells, DNA damage response, cell cycle progression, epithelial- to- mesenchymal transition, and cytokine signaling. In addition, the organoids preserved the cellular composition of the parental glioblastoma tissues, consisting of glioblastoma (stem) cells, macrophages, microglia, lymphocytes, and endothelial cells. Standard treatment decreased viability and invasion of organoids in only 2 of 6 patients, suggesting that organoids recapitulate tumor therapy resistance. The increased expression of MDM2 and CDKN1A in organoids after treatment with irradiation and temozolomide suggests that the p53 pathway and DNA damage response mechanisms may contribute to the therapy resistance. Next, we detected high abundance of secreted cytokines in the culture medium of the organoids, including CXCL12. However, treatment of the organoids with the CXCR4 antagonist Plerixafor, which blocked the interactions between CXCL12 and CXCR4 in the organoids, had no effect on organoid viability and invasion, suggesting resistance of glioblastoma to this immunotherapy. Since high expression of several type I MAGEs correlates with poor prognosis of glioma patients based on TCGA database analysis, we investigate the role of MAGEs in therapy resistance of glioblastoma organoids. Conclusions: Patient-derived tumor organoids provide a valuable tool to 1) identify novel therapeutic vulnerabilities in the context of the tumor microenvironment, 2) evaluate the effect of novel candidate treatments including immunotherapy, and 3) discover novel markers of therapy resistance. Citation Format: Barbara Breznik, Bernarda Majc, Anamarija Habič, Gloria Krapež, Andrej Porčnik, Jernej Mlakar, Tamara Lah Turnšek, Metka Novak, Klementina Fon Tacer. Glioblastoma patient-derived organoids as a model for discovering novel markers of therapy resistance in the context of tumor microenvironment: potential role of melanoma antigens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 170.

Рассматривается дозвуковое турбулентное горение предварительно перемешанной метановоздушной смеси в модельном канале с обратным уступом (P. Magre и др., ONERA, 1975–1989). В экспериментах воспроизведены базовые физические механизмы, характерные для процессов горения в газотурбинных установках. Дан краткий обзор предыдущих работ по численному моделированию этих экспериментов. Описаны новые результаты численного исследования режима стабилизированного горения в данной установке. Проведено сравнение нескольких подходов к описанию турбулентного горения из класса PaSR (partially stirred reactor — модели реактора частичного перемешивания) с квазиламинарным подходом. Представлена модель переменных турбулентных чисел Прандтля и Шмидта, и рассмотрено ее влияние на воспроизведение данного течения в расчете. Subsonic turbulent combustion in a premixed methane—air mixture in a model channel with a backward step is considered (P. Magre et al., ONERA, 1975–1989). The main physical mechanisms characteristic of combustion in gas turbine combustors are reproduced in experiments. A brief overview of previous numerical simulations of these experiments is given. New results from a numerical study of the stabilized combustion regime in this combustor are described. Several partially stirred reactor (PaSR) models for describing turbulent combustion are compared with a quasi-laminar approach. A model of variable turbulent Prandtl and Schmidt numbers is presented, and its influence on the reproduction of this flow in calculations is considered.

Ein innovativer Stromsensor bietet Vorteile gegenüber konventionellen Stromwandlern. Das neusee- ländische Unternehmen Raztec Sensors, bekannt als Spezialist für Halleffekt-Stromwandler, präsentiert einen ganz und gar unkonventionellen Ansatz für die Strommessung, der ohne den üblichen magne- tischen Ringkern auskommt und damit neue Möglichkeiten in der Anwendung und Montage eröffnet.

Clostridium botulinum type A3 str. Loch Maree is a clinically important strain that produces botulinum neurotoxin type A3 and causes foodborne, infant, and wound botulism worldwide. Studying the mechanism underlying the virulence of this organism is imperative to understand its antibacterial resistance and discovering new drugs or inhibitors. The biochemical and molecular characteristics of this organism have been intensively studied, but their gene regulatory mechanisms are unclear. Hence, we reconstructed the transcriptional regulatory network from the complete genome of this organism and analyzed interactive genes from the identified hub module using a knowledge-based bottom-up approach. The biological reliability, topological properties, and robustness of the regulatory network model were validated with network parameters, followed by gene ontology terms and literature support. The reconstructed regulatory network consisted of 12 transcriptional regulators associated with 2369 coding genes. ResD, SpoOA, ComK, CcpC, DinR, DegU, CitT, CodY, GerE, GltC, GltR, IolR, and LevR were identified as transcriptional regulators from this organism homologous to Bacillus subtilis 168. These regulators have been shown to control beta-lactamase, methyl-accepting chemotaxis protein, DNA replication protein DnaD, sensor histidine kinase, and putative membrane proteins of this organism. This study also predicted all possible promoter sites in regulated genes and their associated molecular functions. We conclude that a global regulatory network model of this organism provides insights into its growth physiology and virulence elicitation in the human intestinal environment.

Endometrial receptivity is a state of the endometrium defined by its readiness for embryo implantation. When the receptivity of the endometrium is impaired due to hyperandrogenism or androgen excess, this condition can lead to pregnancy loss or infertility. Hyperandrogenism encompasses a wide range of clinical manifestations, including polycystic ovary syndrome (PCOS), idiopathic hirsutism, hirsutism and hyperandrogaenemia, non-classical congenital adrenal hyperplasia, hyperandrogenism, insulin resistance, acanthosis nigricans (HAIR-AN), ovarian or adrenal androgen-secreting neoplasms, Cushing’s syndrome, and hyperprolactinaemia. Recurrent miscarriages have been shown to be closely related to elevated testosterone levels, which alter the endometrial milieu so that it is less favourable for embryo implantation. There are mechanisms for endometrial receptivity that are affected by excess androgen. The HOXA gene, aVβ3 integrin, CDK signalling pathway, MECA-79, and MAGEA-11 were the genes and proteins affect endometrial receptivity in the presence of a hyperandrogenic state. In this review, we would like to explore the other manifestations of androgen excess focusing on causes other than PCOS and learn possible mechanisms of endometrial receptivity behind androgen excess leading to pregnancy loss or infertility.

CAPTCHAs are strategies to recognize human clients and PC programs naturally. CAPTCHAs shield different sorts of online administrations from beast compel assaults and foreswearing of administration via programmed PC programs. Most CAPTCHAs comprise of mages with misshaped content. Shockingly, visual CAPTCHAs constrain access to the a huge number of outwardly hindered individuals utilizing the Web. Sound CAPTCHAs were made to fathom this openness issue. However the presently accessible sound CAPTCHAs have been broken with differing achievement, utilizing the shortcoming in the techniques utilized. Our system, presents the user with an interface that plays a song using instrumental music (nonvocal) randomly selected from some language of users choice. The user is then asked to kind the music composer and then the device estimates whether it is a human or no longer by means of analyzing the response. A person look at turned into conducted to research the overall performance of our proposed mechanism.

To manage autonomic disorders in adolescents with tuberculosis, 100 people from 12 to 15 years old were examined and randomly divided into two groups: Group 1 received standard anti-tuberculosis therapy, and in Group 2, additionally to standard therapy, comprehensive treatment with Magne B6 was used for 2 months, and then Valerian extract was used for another 2 months. For comparison, 50 healthy adolescents were examined. Before treatment, all adolescents with tuberculosis experienced a decrease in heart rate variability, a pronounced autonomic imbalance with the prevalence of sympathicotonia and overexertion of compensatory mechanisms. In Group 1, during the comprehensive treatment there were an increase in SDNN and Mo and decrease in AMo and tension index at rest. Active orthostatic test (AOT) was accompanied by a significant increase in SDNN, Mo and a decrease in AMo, which indicated an increase in the activity of the parasympathetic autonomic nervous system and decreased activity of the sympathetic autonomic nervous system. In the group receiving standard therapy, hypersympathycotonia persisted and manifested through significant (p < 0.05) reduction in such parameters as SDNN, increased heart rate, AMo, and Mo shift to short intervals. Medication management resulted in the vegetative balance by increasing heart rate variability, reducing hypersympathicotonia, improving the autonomic support of the cardiovascular system at rest and during AOT.

The three-dimensional structure of CD4M33, a mimic of the host-cell receptor-antigen CD4 and a powerful inhibitor of CD4–gp120 (viral envelope glycoprotein 120) interaction and HIV-1 entry into cells [Martin, Stricher, Misse, Sironi, Pugniere, Barthe, Prado-Gotor, Freulon, Magne, Roumestand et al. (2003) Nat. Biotechnol. 21, 71–76], was solved by 1H-NMR and its structure was modelled in its complex with gp120. In this complex, CD4M33 binds in a CD4-like mode and inserts its unnatural and prominent Bip23 (biphenylalanine-23) side-chain into the gp120 interior ‘Phe43 cavity’, thus filling its volume. CD4M33 was specifically labelled with fluorescein and shown by fluorescence anisotropy to bind to different gp120 glycoproteins with dissociation constants in the nanomolar range. Fluorescent CD4M33 was also used in a miniaturized 384-well-plate assay to study direct binding to a large panel of gp120 glycoproteins and in a competition assay to study binding of CD4 or other ligands targeting the CD4 binding site of gp120. Furthermore, by using the fluorescently labelled CD4M33 and the [Phe23]M33 mutant, which possesses a natural Phe23 residue and thus cannot penetrate the gp120 Phe43 cavity, we show that a recently discovered small-molecule-entry inhibitor, BMS-378806, does not target the CD4 binding site nor the Phe43 cavity of gp120. The fluorescently labelled CD4M33 mimic, its mutants and their derivatives represent useful tools with which to discover new molecules which target the CD4 binding site and/or the Phe43 cavity of gp120 glycoproteins in a high-throughput fluorescence-polarization assay and to characterize their mechanism of action.

The 19th International Conference on Chemical Thermodynamics (ICCT-19) took place as part of THERMO International 2006, together with the 16th Symposium on Thermophysical Properties and the 61st Calorimetry Conference, from 30 July to 4 August 2006 at the University of Colorado, Boulder, CO, USA. Dr. W. M. Haynes was President of the Executive Board of THERMO International 2006, and Drs. M. Frenkel, R. D. Chirico, and J. W. Magee were the organizers of ICCT. Overall, 768 speakers submitted the abstracts of their presentations, including about 30 students and 11 exhibitors, from 62 countries (235 from North America, 341 from Europe, 76 from Japan, and 33 from China). About 65 % of the participants were from academia and 15 % from industry, with 20 % from governmental and international organizations.These individual conferences have an overlap of areas of interest, but this was the first time that they have been held jointly at the same site. This provided a unique opportunity for researchers and practitioners worldwide to meet and discuss a broad range of scientific problems in the fields of thermodynamics and thermophysical properties for a wide variety of systems, with applications in chemistry and other scientific and engineering disciplines.After the official opening ceremony, there was an invited keynote presentation by Prof. W. A. Wakeham from the University of Southampton, Southampton, UK, entitled "Thermophysical property measurements: The journey from accuracy to fitness for purpose". The Rossini Award lecture was given by Prof. A. Navrotsky on "Calorimetry of nanoparticles, surfaces, interfaces, thin films, and multilayers".The ICCT program consisted of nine symposia, some of which were held jointly with the other conferences. The plenary lecturers and invited speakers in these symposia, and the titles of the plenary lectures, were as follows:Electrolyte and Non-Electrolyte Solution Thermodynamics: J. M. Prausnitz (plenary), "Some promising frontiers in the thermodynamics of protein solutions"; C. G. Panayiotou, P. R. Tremaine, and T. Kimura (invited)Ionic Liquids: K. Seddon (plenary); "The mark of an educated mind"; L. P. N. Rebelo and C. J. Peters (invited)Molecular Modelling, Including Simulation: D. Evans (plenary), "The fluctuation and non-equilibrium free energy theorems: Theory and experiment"; H. Tanaka, J. Errington, and A. Klamt (invited)Thermochemistry and Molecular Energetics: J. A. de Sousa Martinho Simões (plenary), "Energetics of free radicals: Bridges between gas-phase and solution data"; W. E. Acree, Jr. and J. S. Chickos (invited)Thermodynamics and Properties in the Biological, Medical, Pharmaceutical, Agricultural, and Food Sectors: P. L. Privalov (plenary), "Thermodynamic problems in structural molecular biology"; J. M. Sanchez-Ruiz and H. H. Klump (invited)Databases, Data Systems, Software Applications, and Correlations: M. Satyro (plenary), "Life, data and everything"; R. L. Rowley and R. Sass (invited)Phase Equilibrium, Supercritical Fluids, and Separation Technologies: S. Sandler (plenary), "Computational quantum mechanics: An under-utilized tool for applied thermodynamics"; L. F. Vega and R. P. Danner (invited)Colloid and Interface Science: L. Piculell (plenary), "Controlling structure in associating polymer-surfactant mixtures"; H. K. Yan and K. Lohner (invited)New Materials: V. K. Pecharsky (plenary), "Structure, mechanism, and thermodynamics of novel rare-earth-based inter-metallic materials"; C. Staudt-Bickel and J. Pons (invited)The plenary lectures, with the exception of the lecture by Prof. K. Seddon, are published in this issue.There were workshops on New Experimental Techniques, with Profs. C. Schick and J. P. M. Trusler as invited speakers, on Properties and Processes for a Hydrogen-Based Economy, where Prof. C. J. Peters was the invited speaker, and on Thermodynamic Frontiers and Education, with Profs. R. N. Lichtenthaler and R. Battino as invited speakers.In addition, there was a workshop on the Thermodynamic Properties of Hydration (with Prof. V. Majer as invited speaker), software demonstrations, and two afternoon poster sessions, with over 400 posters. The sessions were held in the well-appointed Stadium Club, against the beautiful backdrop of the Flatirons to the west and the plains stretching across to the east. IUPAC had donated three poster prizes, a framed certificate signed by IUPAC President Brian Henry, a copy of the IUPAC "Gold Book" and a two-year subscription to Chemistry International. These were awarded to Martinez-Herrera Melchor (Mexico), Lisa Ott (USA), and Isabel Marrucho (Spain).Doctorate awards were presented by the International Association of Chemical Thermodynamics (IACT), with sponsorship from Elsevier. The four recipients were M. Fulem (Prague, Czech Republic), Y. U. Paulechka (Minsk, Belarus), E. Asabina (Nizhni Novgorod, Russian Federation), and J. Xu (Trondheim, Norway). They each received a certificate, plus a cash prize of $500, and presented their papers at the conference.All the lectures demonstrated how chemical thermodynamics is making, and will continue to make, very significant contributions to the rapidly developing interdisciplinary fields such as the life sciences, new materials, medicine and pharmacy, new energy resources, the environment, separation technologies, agriculture, green chemistry, and so on. These are all extremely important issues for scientists worldwide, and particularly for those who are in developing or economically disadvantaged countries. The opportunity for face-to-face discussion and communication with scientists from developed countries was a great benefit, which will lead to further research and improved education.The weather was most pleasant for the conference. This, together with the attractive setting of the campus, the welcoming reception, the conference banquet at the National Center for Atmospheric Research, and the high standard of the presentations, made this a memorable conference. In addition, there was a full program of tours for accompanying persons, which included a visit to the mile-high city (Denver). Our thanks are extended to the Conference Chair and Co-chairs, and to all members of the local Organizing Committee, the members of the International Advisory Committee, and the members of the International Scientific Committee. We are most grateful to IUPAC, the International Association of Chemical Thermodynamics, the National Institute of Standards and Technology, the American Society of Mechanical Engineers, and the American Institute of Chemical Engineers, Elsevier, Honeywell, and Mettler Toledo for sponsoring THERMO International 2006.Thermodynamics will continue to be an important area of research for many years to come, with a wide range of applications from chemical engineering to the biosciences. We look forward to the presentation and discussion of the results of further advances in chemical thermodynamics at the next ICCT, which will take place in Warsaw, Poland in August 2008.John H. DymondConference Editor

AbstractThe addition of vegetable to carbohydrate-based meals was shown to contribute to glycaemic management. The aim of this study was to investigate the impact of homogenisation on vegetables added to rice meals in terms of acute glycaemic responses (GR). In a randomised crossover trial, sixteen healthy volunteers completed thirteen test sessions, which included two sessions for glucose control, two for rice and nine for different vegetable-rice mixed meals: cooked pak choi and cooked rice (CP+R); cooked cauliflower and cooked rice (CC+R); cooked eggplant and cooked rice (CE+R); and their homogenised counterparts, both raw or cooked. Postprandial GR tests, in vitro carbohydrate digestion and chemical analyses were carried out for each test meal. Compared with pure rice, CE+R, CP+R and CC+R meals achieved significantly lower glycaemic indexes (GI) of 67, 71 and 73, whereas their homogenised counterparts failed to show significant difference with rice. The hydrolysis indexes (HI) of CE+R, CP+R and CC+R were 69·6, 83·8 and 80·6 % of the HI of the rice control. CE had the greatest effect on lowering the GI, the incremental area under the blood glucose curve from 0 to 120 min, the peak glucose value, the maximum amplitude of glucose excursion in 0–120 min (MAGE0–120), the HI and rapid available starch. Both in vitro and in vivo tests demonstrated that incorporating non-homogenised cooked vegetables into a rice meal could slow the carbohydrate digestion and improve postprandial GR. Texture properties of vegetable may play an important role in underlying glycaemic control mechanisms.

2582 Background: The efficacy of immunotherapy approaches such as vaccines in melanoma appears limited, in part, due to immune suppressive mechanisms in the tumor microenvironment. One of these mechanisms is the presence of CD4+CD25+FoxP3+ regulatory T cells (Tregs). It has been hypothesized that strategies to reduce Treg numbers should improve the efficacy of melanoma vaccines. Methods: The study design was to randomize 24 HLA-A2+ patients to receive vaccine alone or denileukin diftitox (18 mcg/kg i.v.) as a single dose 4 days prior to the first vaccination. The vaccine formulation consisted of 4 melanoma antigen peptides (derived from MelanA, gp100, MAGE3, and NA17A) emulsified in Montanide and GM-CSF, administered i.d./s.c. every 2 weeks. The primary endpoints were assessment of depletion of Tregs from the peripheral blood, and measurement of antigen-specific CD8+ T cell responses by ELISPOT. Secondary endpoints included clinical response and analyses of the tumor microenvironment for changes in Treg infiltration. Results: The treatment was well tolerated. Enrollment was halted at 16 patients when it was clear that immune response endpoints would not be reached. There was no significant decrease in Treg numbers, nor was there an increase in vaccine-induced T cell responses, when patients received denileukin diftitox prior to vaccination. In patients with biopsiable tumors, there was no decrease in Tregs in metastatic lesions post-versus-pre-treatment. Of the 9 patients who showed clinical benefit (1 PR, 8 SD), 4 of them received denileukin diftitox and 5 did not. Estimated time to progression was 146 days in the group that received denileukin diftitox and 131 days in the group that did not. Conclusions: Denileukin diftitox given as a single dose prior to vaccination was not sufficient to deplete Tregs or improve the potency of this melanoma vaccine. Alternative strategies to reduce Tregs, such as multiple doses of denileukin diftitox or anti-CD25 mAbs, should be considered.

Abstract Background Novel immunotherapies based on targeting of specific immune checkpoints enabled a significant improvement of melanoma outcome, but melanoma brain metastases (BM) remain an unmet oncological need with an overall 2-year survival rate lower than 10%. Tumour immune microenvironment has been demonstrated to play a key role in BM establishment and development, but data regarding the specific milieu of melanoma BM is limited. Material and Methods Gene expression profiles of 55 samples of primary melanoma and BM were evaluated using the nCounter PanCancer IO 360 Panel (NanoString Technologies) targeting 770 mRNA involved in tumor immune microenvironment modulation. The case series consisted of 10 primary melanomas and their 10 matched BM, 25 unmatched BM, and 10 locally advanced control melanomas without evidence of BM after &gt;5 year follow up. Results Among BM samples, most patients (25/45) were males and median age at BM diagnosis was 61,2 years with a median time to BM development of 2,1 years. Median OS from BM diagnosis was 1,3 years. Several genes resulted significantly downregulated in BM compared to primary melanomas, including SERPINB5 (p&lt;0.001), ARG1 (p=0.0067), S100A8 (p&lt;0.001), S100A9 (p&lt;0.001), S100A12 (p=0.0037), IL1RN (p=0.0012), CCL21 (p=0.0012), CCL22 (p=0.0012) and CCL13 (p=0.037) and SELE (p=0.026); conversely, C7 was upregulated (p&lt;0.001). Downregulated signatures in BM involved those associated with multiple immune cell populations, including neutrophils, dendritic cells, mast cells and Treg, as well as inflammatory chemokines, the CTLA4 immune checkpoint and ARG1 enzyme function; conversely, MAGEs-related signature was upregulated. Comparison between primary melanomas which developed BM and those which did not showed a significant overexpression of RRM2 (p=0.0247) and TNFRSF1A (p=0.032) genes in the latter group and an upregulation of the PD-1 pathway. Analysis according to tumour mutational status showed an upregulation of signatures associated with inflammatory chemokines, dendritic and myeloid cells and neutrophils. No differences were observed according to time to BM development and survival from BM diagnosis. Conclusion Our findings show that melanoma BM harbor distinct immunosuppressive mechanisms compared to primary tumors: this data elicits the importance of investigating the heterogeneity of BM microenvironment. Genes and pathways selectively overexpressed or downregulated in melanoma BM should be validated to be possibly considered as novel therapeutic targets.

According to long-term surveys of apple plantations in Ukraine, they are da maged by an unpaired western bark beetle (Xyleborus dispar F.). The share of this pest colonizing perennial plantations in Ukraine is about 30%, and there is also a tendency of spreading colonization of this type of production tracts over fruit plantations. The prevalence of bark beetle in production areas of orchards has increased significantly in recent years. Unpaired pest, unlike many other species of bark beetles, completely destroys healthy trees. The increase in the number and harmfulness of odd western bark beetle is associated with climate change, the use of various protection systems, etc. Nowadays measures to reduce the number of this species consisted only of a mechanical technique (cutting and burning dam-aged trees) and spraying of garden plantings in the phase of “swelling of the buds ‒ beginning of budding” with old insecticides of second generation (metaphos, chlorophos, carbophos, etc). Pruning and burning damaged trees are an ineffective measures to reduce the number of pests in orchards. The expediency of testing a number of modern insecticides is urgent and effective against a complex of phytophages in the “green cone” phase ‒ “ Rose bud” (beetle, goose, budworm, apple blossom beetle, aphids, leafworms) ‒ “end of flowering” (leafworms, aphids) and can be effective against the western unpaired bark beetle. The basis was the du-ration of action and the peculiarities of the mechanism of action of these insecticides (contact intestinal action ‒ Aktara 240 SC, HP, Mospilan, P. P., Calypso 480 SC , etc. and contact, par-tially systemic action with fumigation effect ‒ Bi‑58 new, etc., Pirinex Super, etc., Danadim stable, etc., Danadim Mix, etc., Fufanon 570, etc.). The use of the drugs mentioned above (for double treatment of plantings) demonstrated high technical efficiency against openly living phytophages (for the first treatment of plantations ‒ against aphids, leafworms, garden wee-vils and tube worms, for the second treatment ‒ against aphids, leafworms), which amounted to 91,3‒99,2%. The western unpaired bark beetle (Xyleborus dispar F.) is a permanent species in the industrial areas of the apple tree. And therefore, protecting trees from it in industrial plantings should be an integral part of modern technology for obtaining fruit products. Un-doubtedly, the application of the complex of insecticides Bi‑58 new, к.е. (2,0 l/ha), Pirinex Super, к.е. (1,25 l/ha), Danadim stable, etc. (2,0 l/ha), Danadim Mix, etc. (2,0 l/ha), Fufanon 570, E.C. (2,0 l/ha) is a highly effective method for reducing the harmfulness of the western unpaired bark beetle in industrial plantations of apple trees, an economically beneficial meth-od in the modern intensive protection technologies.

Abstract Introduction: Breast cancer metastases (BCM), which cause most breast cancer (BC)-associated mortality, have increased genetic complexity compared to early-stage disease. However, the contribution of genetic alterations to site of BCM is not well-understood. Different breast cancer subtypes have varying patterns of BCM, e.g., lobular carcinoma more frequently spreads to gynecologic (Gyn) organs and the GI tract, perhaps hinting at selection pressures wherein some organs are hospitable to tumors with certain genetic alterations. Methods: Relationships between BCM site and mutations detected by DNA next-generation sequencing (NGS; NextSeq 592 gene panels or NovaSeq whole exome sequencing) were investigated using 12,464 BC samples sequenced at Caris Life Sciences (sample sizes, Table 1). PD-L1 expression was tested through IHC (Clone SP-142 (cut-off ≥1, 1%)). Tumor mutational burden (TMB) was measured by totaling somatic mutations per tumor (high ≥ 10 mutations per MB). Immune cell fractions were calculated by deconvolution of whole-transcriptome data (NovaSeq) using Quantiseq (reference). Statistical significance was determined using chi-square and Wilcoxon rank sum tests adjusted for multiple comparisons. Results: Compared to primary breast tumors, BCM had increased frequency of TMB-H (10.08% vs. 4.94%), decreased PD-L1 positivity (21.09% vs. 35.82%), and were enriched for PIK3CA (34.62% vs 30.53%) and ESR1 mutations (13.34% vs 2.17%) (all P&lt;0.001). PD-L1 positivity was highest in BCM to lymph nodes (43.06%) and axilla (39.77%). BCM to Gyn organs had more lobular histology, the highest rate of hormone receptor (HR)+ tumors (77.17%), and rarely had high TMB (6.73%) or were PD-L1 positive (11.39%). Double dendrogram hierarchical clustering of BCM site by mutation frequency and pathway alterations revealed BCM to Gyn organs as a simplicifolious clade with a unique mutational pattern. Compared to BC in breast, BCM to Gyn organs had higher rates of mutations of PIK3CA, AKT1, and BRAF; more mutations in DNA repair (0.79% vs 0.06%), transcription factor (4.72% vs 0.93%), and Wnt signaling pathways (2.36% vs 1.47%); but no increase in BRCA mutations. BCM to brain had the most p53 pathway and homologous recombination (HR) pathway mutations (64.71% and 14.01%), while Gyn had the least (19.69% and 7.09%). Quantiseq RNA deconvolution revealed differences in tumor immune cell infiltrate by BCM site. Gyn metastases vs breast tumors had increased B cells (6.20% vs 5.40%), M2 macrophages (5.71% vs 4.07%), and NK cells (3.82% vs 3.18%) (all P&lt;0.01) and a M2/M1 macrophage ratio of 22.8:1 vs 1.3:1. Conclusions: BCM to Gyn organs have a unique mutational and immune suppression profile. Integrating the profiling with clinical outcomes may extend this prognostic signature and set the stage for improved treatment strategies for these patients. Confirmation from matched or sequential specimens could clarify tumor evolution. Our data support repeat biopsy of Gyn site metastases since more targetable mutations might be revealed. Targeting mechanisms of immunosuppression in Gyn BCM could expand therapeutic options. Table 1.Breast Cancer TumorsTumor SiteTotalPredominant Breast Cancer SubtypeAll12464HR+/HER2- (51.6%)Breast5014HR+/HER2- (46.5%)Liver2003HR+/HER2- (63.3%)Bone1132HR+/HER2- (69.4%)Axilla1051HR+/HER2- (47.7%)Lung823HR+/HER2- (46.1%)Lymph Node647HR+/HER2- (43.4%)Chest/Chest Wall375HR+/HER2- (44.8%)Brain359TNBC (38.2%)Other315HR+/HER2- (57.1%)Skin282HR+/HER2- (50.7%)Connective Tissue193HR+/HER2- (51.8%)GI Organs143HR+/HER2- (69.9%)Gynecologic Organs127HR+/HER2- (76.4%) Citation Format: Anna D Louie, Rani Chudasama, Sharon Wu, Marzia Capelletti, Daniel Magee, W. Michael Korn, Virginia Kaklamani, Antoinette R Tan, Pavani Chalasani, Wafik S El-Deiry, Don Dizon, Stephanie L. Graff. Mutational landscape and immune infiltration of breast cancer metastases to gynecologic and other organs [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD6-04.

Abstract Introduction: Melanoma-associated antigens (MAGEs) are linked to aggressive disease progression and treatment resistance in many cancers, but their role in pancreatic cancer is not well understood. Our preliminary TCGA data analysis suggested that 10% of pancreatic tumors express MAGEA3/6 and potentially help pancreatic cancer cells survive and grow under nutrient stress. This study aims to uncover the molecular mechanisms of MAGEA3/6 function in pancreatic cancer cell metabolism and disease progression under nutrient-poor conditions. Methods: To understand the role of highly homologous genes MAGEA3 and MAGEA6 in pancreatic cancer, we expressed them in highly glycolytic MIA PaCa-2 pancreatic cancer cells. Cells were then exposed to cancer-related nutritional stressors, including glycolysis inhibition by 2-deoxy-D-glucose (2DG) treatment and glutamine depletion. We analyzed the effect of MAGEA6 expression on cell viability and cell metabolism by Alamar Blue viability assay (BioRad), Seahorse (Agilent), Phenotype MicroArray Mammalian analysis (Biolog), transmission electron microscopy (TEM), and RNA sequencing. To confirm our results, we performed loss-of- function studies in MAGEA3/6-positive cells, including PANC1. Results: We found that, compared to control GFP-expressing cells, MAGEA6-expressing MIA PaCa-2 cells were resistant to glycolysis inhibition with 2-DG. In contrast, they were more susceptible to glutamine depletion, suggesting the important role of MAGEA3/6 in metabolic phenotype and adaptations of pancreatic cancer cells. Further, we showed that MAGEA6-expressing cells exhibited increased ATP production and mitochondrial oxidative phosphorylation, especially under 2DG stress. After 48 hours of 2DG treatment, TEM images revealed significant differences in nuclear, mitochondrial, and ribosomal structures in MAGEA6-cells, further suggesting MAGEA6's role in metabolic adaptation. The Phenotype MicroArray assay demonstrated that α-D-Glucose is the primary nutrient source consumed by both GFP and MAGEA6-expressing cells among the 93 nutrients tested. Additionally, A6 cells utilize Pyruvic Acid as an alternative nutrient source more than GFP cells. To uncover molecular underpinning, we performed RNA sequencing and found that MAGEA6 expression affects transcriptome under glutamine depletion and 2DG treatment. Intriguingly, one of the major differentially regulated biological processes was associated with the upregulation of several signaling pathways, including MAPK and AMPK. Conclusion: Our findings suggest that in pancreatic cancer, MAGEA6 recruits stress and growth factor- dependent MAPK and AMPK pathways to overcome glycolysis inhibition, even in glutamine- depleted conditions, providing a growth advantage. This implies that patients with MAGEA6- positive tumors may benefit from combination therapy targeting metabolism and signaling pathways. Citation Format: Sima Tozandehjani, Barbara Breznik, Klementina Fon Tacer, Miloš Vittori, Juan Sebastian Solano, Sara Uhan. MAGEA6 Oncogene Upregulates MAPK and AMPK Signaling Pathways in Pancreatic Cancer Cells to Promote Survival under Nutrient Stress: Resistance to Glycolysis Inhibition and Increased Susceptibility to Glutamine Depletion [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C035.

Many objects have been observed that are excited by shocks, but nevertheless do not form a shell shape in a variety of astrophysical environments such as in protostellar and circumstellar medium or as seen in optical jets, H-H objects, and GGD objects. The models for these objects, which have been proposed for explaining observed spectrum, can be divided to two types. First one is the cloudlets interaction model (e.g., Schwartz 1978; Norman and Silk 1979; Raga, Böhm, and Solf 1986). Second one is the radiative jet model in which the knotty structure associated in some H-H objects can be suggested as the shock of Mach disks in “underexpanded” jets (e.g., Mundt 1985; Hartigan 1989). However, the wiggles and the irregular knots have been observed in some H-H objects (e.g., knots; HH33, HH40, and wiggles; HH12, HH7-11), which could not be explained well by the two models above. On the other hand, from the observations for maser emissions in star forming regions, the strong magnetic field is inferred(e.g., Fiebig and Gusten 1989). The magnetic field seems to be important for the dynamical motion in star forming regions. Then, a magnetohydrodynamical reconnection model for these phenomena is investigated. We consider the interaction between dense magnetized cloudlet and wind from the central star, through an 2-D MHD code with the MacCormack-Doner cell hybrid scheme to well represent shock phenomena as well as advective phenomena. The density contours and magnetic field lines of numerical results for γ=5/3 and 1.1 are shown in figure (a) and (b), respectively. We present two case for γ=5/3 and 1.1. Results show that the compression effects related to the adiabatic indexes γ are important in the shocked region. The interaction between the cloudlet and wind induces the bow shock around the cloudlet and elongated magnetic structure in the downstream direction, which is similar between two cases. However, the downstream flow patterns behind the cloudlet are different for each other. For γ=1.1, we have a jet-like structure in downstream region. This is induced by magnetic reconnection with strong compressed effects which produces secondary plasma acceleration. This magnetic reconnection is fast (Patscheck) type, which have been well studied for the model of the earth magne-tospheric substorm, related to the slow shock formation and the strong plasma jet generation (e.g. Sato and Hayashi 1979). Furthermore, these behavior is similar to the disconnection events of comets (Alfvén 1957, Niedner 1980), which suggests us the knots in HH objects can be explained as some separated reconnection points in which the magnetic energy is released explosively. For real interstellar medium, γ=5/3, however, we must consider the effect of radiative cooling (Spitzer 1978). This cooling seems to be important for outflows in the star forming regions (Hanami and Sakashita, 1987) Then, we can interpret the compression effect with small γ as that of the cooling, approximately. This compression mechanism related to the cooling effect in interstellar and magnetic reconnections are important for interpreting the formation, and the structure, and the dynamics of optical jets, H-H objects, and GGD objects.

The BCL-2 protein family plays a key role in leukemogenesis via counteracting proapoptotic signals, thereby enhancing leukemia cell survival. Venetoclax, a selective BCL-2 inhibitor, has been proven effective against AML in combination with hypomethylating agents. Since MCL1, another anti-apoptotic protein, is reportedly more abundant than BCL-2 in AML cells and associated with resistance to chemotherapy, development of clinical-grade MCL1 inhibitors has been highly regarded. In fact, a series of MCL1 inhibitors are currently being tested in clinical trials. While relationships between metabolic condition in mitochondria and sensitivity to Venetoclax have been proposed in recent studies, molecular mechanisms underlying MCL1 inhibitor resistance are not well understood. To identify genes/pathways whose loss induce MCL1 inhibitor resistance in AML cells, we performed genome-wide CRISPR-Cas9 screens using a mouse AML cell line in the presence or absence of s63845, a MCL1 inhibitor (Kotschy et al. Nature 2016). To establish AML cell lines with a relatively clean genetic background, we first established AML in mice by transducing the MLL/AF9 leukemia oncogene into mouse bone marrow stem/progenitor cells ex vivo, followed by serial transplants (Yamauchi et al. Cancer Cell 2018). We then harvested AML cells from leukemic mice and established AML cell lines with normal karyotype and intact Trp53 activity. We then performed genome-wide CRISPR-Cas9 screens in the presence or absence of s63845, followed by a second screen with a small-scale library targeting genes that were negatively- or positively-selected upon s63845 treatment in the primary screen. Genes relevant for S63845 resistance were identified using MAGeK MLE (Li et al. Genome Biology 2015) and DrugZ (Colic et al. BioRxiv 2019) programs. Single-guide RNAs (sgRNAs) targeting intrinsic pro-apoptotic genes, such as Bak1, Casp9 and Apaf1, were enriched exclusively in the presence of s63845, attesting to the validity of our experiment. To identify genes relevant to s63845 resistance, we focused on the genes whose sgRNAs were enriched exclusively upon s63845 treatment. We found that loss of Me2, which encodes a mitochondrial malic enzyme that catalyzes the oxidative decarboxylation of malate to pyruvate, promotes AML cell survival only in the presence of s63845, but not in vehicle- or Venetoclax-treated cells. This finding was validated in the second screen, in which 8 independent sgRNAs targeting Me2 were tested. We next generated two independent Me2-null mouse AML cell lines (MLL/AF9 and CALM/AF10) using sgRNAs targeting Me2. Me2-null cells exhibited survival advantage over control cells upon s63845 treatment, revealed by cell proliferation assay. To determine at which step of the apoptotic pathway Me2 deficiency exerts s63845 resistance, we assessed MOMP (mitochondrial outer membrane permeabilization) levels by FACS with or without s63845 treatment. Me2-null cells released less cytochrome C than Me2-wild type (WT) cells upon s63845 treatment. As expected, Me2-null cells exhibited less Annexin-positivity than WT cells upon MCL1 inhibition. Importantly, both mRNA and protein levels of BCL-2 family members, including Bcl-2, Mcl1, Bcl2L1, Bax and Bak1, were comparable regardless of Me2 status. We next performed CRISPR-Cas9 saturation mutagenesis scan of all ME2 exons in the presence or absence of s63845 using Molm-13, a human AML cell line. sgRNAs targeting ME2 coding exons were enriched only in the presence of s63845, while those targeting ME2 3'UTR were unchanged/neutral regardless of experimental conditions. In conclusion, using an unbiased, genome-wide CRISPR/Cas9 screens, we identified Me2, a mitochondrial metabolic enzyme, as a factor relevant for MCL1 inhibitor resistance. Our study may facilitate the understanding of molecular mechanisms underlying acquired resistance to MCL1 inhibitors in AML. Disclosures Akashi: Celgene, Kyowa Kirin, Astellas, Shionogi, Asahi Kasei, Chugai, Bristol-Myers Squibb: Research Funding; Sumitomo Dainippon, Kyowa Kirin: Consultancy.

Abstract Introduction: PrBC is an uncommon malignancy with aggressive behavior. Its pathogenesis involves distinct immune mechanisms associated with maternal-fetal tolerance and tumor-host immunoediting. PrBC displays specific patterns of TILs with increased CD8+ cells. Gaining a comprehensive understanding of the molecular processes underlying this immune synergy is crucial for enhancing PrBC patients’ clinical management. Here, we sought to identify dysregulated immune-related genes in PrBC and explore their association with HR status and TILs. Methods: A total of n=75 PrBC (age range 26-43 years) and n=67 age-matched early-onset breast cancer (EOBC) in non-pregnant women (controls; age range 28-43 years) were selected from our Institutional registry. For all cases TILs were quantified according to the International TILs Working Group recommendations and profiled by IHC for CD4 and CD8. RNA was extracted from representative FFPE tissue blocks to perform the expression analysis of 395 genes involved in tumor-immune interactions using a targeted NGS panel (Oncomine™ Immune Response Research Assay, Thermofisher). Samples with &gt;1,000,000 mapped reads and &gt;800,000 valid reads were considered adequate. R package DESeq2 software 1.38.3 was used for sequencing depth differences normalization and differential gene expression analysis. Differentially expressed genes (DEGs) were identified based on a significant p-value (p&lt; 0.05). Results: The comparison between PrBC and EOBC revealed a total of n=7 DEGs. All of these genes were upregulated and belonged to distinct superfamilies, including Cancer/Testis (CT) Antigen (MAGEA1/3, XAGE1B), Interferons/Cytokines (IFNA17, IFNB1), Chemokines (CXCL13), and Immunoglobulin (PECAM1/CD31). Notably, the upregulation of Chemokines in respect to EOBC was observed exclusively in HR+ PrBC, whereas triple-negative (TN) PrBC did not exhibit this pattern compared to the control group. Hence, the upregulation of ALOX15B, an enzyme involved in fatty acid peroxidation, was specific to TN PrBC. The immune signatures showed significant variations between PrBC and EOBC also based on TILs density and subpopulations. Indeed, the upregulation of CT genes (MAGEA1, XAGE1B) was exclusively observed in PrBC cases characterized by low TILs levels and prevalence of CD8+ or CD4+ cells. Finally, CD4+ TILs were absent or low in PrBC with upregulated Interferons, Cytokines, Chemokines, and Immunoglobulin genes, but present in cases with increased expression of CT and KLRF1 (NK cells). Conclusion: These findings highlight the heterogeneity and distinct molecular characteristics of PrBC and EOBC. The upregulation of specific immune-related gene families, such as CT and Chemokines, in different PrBC subtypes may suggest their potential role as actionable biomarkers (e.g. MAGEA, a well-known oncogene and potential immunotherapy target). The differences in immune signatures and TILs subpopulations further emphasize the importance of the immune microenvironment in PrBC biology and behavior. Future studies could delve deeper into the clinical implications of these gene expression patterns and explore their relevance in personalized treatment strategies for PrBC. Citation Format: Konstantinos Venetis, Elham Sajjadi, Chiara Frascarelli, Mariia Ivanova, Marianna D'Ercole, Concetta Blundo, Massimo Giroda, Eugenia Di Loreto, Giovanna Scarfone, Stefano Ferrero, Paolo Veronesi, Viviana Galimberti, Fedro Alessandro Peccatori, Nicola Fusco, Elena Guerini-Rocco. Decoding the immune landscape of breast cancer (BC) during pregnancy (PrBC): Impact of hormone receptors (HR) and tumor-infiltrating lymphocytes (TILs) phenotype on gene expression signatures [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-16-04.

Abstract Introduction: Rationale for anticancer vaccine therapy is based on humoral and/or cellular response against unique tumor antigens (Ag). Peptide vaccines specific for Ag are under investigation for patients with multiple myeloma (MM). Among cell-based vaccines, monocyte derived dendritic cell (MDDC) fused with myeloma cells serve as Ag presenting cells to develop an immune response against a variety of targets. The purpose of this study is to report clinical response and tolerability of anti-myeloma vaccines. Methods: We included phase I and I/II trials developed between January 2008 to December 2017, where vaccines or viruses were used against MM, irrespective of the geo-location, age, and sex. We performed a comprehensive literature search (last update 3-30-2018) using the following databases: PubMed, Embase, AdisInsight, and Clinicaltrials.gov. Results: The initial search identified 2537 early phase studies. After screening by 2 reviewers and categorization by mechanism of action, 25 clinical trials (CT) that involved vaccines and/or viruses were included. We added 1 CT after the manual search. Therapy was given to 3 distinct classes of patients: patients without prior treatment (high risk smoldering MM or stage I MM, 4 CT), as an adjunct therapy for patients undergoing FDA approved treatments [high dose chemotherapy (HDT), allogeneic (allo-SCT) or autologous stem cell transplant (ASCT), 9 CT], and patients with residual or relapsed/refractory (RR) disease after FDA approved therapies (11 CT). Of the included 25 CT, 14 have published results available for analysis. For patients without prior treatments, PVX-410, a multi-peptide vaccine, resulted in at least minimal response (MR) in 50% of patients when combined with lenalidomiden and achieved stable disease (SD) for 60% of patients when used alone at 12 months follow up. Treatment with Idiotype-pulsed mature MMDC targeting idiotype proteins in MM showed MR in 30% of patients and SD in 43% of patients at 12 months. For patients receiving vaccines as an adjuvant treatment, recMAGE-A3 resulted in complete response (CR) and very good partial response (VGPR) in 46% and 54% respectively, at 3 months post ASCT follow up. By 12 months post ASCT, these responses were 38% CR and 23% VGPR. Treatment with MDDC (MAGE3 + Survivin + BCMA) resulted in SD in 42% of patients at a median of 25 months post vaccination and 55 months post ASCT. ScFv-FrC, a DNA fusion vaccine, resulted in CR in 50% and MR/SD in 21% at 52 weeks post vaccination. Ongoing CR/PR was maintained for 3+ years in 57 % patients, 4+ years in 36%, and 5+ years in 14% of patients following ASCT; OS was 64% after a median follow up of 85.6 months . Patients treated with MDDCs/tumor cells fusion vaccine had 69% SD after vaccination and 20% SD at a median of 26 months. When vaccines were given as a salvage therapy in RR MM, ImMucin vaccine showed a CR in 30% of patients during treatment, 20% maintained CR, and 13% had SD at a median of 24 months. Galinpepimut-S vaccine showed CR or very good partial response (VGPR) in 37% of patients at a median of 12 months, and 26% CR and VGPR at 18 months, with a progression free survival rate of 23.6 months. Patients receiving mHag loaded host MDDC vaccination also showed 8% CR for > 6 years (n=1) and 8% PR for 19 weeks (n=1); 33% had SD. Reolysin (wild-type reovirus), a virus-based vaccine, was used in 3 trials for RR MM patients. When alone, 42% of patients had SD and 58% had PD. When combined with dexamethasone and bortezomib 37% of patients had SD lasting for 3 cycles. Whereas, when combined with dexamethasone and carfilzomib, all patients had decrease in monoclonal proteins, with VGPR reported in 28%, PR in 43%, MR in 8%, and SD in 8% patients after 8 cycles. Most vaccines were well tolerated by patients, only grade (G) 1 and G2 side effects (SE), which were mostly flu-like symptoms and local skin reactions. G3 SE included pneumonia with mHag DC and Bcl2 peptide vaccine, GVHD with hTERT tumor vaccine, DVT and rash seen with scFv-FrC DNA vaccines. G4 SE were rare, but seen with reolysin, requiring 2 patients to be removed from study, and with DC/tumor cell fusion vaccine (1 pulmonary embolism). Conclusion Anti-myeloma vaccination therapy appears to be well tolerated, which makes it a promising adjuvant therapeutic agent against MM. Current data reveals positive immunologic activity in most patients and there is possibility of promising clinical responses with further drug development. Disclosures No relevant conflicts of interest to declare.

Introduction: The principle of precision cancer medicine is to customize therapy based on the genomic profiles of the cancer and the host constitution/response to the cancer. Since RNA expression is influence by many genetic mechanisms, RNA profiling may provide broader coverage of genomic changes and might be a better predictor of response to therapy. However, incorporating the many biological changes of the host and the cancer in the decision of selecting therapeutic approach is not practical without using computer-aided algorithms. This is particularly relevant when combination therapy is used. We explored the potential of developing algorithms for the prediction of complete response (CR) to novel combination therapy in patients with acute myeloid leukemia (AML) using targeted RNA expression profiling. Methods: RNA was extracted from the peripheral blood (PB) and bone marrow (BM) samples from patients with AML being treated on two different protocols: FLAG-IDA+venetoclax (F-I-V)( Abou Dalle I et al, ASH 2019; the NCT # is NCT03214562) and ivosidenib+venetoclax (I-V). In the initial study, 22 samples (9 PB and 13 BM) were used as training set. Subsequently 16 PB samples from the F-I-V arm and 4 from the I-V arm were collected and tested blindly as testing set after the development and locking of the algorithm. RNA was sequenced using NGS panel composed on compared of 1408 genes. The RNA sequencing is based on hybrid capture and the number of reads ranged from 5 to 10 million. RNA quantification was performed using Cufflinks. The RNA levels were normalized to ABL1 mRNA levels. Each gene is normalized by the mean and standard deviation of the gene. To develop a model for predicting CR, we used the training set in each arm and first evaluated the performance of each of the 1408 genes using receiver operating characteristic (ROC) curve. Then used the following mathematical methods for developing algorithms for predicting CR: Support Vector machine (SVM), Bayesian modeling with Gaussian Processes (GP), and Naïve Bayesian (NB). Results: In univariate analysis, multiple genes showed very high AUC. In the F-I-V arm, top genes in predicting CR were GLI3, SETBP1, SH3D19, ARHGAP20, ETS1, IKZF2, GNG4 and MAGEE1 with AUC ranged from 0.74 to 0.85. In the I-V arm, the top genes in predicting CR were STL, TNFRSF10D, PTGS2, RET, TFRC, NAV3, WSB1, and GAS1 with AUC varied from 0.91 to 0.96. Using the training samples we developed algorithms for predicting CR by SVM, NB and Bayesian GP. Upon testing these models using leave-one-out (LOO), the three algorithms performed similarly with AUC around 0.97 for the I-V arm and around 0.96 for the F-I-V arm. There was no difference between BM and PB in predicting CR. Therefore, we collected and sequenced only peripheral blood for blind testing. The three algorithms were tested using 16 PB samples from the F-I-V arm and 4 samples from the I-V arm. The SVM and NB algorithms predicted CR correctly in 15 of the 16 samples (94%) while Bayesian GP missed 4 of the 16 samples. As for the I-V arm, the NB predicted CR correctly in the 4 samples, while both SVM and Bayesian GP missed 3 of 4. Conclusions: While the data is limited and further validation is need, algorithms using RNA expression profiling of peripheral blood using targeted RNA NGS may provide an excellent tool for customizing therapeutic approach, especially in the age of combination therapy when number of cases for training can be limited. Furthermore, this study suggests that modeling using Nave Bayesian is reliable approach in developing prediction algorithms. Disclosures Albitar: Genomic Testing Ccoperative: Employment, Equity Ownership. Konopleva:Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Ablynx: Research Funding; Agios: Research Funding; Amgen: Consultancy, Honoraria; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding; Genentech: Honoraria, Research Funding; Astra Zeneca: Research Funding; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding. Loghavi:MDACC: Employment; AlphaSights: Consultancy; GLG Consultants: Consultancy. Takahashi:Symbio Pharmaceuticals: Consultancy. Kantarjian:Daiichi-Sankyo: Research Funding; Ariad: Research Funding; Cyclacel: Research Funding; AbbVie: Honoraria, Research Funding; Agios: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; BMS: Research Funding; Jazz Pharma: Research Funding; Novartis: Research Funding; Takeda: Honoraria; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Astex: Research Funding. DiNardo:syros: Honoraria; daiichi sankyo: Honoraria; celgene: Consultancy, Honoraria; jazz: Honoraria; abbvie: Consultancy, Honoraria; agios: Consultancy, Honoraria; medimmune: Honoraria; notable labs: Membership on an entity's Board of Directors or advisory committees.

Green gram (Vigna radiata) is considered the chief legume in Pakistan. Thus, current study was conducted to examine the ameliorating effect of phytohormones pre-treatments under salt stress on proteome profile of green gram by sodium-dodecyl-sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The soluble green gram seedlings proteins were resolved on 4% stacking and 12% resolving gels. The SDS-PAGE resolved 24 polypeptide bands ranging from 200 to 17kDa. Among these, 12 out of 24 bands of proteins were essentials house-keeping or growth proteins of green grams. While, 120, 114.6, 51.8, 29.1, and 22.8 kDa bands were over-expressed under 50 to 350mM salt with phytohormones treatments. The others 104.5 kDa, 99.8 kDa, 95.3 kDa, 91.0 kDa, 55 kDa, 46 kDa, and 17kDa bands were related to the GAᴣ, IAA, and SA induced tolerance. Overall 120 kDa, 114.6 kDa, 104.5 kDa, 99.8, 95.3 kDa, 51.8 kDa, 29.1 kDa and 22.8kDa bands were first time identified in the current study. The information retrieved from NCBI protein database, the resolved peptides were principally belonging to 7S and 8S vicilin, 2S, 8S, 11S, and 16.5S globulins. It is determined that seed priming with SA enhanced tolerance in green gram by rapidly synthesizing stress alleviating peptides.Key word: Cluster analysis, dendrogram, mungbean, salt stress, SDS-PAGEINTRODUCTIONVarious world-wide health concerning organization recommended the use of high graded plant protein such as legumes to prevent the risk of metabolic disorder (Hou et al., 2019). Legumes are most important protein crop on the earth. Among the legumes, the green gram is the major pulses. Its seeds are rich in superior quality storage protein, which account 85% of the total protein while, another 15% have not been broadly studied (Yi-Shen et al., 2018). The soluble storage protein comprises of 60% globulins, 25% albumin and 15% prolamins. Globulins are further divided into 3.4% basic-type (7S), 7.6% legumin-type (11S), and 89% vicilin-type (8S) (Mendoza et al., 2001; Itoh et al., 2006). Other than proteins, the green gram seeds also contain starch, fiber, phenolic compound, saponins, vitamins, calcium zinc, potassium, folate, magnesium, manganese and very low in fat that made it meager man’s meat (Hou et al., 2019). It is also a good source of green manure and fodder (El-Kafafi et al., 2015). Its root has ability to fix 30 to 50 Kg/ha atmospheric nitrogen in the soil which is essential for maintaining soil fertility (Chadha, 2010). The green gram is the valuable and the major Rabi pulse crop of Pakistan. Its cultivation area in 2016-2017 was about 179,000 hectares with seed yield of 130,000 tones. In comparison during 2017-2018, it was cultivated on 161,800 hectares land with 118,800 tones seed yield (GOP, 2018). One of the reasons of this 9% decrease in both land and productivity is the shortage of irrigated land due to soil salinity. The salinity induce oxidative bust in the mungbean cells, caused by responsive oxygen species (ROS) such as hydrogen peroxide, singlet oxygen, hydroxyl radical and superoxide radical. The ROS create hindrance in various metabolic processes of plant via interacting with macromolecules like proteins (Alharby et al., 2016). However, phytohormones like gibberellic acid (GAᴣ), indole acetic acid (IAA), and salicylic acid (SA) take part in the biosynthesis of salt tolerance proteins under salinity. These salt tolerance proteins acclimate plants under salinity stress. Application of biotechnology plays a significant role in agriculture (Khan et al., 2017). Therefore, production of particular proteins under salt stress is a specific response of cell which can be analyzed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE is the simple, valid, and cost-effective biochemical marker (Mushtaq et al., 2018). This marker has been widely used to determine the extent of evolutionary variations in crops (El-Kafafi et al., 2015).OBJECTIVES The present study was directed first time with the aim to investigate the toxic effect of sodium chloride (0-350 mM) and stress acclimation by pre-treatment of GAᴣ, IAA, and SA on the proteome profile of NM-92 cultivar of a Pakistani green gram.MATERIALS AND METHODSThe present study was replicated thrice in the plant laboratory of Department of Genetics, Faculty of Science, and University of Karachi. The seeds of mung bean cultivar NM-92 were acquired from National Agricultural Research Centre (NARC), Islamabad. These freshly collected 15 seedsˉ1 treatment / replication were divided into two sets. The first was named as sodium chloride (SC) stress treatments were imbibed in sterile distilled water (DW) whereas, second set soaked in gibberellic acid (GAᴣ) (BDH Chemicals, England), indole acetic acid (IAA) (Fluka, Switzerland), and salicylic acid (SA) (J.T. Baker, Holland) in the separate beaker for 24 hours under dark condition. After 24 hours, given ample time to both the sets at room temperature. After recovery, all 20 treatments were sown in the 150 X 30 mm sized petri-dishes containing 0, 50, 150, 250, and 350 millimolar (mM) sodium chloride solution (Fisher Scientific, UK) for 72 hours.Protein extraction: Protein extraction was done by taking 0.3g of seedlings in an ice chilled mortar and crushed by adding 600µL 0.2 M Tris-HCl buffer having pH 7.5 contained 5% SDS (w/v) and 5% 2-mercaptoethanol (v/v). The homogenate was incubated at 0oC for 30 min., boiled in the water bath for 3 min. at 100oC. Samples were centrifuged in Heraeus Biofuge D-37520, Germany for 30 min. at 8000 rpm. The protein supernatant was saved at below 0°C for quantitative and qualitative determination with minor modifications. The total soluble protein content of the samples was estimated via “Bovine Serum Albumin (BSA) standard curve” and explicit in µg protein milligramˉ1 fresh weight of mung seedlings.Bovine serum albumin standard curve (2000 μg/mL): Total protein standard curve was made by dissolving 0.05g of Bovine Serum Albumin (BSA) in 25mL of distilled water. Ten serial dilutions were made from 0.1 mL to 1mL by BSA solution then performed Lowry. A standard curve of total proteins was plotted by taking BSA absorbance at Y-axis and 2000 μg BSA / mL at X-axisSample preparation for SDS-PAGE: For qualitative assessment of total proteins; the 35μL of saved protein supernatant was combined with 15μL of sample diluting buffer (SDB). The SDB was made up of 0.0625 M Tris-HCl pH 6.8 with 2% of SDS, 10% of glycerol, 0.003% of bromophenol blue dye and 5% of 2-mercaptoethanol. Boil the 50μL protein SDB supernatant at 100oC in water bath for 3 min., centrifuged at 6000 rpm for 4 min. The supernatant was loaded on SDS-PAGE gel with the given formulae. The SDS- PAGE: Total proteins were fractionated via SDS-PAGE with 4% stacking and 12% resolving gel. The resolving gel of 12% was made by taking 6mL solution A, 1.8 mL 3 M Tris 1 M HCl buffer pH 8.8, 144μL 10% SDS, 5.74 mL sterile distilled water, 720μL 1.5% ammonium persulphate (APS) in deionized water and 10μL TEMED. While, stacking was composed of 1.25mL of solution A, 2.5mL of 0.5M Tris 1M HCl buffer pH 6.8, 100μL 10% SDS, 1.8 mL of distilled water, 500μL 1.5% APS and 12μL TEMED. Solution A was prepared by conjoining 30% acrylamide and 0.8% N, N’-methylene-bisacrylamide in deionized water. To avoid polymerization in the beaker; the prepared solution was quickly poured into the 3 mm thick gel plates after adding TEMED. The stacking was lined over resolving gel, then combs were inserted between the gel plates of SCIE-PLAS TV-100 separation system, UK, and allowed to polymerize for ½ an hour. After polymerization gel was placed in the tank which were filled with Tris-Glycine buffer (electrode buffer) pH 8.4 then combs were removed. The electrode buffer contained 0.3% Tris, 1.41% Glycine and 0.1% SDS in 2000mL d/w. The gel was pre-run for 15 min. at 60 volts and 120 mA currents. The prepared SDS-PAGE samples were loaded in wells with BlueStepTM Broad Range Protein Marker, AMRESCO, USA as standard and run at 60 volts & 120 mA for about 45 min. When samples entered in resolving gel, and then gave 100 volts and 200 mA currents for around 2.5 hours. Furthermore, electrophoresis was carried out at a constant watt.The Gel was washed with 30% ethanol on Uni Thermo Shaker NTS-1300 EYELA, Japan at the constant shaking for 30 min. Then gels were placed in 10% glacial acetic acid in 50% methanol solution (Fixative) for 24 hours. SDS Gel was stained until protein bands were visible thereat placed as 5% of Methanol in 7.5% acetic acid glacial solution to destain the bands background. SDS-PAGE stain composed of 0.125% coomassie brilliant blue R-250 dissolved in 40% of Methanol and 7% acetic acid glacial solution. The stain was stirred on Magnetic stirrer & hot plate M6/1, Germany for 6-10 hours before used. Photographs were taken by Sanyo digital camera VPC-T1284BL and bands were scored through numbering pattern. Gels preserved in 10% acetic acid solution at 4°C.Interpretation of bands and data analysis: The total soluble protein bands relative mobility calculated by below formulae and Dendrogram was constructed via SPSS v. 20Where,F=(Migrated distance of protein band)/(Migrated distance of dye front)Slop=(Log MW of protein marker lower limit band–log〖MW of protein marker upper limit band )/(RF protein marker lower limit band –RF of protein marker upper limit band)RESULTS:The total soluble proteins extracted from green gram were perceived by SDS-PAGE Blue StepTm broad range biochemical markers. The protein-based marker was used to evaluate the toxic effect of sodium chloride along with pre-treatments of GAᴣ, IAA, and SA on proteome assay. In the current work, seedlings total soluble proteome resolved 24 polypeptide bands ranging from 200 to 17.1 kDa were recognized by using SDS-PAGE. The figure 1 showed Dendrogram assay, which classified the 20 treatments of SC, GAᴣ, IAA and SA into two major clusters where, the cluster I was the largest one (figure 1). Cluster I consisted of 15 treatments that further divided into I-A, and I-B. The pre-treatments of SC50+SA, SC150+SA, SC250+SA, and SC350+IAA were grouped together into C-1 of sub-cluster I-A. The C-2 of sub-cluster I-A, pre-treatment SC350+SA was most diverse among 20 treatments. The C-1 treatments showed 99% homology when compared with each other while, it was 97% similar with C-2. The sub-cluster I-B comprised another 10 treatments, SC0+GAᴣ, SC50+GAᴣ, SC150+GAᴣ, SC250+GAᴣ, SC350+GAᴣ, SC0+IAA, SC50+IAA, SC150+IAA, SC250+IAA, and SC0+SA that were also 99% similar for total proteins. Sub-cluster I-B pre-treatments was exhibiting 94% homology with the sub-cluster I-A. The second cluster was the smallest one that was divided into two sub-clusters, II-A and II-B. The II-A was comprised of SC50, SC150, and SC250 while, sub-cluster II-B consisted of SC0 and SC350. Within each sub-cluster, pre-treatments expressed 99% homology whereas, II-A was 97 different from II-B. Furthermore, cluster I showed 75% similarities with cluster II (figure 1). The seedlings storage proteome profile of green gram was shown in table 1.The results showed that 120kDa, 114.6 kDa, 51.8 kDa, 29.1 kDa and 22.8 kDa proteins bands were not induced at 0 mM SC, GAᴣ, IAA, and SA. The table 1 depicted the presence of 120 kDa and 114.6 kDa bands only at 350 mM SC level with all phytohormones treatments. Similarly, 51.8 kDa protein bands were appearing at 150SC, 250SC and 350SC stress with phytohormones. Based on the information collected from the NCBI protein database, this peptide was related to the 8S globulin alpha subunits. The two other, 7S globulins sub-units having 29.1kDa and 22.8 kDa molecular weights bands were synthesized under 50mM, 150mM, 250mM, 350mM SC stress with phytohormones. Concerning protein polypeptide of molecular weight 104.5 kDa, 99.8 kDa, 91.0 kDa, 55.0 kDa, and 46.0 kDa, those were induced by GAᴣ, IAA and SA at 0 to 350 mM SC. While, 17kDa protein band was appearing in SA, and IAA treated samples and 95.3kDa band was only present in SA treatment. Other 12 protein bands were present in all treatments proved as house-keeping proteins of green gram (table 1).DISCUSSIONThe SDS-PAGE profiling for proteome is the reliable and applied biochemical approach that has been used as biochemical marker in various crop differentiation, and characterization. In the current study, first time SDS-PAGE was utilized to investigate the impact of GAᴣ, IAA, and SA pre-soaking on green gram under salt toxicity. The salt toxicity adversely affects all seed, seedling, and plant metabolic process (Parveen et al., 2016). At salt toxicity, the endogenous GAᴣ, IAA, and SA levels markedly decrease (El-Khallal et al., 2009). In such condition, exogenous application of GAᴣ, IAA, and SA enhance seedlings survival rate by increasing synthesis of seed storage proteins. Likewise, our Dendrogram characterization based on 20 treatments showed significant diversity under 0 to 350 mM SC stress. The salicylic acid treatments were grouped together except SC0+SA treatment, exhibiting a close relationship, which proved its acclimating role under salt stress. These findings will help plant breeder toward enhancing food quality and quantity of green gram in future breeding programme on saline sodic land.The SDS-PAGE assay revealed 200. kDa, 109.4 kDa, 77 kDa, 68 kDa, 49 kDa, 38 kDa, 33 kDa, 26 kDa, 24 kDa, 22 kDa, 21 kDa and 19 kDa fractions as essential green gram proteins. Among these, 68 kDa, 49 kDa, 33 kDa, 26 kDa, 24 kDa and 21 kDa peptides were seed biotinylated isoform protein (Riascos et al., 2009), putative NADH-ubiquinone oxidoreductase subunit H (Gostinčar et al., 2019), heat shock protein 33 (Hamidian et al., 2015), globulin protein, seed coat / maturation protein (Dhaubhadel et al., 2005), and protein for dimerization. While, 22 kDa proteins belonged to the class of prolamin alpha zein Z1C1_2, Z1C1_4, and Z1C1_8 precursors, and 19kDa peptide was related with Z1A1_2, Z1A2_2, and Z1B_6 precursors (Miclaus et al., 2011). Further, the 91 kDa peptide is sucrose synthase SS1 protein, and 77kDa protein is the NADPH-cytochrome P450 reductase (Wang et al., 2004). Also, the phosphatase-associated two other proteins having 46 and 55 kDa molecular weight were reported earlier in Mucuna pruriens. Hameed et al. (2012) and Malviya et al. (2008) found 55 and 46kDa peptides as 7S vicilin small sub-units and 17kDa as 11S globulins sub-unit in the studied Vigna radiata. Some other molecular weight proteome such as 68 kDa and 49kDa are 7S vicilin, 33kDa is 8S vicilin, 38 and 26kDa 8S globulins, 24kDa 11S globulins, and 22kDa 16.5S globulins. These proteins required for germination and seed establishment of green gram plant (Hameed et al., 2012).The vast accumulation of 23kDa and 22kDa peptides under salt stress by salicylic acid, were reported previously in the mangrove Bruguiera parviffora and Zea mays (El-Khallal et al., 2009). Correspondingly, El-Kafafi et al. (2015) reported the presence of 115kDa, 23kDa, and 22kDa bands in the salt tolerant lines of green gram. These proteomes induced under salt stress may play a pivotal part in the stress acclimation and osmotic adjustment. Similarly, the induction of 104 kDa and 100kDa MW polypeptide by SC stress in the salt tolerant genotypes of green gram indicated the functional role of phytohormones in various metabolic and defense response El-Kafafi et al. (2015); Alharby et al. (2016), El-Khallal et al. (2009), Qados (2010). Ali et al. (2007), Alharby et al. (2016), and El-Kafafi et al. (2015) observed 17kDa, 26kDa, 33kDa and 77kDa bands involving in salt tolerance and can be considered as a positive biochemical marker for salt stress. Further, 26 kDa MW peptide also functions as osmotin under the salt stress that involved in enhancing the accumulation of glycine betaine and proline in the cells. Hence, proteome assay of green gram showed that GAᴣ, IAA, and SA could regulate the expression of salt stress proteins that are anticipated to play a crucial part in the salt tolerance mechanism. Likewise, the involvement of phytohormones in the induction of changes in the proteome profile pattern was attributed to their part in managing cell division by regulating some genes of apical meristems.CONCLUSIONFinally, the results revealed the presence of the ten new bands with MW of 200kDa, 120 kDa, 114.6 kDa, 109.4kDa, 104.5kDa, 99.8kDa, 95.3kDa, 51.8kDa, 29.1kDa and 22.8kDa have not reported previously under salt stress with phytohormones treatments in green gram. Furthermore, it was observed that phytohormones alleviate the negative impact of salt stress on green gram by enhancing synthesis of salt defense polypeptides. Hence, higher accumulation of proteins was observed in salicylic acid treated seedlings. Thus, present work recommended the pre-soaking of phytohormones to overcome the toxic impact of sodium chloride on green gram. Further research is needed on a biomolecular level to reveal the mechanism of signalling pathways under sever salt stress.CONFLICT OF INTERESTBoth authors have declared that no disagreement of interest regarding this research.REFERENCES Alharby, H. F., E. M. Metwali, M. P. Fuller and A. Y. Aldhebiani, 2016. The alteration of mRNA expression of sod and gpx genes, and proteins in tomato (Lycopersicon esculentum Mill) under stress of Nacl and/or ZnO nanoparticles. Saudi journal of biological sciences, 23(6): 773-781.Ali, A., M. Mageed, I. Ahmed and S. Mariey, 2007. Genetic and molecular studies on barley salt tolerance. In: African crop science conference proceedings. pp: 669-682.Chadha, M., 2010. Short duration mungbean: A new success in South Asia. Asia-Pacific association of agricultural research institutions.Dhaubhadel, S., K. Kuflu, M. C. Romero and M. Gijzen, 2005. A soybean seed protein with carboxylate-binding activity. 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Melanoma associated antigen (MAGE) is an extensively studied family of tumor-associated genes that share a common MAGE homology domain (MHD). Based upon their expression pattern, MAGE genes have been broadly classified into type 1 MAGEs (T1Ms) and type 2 MAGEs (T2Ms) categories. Interestingly, several T2Ms are highly expressed in the brain and involved in the regulation of neuronal development, differentiation, and survival. Available literature suggests possible tumor suppressor functions of a few T2Ms, while information available about their expression, regulation, and clinical significance in glioma is scanty. This prompted us to perform a comprehensive analysis of T2M expression in glioma. Gene expression data from glioma datasets: Oncomine, TCGA, and REMBRANDT study, were used to assess the mRNA expression of T2M genes (MAGED1, MAGED2, MAGED3, MAGED4, MAGED4B, MAGEE1, MAGEE2, MAGEF1, MAGEH1, MAGEL2, NSMCE3, and NDN), and their association with clinical characteristics and composition of the tumor microenvironment. Further, mutation, copy number alteration, and DNA methylation data from TCGA were assessed for determining potential mechanisms of T2Ms expression in glioma. Expression analysis revealed overexpression of MAGED subfamily genes in glioma, while other genes of this family exhibited reduced expression in advanced grades of this malignancy. Further, the expression of T2Ms exhibited varying extent of positive correlations with each other. Amongst downregulated T2Ms, MAGEH1 expression exhibited negative correlations with DNA methylation. Additionally, genes associated with MAGEH1 were enriched in Myc and Hedgehog signaling. Furthermore, T2Ms downregulation was associated with immune infiltration in glioma tissues and poor overall survival of glioma patients. In multivariate Cox regression analysis, MAGEH1 emerged as an independent prognosticator in lower grade glioma. Conclusively, these results suggest that expression of T2Ms is associated with important clinical and molecular features in glioma. Mechanistic studies may further provide novel insights into their role in glioma progression.

Preeclampsia (PreE), a hypertensive disorder in pregnancy, contributes to long-term maternal cardiovascular disease risk. By 2025, it is estimated that more women than men will have hypertension (HTN), yet the mechanisms contributing to the development of HTN in women are less understood. One potential mechanism underlying HTN in women is a persistent imbalance of anti- and pro- inflammatory T H cells following PreE. Consistent with this, anti-inflammatory T helper (T H ) cytokines are reduced and pro-inflammatory T H cytokines are increased during a PreE pregnancy. De-identified and coded plasma samples and clinical data were obtained from the Magee-Women’s Research Institute & Foundation or the University of Iowa Maternal-Fetal Tissue Bank (IRB 201808705) from women 1-3 (N=93) or 8-10 (N=58) years (yrs) following a normotensive (CTL) or PreE-affected pregnancy. Postpartum (PP) HTN was defined as having stage 1 or higher HTN as designated in the updated 2017 ACC/AHA guidelines. Women with PreE had higher rates of HTN at 1-3 years and at 8-10 years PP, (24% vs. 5% and 65% vs. 17%, all p<0.05) compared to women with a normotensive pregnancy. To determine if T H cells play a role in the future development of HTN, we investigated if the T H cytokine changes observed in PreE persist 1-3 yrs and 8-10 yrs PP. Cytokine concentrations were determined via ELISAs and normalized to total protein. Average cytokine concentrations are reported in pg/g. At 1-3 yrs PP, concentrations of anti-inflammatory cytokines IL-4 (47 vs. 6819, p<0.05), IL-10 (1204 vs. 15042, p<0.05) and TGFβ (8.2x10 5 vs. 4.4x10 6 , p<0.05) were reduced in women with a prior PreE pregnancy vs. women with a CTL pregnancy. At 8-10 yrs PP, pro-inflammatory IL-6 (86 vs. 18, p<0.05) and TNFα (298 vs. 53, p<0.05) were both significantly increased in women with prior PreE compared to women with a CTL pregnancy. Here, we confirm women with a prior PreE pregnancy present with a higher prevalence of HTN early (1-3 yrs) and later (8-10 yrs) PP compared to women with a normotensive pregnancy. Further, we show an altered T H cytokine milieu persists following delivery in women with PreE. This pro-inflammatory milieu is associated with increased rates of HTN and thus, may underlie the future development of HTN in women with a history of PreE.

Abstract Background MAGnetic Expansion Control (MAGEC) rods can prevent repeated lengthening operations for scoliosis patients. However, there have been several Field Safety Notices issued, including a worldwide product recall due to actuator endcap separation. We aimed to review adverse events reported to the Food and Drug Administration (FDA) regarding MAGEC rods, focusing on MAGEC X. Methods Reports submitted to the Manufacturer and User Facility Device Experience database in relation to MAGEC devices were accessed and analysed using R Statistical Software. Exclusion criteria included duplicate and literature review reports (n = 54). Free-text data were analysed using inductive content analysis. Results 1016 adverse events were reported to 11/30/2023. 99.0% (1006) were submitted by the manufacturer. Reports primarily arose from the UK (465, 45.8%) or US (421, 41.4%). From free-text data the most frequent adverse events were distraction mechanism failure (573), device wear (272), and actuator seal damage (180). Rod fracture (n = 48) was not significantly associated with rod diameter (≤ 5.0 mm or > 5.0 mm), p = 0.736. 234 reports referenced MAGEC X devices; actuator endcap separation was identified in 41.9% (99). Other events include failure of distraction (63), surface damage (31), and rod fracture (15). On 06/30/2020 MAGEC X2 received FDA approval. Twenty reports reference devices manufactured after this date, seven describe distraction mechanism failure; notably there are no reports of endcap separation. Conclusion These data represent the largest series of adverse events reported for MAGEC rods, including significant new data regarding MAGEC X. As well as endcap separation, failure of distraction, surface damage, and rod fracture were reported.

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is characterised by limited responses to chemoimmunotherapy attributed to highly desmoplastic tumor microenvironment. Disrupting the tumor-stromal cell crosstalk is considered as an improved PDAC treatment strategy, whereas little progress has been made due to poor understanding of its underlying mechanism. Here, we examined the cellular role of melanoma associated antigen A isoforms (MAGEA) in regulating tumor-stromal crosstalk mediated chemoresistance. Methods We used clinical samples to explore the correlation between MAGEA expression and patient prognosis in multiple cancers. We utilized cancer cell lines, patient derived organoids and orthotopic PDAC model to examine the function of MAGEA in chemoresistance. We performed biochemical, proteome profiler array and transcriptional analysis to uncover a mechanism that governs tumor-stromal crosstalk. We developed a multi-MAGEA antigen targeted DNA vaccine and tested its effect on PDAC tumor growth. Results We establish MAGEA as a regulator of the tumor-stromal crosstalk in PDAC. We provide strong clinical evidence indicating that high MAGEA expression, including MAGEA2, MAGEA3 and MAGEA10, correlates with worse chemotherapeutic response and poor prognosis in multiple cancers, while their expression is up-regulated in chemoresistant PDAC patient derived organoids and cancer cell lines. Mechanistically, MAGEA2 prohibits gemcitabine-induced JNK-c-Jun-p53 mediated cancer cell apoptosis, while gemcitabine stimulated pancreatic stellate cells secretes GDF15 to further enhance the gemcitabine resistance of MAGEA2 expressing cells by activating GFRAL-RET mediated Akt and ERK1/2 dependent survival pathway. Strikingly, immunization with a DNA vaccine that targeting multiple MAGEA antigens, including MAGEA2, MAGEA3 and MAGEA10, elicits robust immune responses against the growth of gemcitabine resistant tumors. Conclusions These findings suggest that targeting MAGEA-mediated paracrine regulation of chemoresistance by immunotherapy can be an improved pancreatic cancer treatment strategy.

Current adhesives bond to dentin via a micro-interlocking mechanism within the hybrid layer. Besides such mechanical retention, bonding to dentin would benefit from additional chemical interaction between collagen and resin. This study aims to synthesize a novel light-curable collagen crosslinker methacrylate (MA) functionalized grapeseed extract (GSE) and to assess MAGSE’s ability to crosslink dentin collagen in a clinically relevant setting as well as its role in light-cure as a resin. MA functionalization was accomplished by reacting GSE with methacryloyl chloride to obtain MAGSE, which was characterized by 1H-NMR and Fourier transformed infrared spectroscopy (FTIR). The 6-µm-thick dentin films were microtomed from dentin slabs of third molars. Following demineralization, they were treated for 30 s by 1% MAGSE. Collagen crosslinking and resistance to digestion of MAGSE were evaluated by FTIR, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) assay of films, and scanning electron microscopy (SEM)/transmission electron microscopy (TEM) on slabs. Meanwhile, 1% MAGSE or GSE was added to an experimental adhesive formulated with 2-hydroxyethyl methacrylate and a tricomponent photoinitiator system. Polymerization kinetics were monitored continuously in real time for 10 min using FTIR–attenuated total reflection. The results indicated that MAGSE could bind to dentin collagen and protect it from collagenase degradation as strong as GSE. Dentin collagen treated by 1% MAGSE for 30 s was scarcely digested (1.6 ± 1.6%) after 1 h in 0.1% collagenase, while untreated collagen was completely digested (100.9 ± 20.2%). SEM/TEM images indicated MAGSE efficiently crosslinked dentin collagen in 30 s and rendered it almost inert to digestion under clinically relevant settings. Unlike GSE that hindered light-curing of HEMA, MAGSE accelerated the rate of polymerization and exhibited typical traits of a resin monomer with multiple polymerizable units. In conclusion, a novel collagen crosslinking resin MAGSE is synthesized, which inherits collagen crosslinking ability from GSE and polymerization function from MA. Inclusion of this light-curable collagen crosslinker into adhesives might be a revolutionary way to improve durability of dentin bonding in composite restorations.

Evelyn Neppelberg disputerte den 4. mai 2007 for PhD-graden ved Det odontologiske fakultet, Universitetet i Bergen med avhandlingen: «Pathological Mechanisms in Oral lichen planus: A Study of Apoptosis – Regulatory Proteins and Risk Markers for Malignant Transformation». Bedømmelseskomiteen bestod av professor dr.odont. Jesper Reibel, Københavns Universitet, professor dr.odont. Magne Bryne, Universitetet i Oslo, og professor dr.odont. Anne Isine Bolstad, Universitetet i Bergen.

AbstractMicrobial carbonates are common components of Quaternary tropical coral reefs. Previous studies revealed that sulfate-reducing bacteria trigger microbial carbonate precipitation in supposedly cryptic reef environments. Here, using petrography, lipid biomarker analysis, and stable isotope data, we aim to understand the formation mechanism of microbial carbonate enclosed in deep fore reef limestones from Mayotte and Mohéli, Comoro Islands, which differ from other reefal microbial carbonates in that they contain less microbial carbonate and are dominated by numerous sponges. To discern sponge-derived lipids from lipids enclosed in microbial carbonate, lipid biomarker inventories of diverse sponges from the Mayotte and Mohéli reef systems were examined. Abundant peloidal, laminated, and clotted textures point to a microbial origin of the authigenic carbonates, which is supported by ample amounts of mono-O-alkyl glycerol monoethers (MAGEs) and terminally branched fatty acids; both groups of compounds are attributed to sulfate-reducing bacteria. Sponges revealed a greater variety of alkyl chains in MAGEs, including new, previously unknown, mid-chain monomethyl- and dimethyl-branched MAGEs, suggesting a diverse community of sulfate reducers different from the sulfate-reducers favoring microbialite formation. Aside from biomarkers specific for sulfate-reducing bacteria, lipids attributed to demosponges (i.e., demospongic acids) are also present in some of the sponges and the reefal carbonates. Fatty acids attributed to demosponges show a higher diversity and a higher proportion in microbial carbonate compared to sponge tissue. Such pattern reflects significant taphonomic bias associated with the preservation of demospongic acids, with preservation apparently favored by carbonate authigenesis.

AbstractNon-enzymatic modification of proteins by carbohydrates, known as glycation, leads to generation of advanced glycation end-products (AGEs). In our study we used in vitro generated AGEs to model glycation in vivo. We discovered in vivo analogs of unusual melibiose-adducts designated MAGEs (mel-derived AGEs) synthesized in vitro under anhydrous conditions with bovine serum albumin and myoglobin. Using nuclear magnetic resonance spectroscopy we have identified MAGEs as a set of isomers, with open-chain and cyclic structures, of the fructosamine moiety. We generated a mouse anti-MAGE monoclonal antibody and show for the first time that the native and previously undescribed analogous glycation product exists in living organisms and is naturally present in tissues of both invertebrates and vertebrates, including humans. We also report MAGE cross-reactive auto-antibodies in patients with diabetes. We anticipate our approach for modeling glycation in vivo will be a foundational methodology in cell biology. Further studies relevant to the discovery of MAGE may contribute to clarifying disease mechanisms and to the development of novel therapeutic options for diabetic complications, neuropathology, and cancer.

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ABSTRACTA systematic study on magnetic properties of (Fc/Pt) compositionally-modulated films as a function of substrate-deposition-temperature has been carried out. For films with (2.5ÅFe/18ÅPt)×40, the intrinsic perpendicular magnetic anisotropy Ku is found to increase with decreasing temperature for all the sarmples deposited at temperatures from -114 °C to 220°C during deposition. The higher the substrate-deposition-temperature, the larger the Ku values become. This result implies a possible contribution from the magne to-elastic effect to the total anisotropy. However, a major part responsible for the perpendicular anisotropy may be found in other mechanisms. The saturation magnetization is found to exceed the value for pure Fe at temperatures lower than 180K.

ABSTRACTAn attempt is made to study the thermoelectric power in ultrathin films of semiconductors under magnetic quantization by including all types of aniso tropies in the energy spectrum within the domain of theory, and taking n-Cd3As2 as an example. It is found that, the magne to-thermopower decreases with increasing surface electron concentration and also changes in an oscillatory manner with film thickness respectively. The theoretical results are in agreement with the experimental observations as reported elsewhere.

Unlike the presentation format in a typical delay discounting task (e.g., “Would you prefer [A] US$4.3 today OR [B] US$7.5 in 22 days?”), Magen et al. inserted a zero to each alternative (e.g., “Would you prefer [A] US$4.3 today and US$0 in 22 days OR [B] US$0 today and US$7.5 in 22 days?”) and found this manipulation effectively reduced delay discounting ( d = .84), which was referred to as the hidden-zero effect. Study 1 was a direct replication of this effect. In Study 2, we tested whether the explicit-zero format could buffer against the detrimental effect of exposure to sexy cues on delay discounting. In Study 3, we explored the mechanism underlying the hidden-zero effect. Taken together, the hidden-zero effect was consistently found across all studies ( N = 2,440) and our internal meta-analysis yielded a medium to large effect size ( d = .52).

AbstractMAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.

Abstract Disclosure: X. Zhang: None. A. Majumdar: None. B. Kleiboeker: None. K.L. Magee: None. B.S. Learman: None. S.A. Thomas: None. I.J. Lodhi: None. O.A. MacDougald: None. E.L. Scheller: None. Adipocytes classically store or release energy in response to changes in metabolic status. However, several fat depots throughout the body are resistant to these metabolic cues. Identification of novel mechanisms that drive energy release from metabolically inert adipocytes is needed to inform strategies to deplete stubborn fat and, conversely, to preserve these depots in pathologic settings of wasting and cachexia. To address this question, we focused on the constitutive bone marrow adipose tissue (cBMAT), a well-established site of metabolically inert fat that remains unchanged with fasting, exercise, and cold exposure and is depleted only in certain extreme conditions such as cachexia or starvation. To study this unique depot, we developed a mouse model of rapid, complete depletion of all fat, including cBMAT, within 9 days using central intracerebroventricular (ICV) injection of leptin. Surgical denervation, chemical sympathectomy, or genetic dopamine beta-hydroxylase (DBH) deletion did not prevent the ICV leptin-induced depletion of cBMAT, revealing this process to be independent of catecholamines and the sympathetic nervous system (SNS). Vossicle implantation defined the mediators as transported through the circulation, and BMAT-specific ablation of adipose triglyceride lipase (ATGL) identified dependence on basal lipolysis. Gene expression and a radioisotope-based de novo lipogenesis assay pinpointed a concurrent suppression of lipogenesis, and subcutaneous insulin supplementation prevented leptin-induced cBMAT depletion, but not other depots. Together, this defines the differential regulation of metabolism in metabolically inert fat depots such as cBMAT. Specifically, leptin-responsive circuits in the brain can deplete inert adipocytes by suppressing lipogenesis while maintaining basal lipolysis independent of the SNS. Presentation: Saturday, June 17, 2023

ABSTRACTThin films of calcium doped lanthanum manganites Lal-xCaxMnO3 (LCMO) with x ∼ 0.41 have been prepared on LaAlO3(001) (LAO) Y-stablized ZrO2(001) (YSZ), and Al2O3(0001) (SAP) substrates by liquid delivery metal-organic chemical vapor deposition (LD-MOCVD). The films on YSZ and SAP substrates have a textured, polycrystalline morphology with a preferred orientation of (110). The films on LAO show a single-crystalline morphology and a (100) orientation. Transport measurements show the polycrystalline films have a resistance peak approximately 60K lower than the films on LAO and, in general, have a much higher overall resistance. The magne-toresistance (MR) ratio ([R(H) - R(0)]/R(H)) is sharply peaked near the maximum in resistance for the films on LAO, while the polycrystalline films show a noticeable absence of this sharply peaked behavior and a flat, rather large (∼ 100%) MR ratio over a large temperature range. These results will be discussed in terms of grain boundary scattering, crystallite size, and magnetization.