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The objective of this work is to develop machine learning (ML) methods that can accurately predict adverse drug reactions (ADRs) using databases like SIDER (Side Effect Research) and OFFSIDES (Government medical records). In this paper, three Machine Learning algorithms SVM (Support Vector Machine), Random Forest, and Gradient boosted trees are implemented on the datasets to predict various disorders caused due to adverse effects, and the performance is evaluated based on performance metrics such as average precision, recall, accuracy, f1 binary, f1 macro, and f1 micro. Finally, the two datasets were merged to understand and compare the performance of a combined dataset to a single dataset.

Tumor hypoxia is a negative prognostic factor that is implicated in oncogenic signal activation, immune escape, and resistance to treatment. Identifying the mechanistic role of hypoxia in immune escape and resistance to immune-checkpoint inhibitors may aid the identification of therapeutic targets. We and others have shown that V-domain Ig suppressor of T-cell activation (VISTA), a negative checkpoint regulator in the B7 family, is highly expressed in the tumor microenvironment in tumor models and primary human cancers. In this study, we show that VISTA and HIF1α activity are correlated in a cohort of colorectal cancer patients. High VISTA expression was associated with worse overall survival. We used the CT26 colon cancer model to investigate the regulation of VISTA by hypoxia. Compared with less hypoxic tumor regions or draining lymph nodes, regions of profound hypoxia in the tumor microenvironment were associated with increased VISTA expression on tumor-infiltrating myeloid-derived suppressor cells (MDSC). Using chromatin immunoprecipitation and genetic silencing, we show that hypoxia-inducible factor (HIF)-1α binding to a conserved hypoxia response element in the VISTA promoter upregulated VISTA on myeloid cells. Further, antibody targeting or genetic ablation of VISTA under hypoxia relieved MDSC-mediated T-cell suppression, revealing VISTA as a mediator of MDSC function. Collectively, these data suggest that targeting VISTA may mitigate the deleterious effects of hypoxia on antitumor immunity.

Supplementary Methods and Figure Legends

Supplementary Figure Legends

Figure S2. Vista expression is correlated with myeloid infiltration in the TME of colon cancer patients. Volcano plot of CLS differences (granulocytic, monocytic, and αβ T cells) between VISTAhigh and VISTAlow cohorts and the associated significance values obtained using Mann-Whitney tests.

V domain immunoglobulin suppressor of T-cell activation (VISTA) is a premier target for cancer treatment due to its broad expression in many cancer types and enhanced expression upon development of adaptive immune checkpoint resistance. In the CT26 colorectal cancer model, monotherapy of small tumors with anti-VISTA resulted in slowed tumor growth. In a combination therapy setting, large CT26 tumors showed complete adaptive resistance to anti–PD-1/CTLA-4, but inclusion of anti-VISTA led to rejection of half the tumors. Mechanisms of enhanced antitumor immunity were investigated using single-cell RNA sequencing (scRNA-seq), multiplex image analysis, and flow cytometry of the tumor immune infiltrate. In both treatment models, anti-VISTA upregulated stimulated antigen presentation pathways and reduced myeloid-mediated suppression. Imaging revealed an anti-VISTA stimulated increase in contacts between T cells and myeloid cells, further supporting the notion of increased antigen presentation. scRNA-seq of tumor-specific CD8+ T cells revealed that anti-VISTA therapy induced T-cell pathways highly distinct from and complementary to those induced by anti–PD-1 therapy. Whereas anti–CTLA-4/PD-1 expanded progenitor exhausted CD8+ T-cell subsets, anti-VISTA promoted costimulatory genes and reduced regulators of T-cell quiescence. Notably, this is the first report of a checkpoint regulator impacting CD8+ T-cell quiescence, and the first indication that quiescence may be a target in the context of T-cell exhaustion and in cancer. This study builds a foundation for all future studies on the role of anti-VISTA in the development of antitumor immunity and provides important mechanistic insights that strongly support use of anti-VISTA to overcome the adaptive resistance seen in contemporary treatments involving PD-1 and/or CTLA-4.See related Spotlight by Wei, p. 3

Supplementary Figure S4. Additional data supporting Figure 6. In the I/V model, BALB/c mice were administered 100k CT26 cells and treated when tumors reached a size of 40mm3 with isotype (I) or anti-VISTA (V). In the CPV model, BALB/c mice were administered 100k CT26 cells and treated with anti-CTLA-4 and anti-PD-1 plus isotype (CP) or plus anti-VISTA (CPV) when tumors reached a size of 600mm3. CD45 + (A-C, E) or CD8+ AH1-tetramer+ (D) cells were isolated from the TME and analyzed by scRNAseq. (A) Stacked bar graph showing all lymphoid cluster frequencies as a proportion of the lymphoid infiltrate for I/V and CP/CPV. (B) Stacked bar graph showing NK cluster frequencies as a proportion of the NK infiltrate for I/V and CP/CPV. (C) Stacked bar graph showing CD8+ T cell cluster frequencies as a proportion of the CD8+ T cell infiltrate for I/V and CP/CPV. (D) Mean cluster frequencies as a proportion of CD8+ AH1-tetramer+ T cells in I/V or CP/CPV treatment. (E) Violin plots showing enrichment of a quiescence gene signature in CD8+ T cell clusters in the CD45 + sorted I/V dataset[58]. *P < 0.05, **P < 0.01, ***P < 0.001.

Methods for Real-Time Quantitative RT-PCR, Assessment of medal accuracy, Gene expression, copy number and survival analysis

Molecular networks governing responses to targeted therapies in cancer cells are complex dynamic systems that demonstrate nonintuitive behaviors. We applied a novel computational strategy to infer probabilistic causal relationships between network components based on gene expression. We constructed a model comprised of an ensemble of networks using multidimensional data from cell line models of cell-cycle arrest caused by inhibition of MEK1/2. Through simulation of a reverse-engineered Bayesian network model, we generated predictions of G1–S transition. The model identified known components of the cell-cycle machinery, such as CCND1, CCNE2, and CDC25A, as well as revealed novel regulators of G1–S transition, IER2, TRIB1, TRIM27. Experimental validation of model predictions confirmed 10 of 12 predicted genes to have a role in G1–S progression. Further analysis showed that TRIB1 regulated the cyclin D1 promoter via NFκB and AP-1 sites and sensitized cells to TRAIL-induced apoptosis. In clinical specimens of breast cancer, TRIB1 levels correlated with expression of NFκB and its target genes (IL8, CSF2), and TRIB1 copy number and expression were predictive of clinical outcome. Together, our results establish a critical role of TRIB1 in cell cycle and survival that is mediated via the modulation of NFκB signaling. Cancer Res; 77(7); 1575–85. ©2017 AACR.

Supplemental Figure Legends

Legend Figure S1: Schematic of the experimental framework Legend Figure S2: Predicted phenotypic value and assessment of predictive power of the model. Legend Figure S3: Taget knock-down. Legend Figure S4: Treatment with individual siRNAs against TRIB1 leads to G1-S arrest. Legend Figure S5: RNA levels of cell cycle genes. Legend Figure S6: TRIB1 knockdown results in inhibition of NFκB-responsive promoter activity: comparison of individual siRNA duplexes. Legend Figure S7: TRIB1 resistant to siRNA (TRIB1-siR) rescues the phenotype induced by TRIB1-siRNA. Legend Figure S8: Downregulation of NFkB target genes after treatment with TRIB1 siRNA. Legend Figure S9: TRIB1 knock down leads to inhibition of NFκB pathway. Legend Figure S10: TRIB1 siRNA promotes TRAIL-induced apoptosis through activation of caspase-8 cleavage. Legend Figure S11: TRIB1 knockdown results in decrease of YY1gene expression. Legend Figure S12: Kaplan-Meier plots Legend Figure S13: Hazard Ratios for BCCS distribution. Legend Figure S14: Hazard Ratios for OS distribution.

Introduction Wagholi, a historical village, that prospered in the Maratha period is situated 16 km east of Pune city. It is surrounded by a stone rampart with two entrances. The traces of which are still visible. This is the village of Pilajirao Jadhav, one of the eminent warriors who played an important role in the spread of the Maratha Empire. He constructed many buildings in and around this village. Since the beginning, Wagholi was facing severe scarcity of drinking water. Thus, Pilajirao Jadhav dug two large lakes in the village premises.1 Also, there is a beautiful barav (step-well) along with an inscription near the northern entrance. In the centre of the village, the remains of the palace are still visible. Besides, probably he built beautiful temples of Hanuman, Shiva and Ram apart from Wagheshwar in this village. Another striking feature of Wagholi is the very important and rare blue metallic stone (Cavansite) found in the quarries here. All over the world, this metal is found in large quantities only in Wagholi.2

Insect herbivory can seriously hinder plant performance and reduce crop yield. Thrips are minute cell-content-feeding insects that are important vectors of viral plant pathogens, and are serious crop pests. We investigated the role of a sweet pepper (Capsicum annuum) lipoxygenase gene, CaLOX2, in the defense of pepper plants against Western flower thrips (Frankliniella occidentalis). This was done through a combination of in-silico, transcriptional, behavioral and chemical analyses. Our data show that CaLOX2 is involved in jasmonic acid (JA) biosynthesis and mediates plant resistance. Expression of the JA-related marker genes, CaLOX2 and CaPIN II, was induced by thrips feeding. Silencing of CaLOX2 in pepper plants through virus-induced gene silencing (VIGS) resulted in low levels of CaLOX2 transcripts, as well as significant reduction in the accumulation of JA, and its derivatives, upon thrips feeding compared to control plants. CaLOX2-silenced pepper plants exhibited enhanced susceptibility to thrips. This indicates that CaLOX2 mediates JA-dependent signaling, resulting in defense against thrips. Furthermore, exogenous application of JA to pepper plants increased plant resistance to thrips, constrained thrips population development and made plants less attractive to thrips. Thus, a multidisciplinary approach shows that an intact lipoxygenase pathway mediates various components of sweet pepper defense against F. occidentalis.

Background AMV564 is a potent bispecific T cell engager that binds CD3 and CD33. Due to its bivalent structure, AMV564 is selective for MDSCs via clustered CD33 expressed on the cell surface both in vitro and in patients. MDSCs are responsible for local and systemic suppression of the immune response to both circulating and solid cancers. Targeting MDSC suppression allows T cell priming to be restored in both the lymph nodes and tumor microenvironment, and expands previously activated tumor-specific T cells. Here we report clinical observations and results of our ex vivo assay development. Methods Cell lines, primary human cells, and patient samples were analyzed using flow cytometry with appropriate marker panels including AMV564 directly labeled (phycoerythrin) or detected with labeled anti-AMV564 antibodies. T cell cytotoxicity assays were conducted using primary human T cells and leukemic blast or other target cells (3:1 ratio) for 48 or 72 hours. Patient peripheral blood was sequenced for TcRbeta CDR3 variable chain on the hsTCRBv4b. Results AMV564 is currently under investigation in a Phase 1 clinical trial (NCT04128423). There have been no dose-limiting toxicities and clinical activity has been observed (RECIST complete response in an ovarian cancer patient) when dosed once daily as a subcutaneous injection. In patients, T cell redistribution is consistent with activation and depletion of both monocytic and granulocytic MDSCs. Immune profile changes consistent with CD8 and Th1 cell activation are observed (figure 1). Furthermore, TCR sequencing data indicate that one cycle of treatment is sufficient to expand and generate de novo clones (figure 2). We developed a primary cell cytotoxicity assay and observe that cytotoxic potency is target dependent. Target cell killing and T cell activation/proliferation depend on CD33 clustering, and both CD4 and CD8 T cells can engage and kill target cells. This is illustrated in assays with KG-1 (M2, clustered) and KG-1a (M0, not clustered) cell lines, in which the KG-1 cells have an EC50 15–20 fold lower than the M0 cell line (figure 3). In addition, there is little to no detectable binding or killing of monocytes or neutrophils, which is consistent with the absence of neutropenia in patients enrolled in the trial to date. Conclusions AMV564 is a potent conditional T cell agonist which is clinically active. We demonstrate that the combination of T cell activation, increased T cell diversity, and target specificity allow AMV564 to deplete MDSCs and restore a native immune response to cancer. Ethics Approval This study was approved by the Institutional Review Board (IRB) or Independent Ethics Committee (IEC) at each participating institution.

The influence of electromagnetic fields on bacterial denitrification has been tested on synthetic media with sludges from wastewater treatment stations, in batch mode. The effects of the intensity of the magnetic induction ratio B (mT), reaction volume and initial biomass concentration on the kinetics of the denitrification process were studied. Magnetic field had both an optimal stimulating effect on the activity of the denitrifying flora for B (mT)/mgx values of the order of 0.212, and an inhibitory effect for the values beyond the latter.Sludges underwent multiple exposure cycles to magnetic fields. It was shown that, after three exposure cycles, denitrification kinetics went from 6.5 to 12.7 mg N–NO-3.L−1.h−1 which corresponds to a 2.7 fold improvement. The improved performance persists even after the cessation of the magnetic field. Observation of the sludge by the environmentalelectron microscope shows that the microbial population forming the starting sludge; changed following exposure to the magnetic field. The action of the; electromagnetic field on the microbial populations in denitrification resulted in the modification of the diversity of the flora that is initially present, favoring the development of Proteo bacteria, particularly the Betaproteo bacteria subclass, which results in improved denitrification.

Key message Lipoxygenases mediate important biological processes. Through comparative genomics, domain-scan analysis, sequence analysis, phylogenetic analysis, homology modelling and transcriptional analysis the lipoxygenase gene family of pepper (Capsicum annuum) has been identified. Abstract Lipoxygenases (LOXs) are non-heme, iron-containing dioxygenases playing a pivotal role in diverse biological processes in plants, including defence and development. Here, we exploited the recent sequencing of the pepper genome to investigate the LOX gene family in pepper. Two LOX classes are recognized, the 9- and 13-LOXs that oxygenate lipids at the 9th and 13th carbon atom, respectively. Using two main in-silico approaches, we identified a total of eight LOXs in pepper. Phylogenetic analysis classified four LOXs (CaLOX1, CaLOX3, CaLOX4 and CaLOX5) as 9-LOXs and four (CaLOX2, CaLOX6, CaLOX7 and CaLOX8) as 13-LOXs. Furthermore, sequence similarity/identity and subcellular localization analysis strengthen the classification predicted by phylogenetic analysis. Pivotal amino acids together with all domains and motifs are highly conserved in all pepper LOXs. Expression of 13-LOXs appeared to be more dynamic compared to 9-LOXs both in response to exogenous JA application and to thrips feeding. Bioinformatic and expression analyses predict the putative functions of two 13-LOXs, CaLOX6 and CaLOX7, in the biosynthesis of Green Leaf Volatiles, involved in indirect defence. The data are discussed in the context of LOX families in solanaceous plants and plants of other families. Electronic supplementary material The online version of this article (10.1007/s11103-018-0785-y) contains supplementary material, which is available to authorized users.

The concentrations of Te, As, Bi, Sb and Se (TABS+) in magmas from large igneous provinces (LIPs) are of interest because these elements are important in the formation of platinum-group minerals (PGM) found in magmatic Ni Cu platinum-group element (PGE) deposits. Furthermore, the TABS+ are volatile and hence they may be lost in degassing and have a role to play in the mass extinctions associated with LIPs events because these are mostly toxic elements. However, the concentrations of TABS+ in magmas from LIPs are not well documented due in part to the analytical difficulties and in part to the numerous processes that affect their distribution. We have determined the concentration of TABS+ and PGE in rocks from the Parana Magmatic Province (PMP) in order to assess the effects of fractional crystallization, sulfide segregation and magma degassing. Decrease in the Rh, Ru and Ir concentrations with Mg and Cr indicate that these elements behaved as compatible elements during crystal fractionation. Based on changes in the Cu/Pd sulfide saturation occurred in some cases resulting into a decrease in Pd and Pt. Arsenic and Sb behave as incompatible elements, whereas Se, Te and Bi show variable behaviour. Most lavas from the PMP display negative As, Se and Te anomalies on mantle-normalized patters coupled with Te/Cu, Se/Cu and As/Th and Sb/Th ratios lower than mantle values, which we attribute to loss of the TABS+ during degassing.

The biomedical field still requires composite materials for medical devices and tissue engineering model design. As part of the pursuit of non-animal and non-proteic scaffolds, we propose here a cellulose-based material. In this study, 9%, 18% and 36% dialdehyde-functionalized microcrystalline celluloses (DAC) were synthesized by sodium periodate oxidation. The latter was subsequently coupled to PVA at ratios 1:2, 1:1 and 2:1 by dissolving in N-methyl pyrrolidone and lithium chloride. Moulding and successive rehydration in ethanol and water baths formed soft hydrogels. While oxidation effectiveness was confirmed by dialdehyde content determination for all DAC, we observed increasing hydrolysis associated with particle fragmentation. Imaging, FTIR and XDR analysis highlighted an intertwined DAC/PVA network mainly supported by electrostatic interactions, hemiacetal and acetal linkage. To meet tissue engineering requirements, an interconnected porosity was optimized using 0–50 µm salts. While the role of DAC in strengthening the hydrogel was identified, the oxidation ratio of DAC showed no distinct trend. DAC 9% material exhibited the highest indirect and direct cytocompatibility creating spheroid-like structures. DAC/PVA hydrogels showed physical stability and acceptability in vivo that led us to propose our DAC 9%/PVA based material for soft tissue graft application.

Myeloid derived suppressor cells (MDSC) are elevated in tumors and draining lymph nodes of cancer patients and produce a variety of immune suppressive factors that impair T cell activation and anti-tumor immunity. MDSC are also recruited from the bone marrow in response to T cell activation, including in response to immunotherapies that activate T cells. As MDSC and immune suppressive monocytes are significant producers of IL6, IL1β and TNFα, their elevation and activation in response to T cell activation can promote pathologic levels of these cytokines leading to potentially serious conditions such as cytokine release syndrome. Furthermore, patients deficient in functional antigen-specific T cells have elevated MDSC, consistent with their described suppression of antigen presentation and T cell activation. Therapeutic agents that deplete MDSC may offer both an improved therapeutic index for maintenance of T cell activation while promoting expansion of anti-tumor T cells. AMV564, a bivalent T cell engager that selectively depletes MDSC, is in clinical development in solid tumor patients. The cytokine profiles of patients treated with AMV564 as monotherapy or in combination with pembrolizumab were assessed using multiplex immunoassays, and the peripheral T cell repertoire was assessed by deep sequencing of the TCRβ locus. While CD33 has no apparent functional role on differentiated myeloid cells, on MDSC and immature monocytes, CD33 signaling promotes the production of immune-suppressive factors. Avid binding of clustered CD33 in this active signaling configuration drives the selectivity of AMV564 for these cell types. Analysis of patient samples demonstrates that AMV564 selectively depletes MDSC and promotes T cell activation and increases in effector CD8 and CD4 populations1. AMV564 promotes strong induction of IFNγ and other proinflammatory cytokines and chemokines, but with comparatively limited induction of IL6, IL1β and TNFα, consistent with the role of MDSC in producing these factors. This cytokine profile was maintained in combination with pembrolizumab. Dynamic changes in the peripheral T cell repertoire were apparent for patients treated with AMV564 monotherapy or in combination with pembrolizumab. Observations include significant expansion after one cycle of therapy (p = 0.008) including T cell clones not detectable at baseline, changes in diversity that reflect increases in middle-frequency clones, and oscillation in the peripheral T cell fraction consistent with T cell trafficking to tissues and tumors. Repertoire expansion correlated with increases in effector memory CD8 T cells. These findings in patients treated with AMV564 support the role of MDSC in both limiting the priming and activation of anti-tumor T cells as well as contributing to an immune suppressive and pathologic cytokine response to T cell activation. 1. Smith, V et. al. Cancer Res. 2020;80(16):5699 Citation Format: Sterling Eckard, Aurelien Sarde, Li Mei, Curtis Ruegg, Patrick Chun, Victoria Smith. MDSC suppress the T cell repertoire and contribute to a pathologic cytokine milieu in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 528.