Whole-genome Microarray Research Articles

Phenolic Compounds Reduce the Fat Content in Caenorhabditis elegans by Affecting Lipogenesis, Lipolysis, and Different Stress Responses.

Supplementation with bioactive compounds capable of regulating energy homeostasis is a promising strategy to manage obesity. Here, we have screened the ability of different phenolic compounds (myricetin, kaempferol, naringin, hesperidin, apigenin, luteolin, resveratrol, curcumin, and epicatechin) and phenolic acids (p-coumaric, ellagic, ferulic, gallic, and vanillic acids) regulating C. elegans fat accumulation. Resveratrol exhibited the strongest lipid-reducing activity, which was accompanied by the improvement of lifespan, oxidative stress, and aging, without affecting worm development. Whole-genome expression microarrays demonstrated that resveratrol affected fat mobilization, fatty acid metabolism, and unfolded protein response of the endoplasmic reticulum (UPRER), mimicking the response to calorie restriction. Apigenin induced the oxidative stress response and lipid mobilization, while vanillic acid affected the unfolded-protein response in ER. In summary, our data demonstrates that phenolic compounds exert a lipid-reducing activity in C. elegans through different biological processes and signaling pathways, including those related with lipid mobilization and fatty acid metabolism, oxidative stress, aging, and UPR-ER response. These findings open the door to the possibility of combining them in order to achieve complementary activity against obesity-related disorders.

KRAS K104 modification affects the KRASG12D-GEF interaction and mediates cell growth and motility

Mutant RAS genes play an important role in regulating tumors through lysine residue 104 to impair GEF-induced nucleotide exchange, but the regulatory role of KRAS K104 modification on the KRASG12D mutant remains unclear. Therefore, we simulated the acetylation site on the KRASG12D three-dimensional protein structure, including KRASG12D, KRASG12D/K104A and KRASG12D/K104Q, and determined their trajectories and binding free energy with GEF. KRASG12D/K104Q induced structural changes in the α2- and α3-helices, promoted KRAS instability and hampered GEF binding (ΔΔG = 6.14 kJ/mol). We found decreased binding to the Raf1 RBD by KRASG12D/K104Q and reduced cell growth, invasion and migration. Based on whole-genome cDNA microarray analysis, KRASG12D/K104Q decreased expression of NPIPA2, DUSP1 and IL6 in lung and ovarian cancer cells. This study reports computational and experimental analyses of Lys104 of KRASG12D and GEF, and the findings provide a target for exploration for future treatment.

The efficacy of chemotherapeutic drug combinations may be predicted by concordance of gene response to the single agents.

Determining the expression of genes in response to different classes of chemotherapeutic drugs may allow for a better understanding as to which may be used effectively in combination. In the present study, the human colorectal cancer cell line HCT116 was cultured with equi-active concentrations of a series of anti-cancer agents. Gene expression profiles were then measured by whole-genome microarray. Although each drug induced a unique signature of gene expression in tumour cells, there were marked similarities between certain drugs, even in those from different classes. For example, the antimalarial agent artesunate and the platinum-containing alkylating agent, oxaliplatin, produced a very similar mRNA expression pattern in HCT116 cells with ~14,000 genes being affected by the two drugs in the same way. Furthermore, the overall correlation of gene responses between two agents could predict whether their use in combination would lead to a greater or lesser effect on cell number, determined experimentally, than predicted by single agent experiments. The results indicated that even when working through different mechanisms, combining drugs that initiate a similar transcriptional response may constitute the best option for determining drug-combination strategies for the treatment of cancer.

Integrin α9 is involved in the pathopoiesis of acute aortic dissection via mediating phenotype switch of vascular smooth muscle cell

Acute aortic dissection (AAD) is a devastating disease with high mortality; however, the pathogenic mechanisms of AAD remain poorly understood. Our present study aimed to identify genes associated with AAD and explore the molecular function of candidate genes in the pathogenesis of AAD. We used a whole-genome transcriptional microarray to identify putative AAD genes using ascending aortic tissues from four patients with AAD and four healthy organ donors. The differentially expressed genes were further validated in eight patients with AAD and eight healthy organ donors. Functional assessments were conducted to analyze the effects of the identified AAD genes on the phenotype of aortic vascular smooth muscle cells (VSMCs). The whole-genome transcriptional microarray analysis found 129 dysregulated genes in the ascending aortic tissues of AAD (fold change≥2), which were mainly associated with the focal adhesion pathway and actin cytoskeleton regulation pathway. Among these genes, integrin α9 (ITGA9) was identified to be involved in both pathways and downregulated by 50% in AAD patients. The association of ITGA9 with AAD was confirmed by Western blotting analysis (P = 0.003). Functional studies showed that knocking down ITGA9 in VSMCs resulted in a decrease in contractile markers (SM22α and α-SMA) and an increase in synthetic markers (OPN and SMemb), suggesting that the VSMCs switched from a contractile to a synthetic phenotype. After overexpression of ITGA9 by a recombinant adenovirus vector in VSMCs, SM22α and α-SMA were upregulated, while SMemb was downregulated, indicating a phenotypic switch from the synthetic to contractile phenotype of VSMCs. In conclusion, our study identified ITGA9 as a novel AAD gene. This gene is downregulated in patients with AAD and is involved in the regulation of the phenotypic switch of VSMCs from a contractile to a synthetic phenotype.

Safety Assessments of Nickel Boride Nanoparticles on the Human Pulmonary Alveolar Cells by Using Cell Viability and Gene Expression Analyses.

Nickel boride is generally used in the steel industry as a melting accelerator due to its feature of creating a protective and stable attribute at high temperatures. It is also used to improve the hardenability of the steel with boron addition in the production. Thus, safety studies and biocompatibility analysis of nickel boride should be performed comprehensively to understand the limitations of use in various areas. In the present study, nickel boride nanoparticles (Ni2B NPs) were synthesized by a single-step method and molecule characterizations were performed via the use of X-ray diffraction analysis (XRD), transmission electron microscopy (TEM), scanning electron microscopy (SEM), and energy dispersive X-ray (EDX) analyses. Cytotoxicity properties of Ni2B NPs were identified on human pulmonary alveolar epithelial cells (HPAEpiC) by using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), neutral red (NR), and lactate dehydrogenase (LDH) assays. Illumina human ht-12 v4.0 whole-genome microarray analysis was conducted to investigate NiB2 NPs effects on gene expression regulations of HPAEpiC cells. The database for annotation, visualization, and integrated discovery (DAVID) analysis was performed to reveal the relationship between Ni2B NP application and cellular pathway alterations. According to cytotoxicity analysis, the IC50 value for Ni2B NP application was found as 81.99mg/L concentration. Microarray analysis of Ni2B NP application was shown for the first time that 693 gene expression changes (FC ≥ 2) occurred significantly over 40.000 gene probes and Ni2B NPs were observed to affect microtubule regulation, centrosome organization, and phosphoprotein synthesis.

Gamma Knife Radiosurgery does not alter the copy number aberration profile in sporadic vestibular schwannoma

IntroductionIonizing radiation is a known etiologic factor in tumorigenesis and its role in inducing malignancy in the treatment of vestibular schwannoma has been debated. The purpose of this study was to identify a copy number aberration (CNA) profile or specific CNAs associated with radiation exposure which could either implicate an increased risk of malignancy or elucidate a mechanism of treatment resistance.Methods55 sporadic VS, including 18 treated with Gamma Knife Radiosurgery (GKRS), were subjected to DNA whole-genome microarray and/or whole-exome sequencing. CNAs were called and statistical tests were performed to identify any association with radiation exposure. Hierarchical clustering was used to identify CNA profiles associated with radiation exposure.ResultsA median of 7 (0–58) CNAs were identified across the 55 VS. Chromosome 22 aberration was the only recurrent event. A median aberrant cell fraction of 0.59 (0.25–0.94) was observed, indicating several genetic clones in VS. No CNA or CNA profile was associated with GKRS.ConclusionGKRS is not associated with an increase in CNAs or alteration of the CNA profile in VS, lending support to its low risk. This also implies that there is no major issue with GKRS treatment failure being due to CNAs. In agreement with previous studies, chromosome 22 aberration is the only recurrent CNA. VS consist of several genetic clones, addressing the need for further studies on the composition of cells in this tumor.

Abstract 3377: NSCLC subtype-associated molecular and cellular alterations in the normal-appearing bronchial epithelium

Abstract Rationale: We have previously identified lung cancer-associated gene expression alterations in normal bronchial airway epithelial cells of ever smokers with and without lung cancer. It is unclear, however, whether or not gene expression in the bronchial airway also reflects non-small cell lung cancer (NSCLC) subtypes. In this study, we sought to identify airway gene expression differences between patients with squamous (LUSC) and adenocarcinoma (LUAD) NSCLC and determine if these reflect changes in cell type composition or cell type-specific expression. Methods: Bronchial brushings from ever smokers with NSCLC from the AEGIS I (153 LUSC; 148 LUAD; 154 benign) & II (82 LUSC; 102 LUAD; 74benign) trials were profiled by whole-genome microarrays. The NSCLC-associated gene expression alterations were identified using a linear model adjusting for smoking status in AEGIS I and confirmed in AEGIS II via GSEA. Enriched biological pathways were identified using EnrichR. In parallel, bronchial airway brushings collected from ever smokers undergoing bronchoscopy for suspect lung cancer (17 subjects) were profiled via scRNA-seq using the CEL-Seq2 protocol (3264 cells). Seurat was used to correct for batch effects and identify distinct cell populations. Genes differentially expressed between cell populations using a negative binomial model were clustered into gene sets using WGCNA. Per patient metagene scores were computed across the datasets for each gene set, and differences in these scores between NSCLC subtypes were evaluated using a linear model adjusting for smoking status. Results: 451 differentially expressed genes were found between LUSC v LUAD patients (FDR < 0.05). Genes involved in ciliary function were enriched among genes up-regulated in LUAD whereas keratinization and cancer-associated pathway genes were enriched among LUSC up-regulated genes (FDR < 0.05). In the scRNA-seq data, 15 populations of cells were identified that expressed known epithelial or immune cell type markers. Using WGCNA to cluster genes that distinguish the cell populations (5148 genes, FDR < 0.05) identified 12 gene sets (GS). In AEGIS I, LUSC patients had higher expression of a mitochondrial GS expressed highly in basal cells, a secretory-cell GS, and lower expression of a ionocyte-specific GS than LUAD patients (p = 0.041, 0.0043 and 2.06e-06 respectively). Both LUAD and LUSC patients have lower expression levels of the immune GSs than the benign patients (p = 0.00034 and 0.0008). Concordant results were found in the AEGIS II samples with the same methods. Conclusion: We leveraged bulk and single cell gene expression data and identified molecular differences in the airway field of injury among ever smokers with LUSC versus LUAD that are highly expressed specific cell populations. Our results suggest that there are different responses to injury in the normal-appearing airway that are associated with NSCLC subtype. Citation Format: Xingyi Shi, Jiarui Zhang, Steven Callori, Joshua Campbell, Denise Fine, Yaron Gesthalter, Gang Liu, Xiaohui Xiao, Avrum Spira, Marc Lenburg, Jennifer Beane. NSCLC subtype-associated molecular and cellular alterations in the normal-appearing bronchial epithelium [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3377.

Abstract 985: SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of early-stage myeloid-derived suppressor cells via the miR-155/C/EBPβ/Wnt axis

Abstract Early-stage myeloid-derived suppressor cells (eMDSCs) are a newly defined subset of myeloid-derived suppressor cells (MDSCs) that accumulate densely in tumors and potently promote tumor growth and metastasis by suppressing antitumor immune responses in vitro and in vivo. We previously identified a subset of eMDSCs in human breast cancer with a characteristic phenotype of Lin-HLA-DR-CD33+. We also found that SOCS3 deficiency and sustained activation of the JAK/STAT signaling pathway are critical molecular events coordinating the differentiation of eMDSCs, although the distinct molecular regulation has not been fully elucidated. Herein, we genetically constructed conditional SOCS3 knockout mice with SOCS3 deficiency specifically in the myeloid linage (SOCS3MyeKO). We delineated that the number of eMDSCs in SOCS3MyeKO mice (eMDSCsSOCS3KO) with the murine GMP phenotype increased significantly, and these cells suffered from a differentiation block in the myeloid lineage. To explore which pathways participated in dysfunctional eMDSC differentiation, we performed whole-genome RNA sequencing and miRNA microarray on CD11b+Gr-1+ cells, eMDSCsfl/fl and eMDSCsSOCS3KO to screen the potential regulatory ceRNA network in eMDSCsSOCS3KO. CD11b+Gr-1+ cells isolated from SOCS3fl/fl mouse spleens were used as mature myeloid cell controls. The transcriptome results and corresponding intervention experiment revealed that the differentiation block in eMDSCsSOCS3KO was caused by SOCS3 deficiency-mediated limited autophagy activation in an AMPK-independent manner. The results of miRNA microarray and RNA sequencing demonstrated that miR-155 overexpression and Wnt/β-catenin pathway activation were involved in the SOCS3 knockout-mediated myeloid differentiation block and autophagy repression. Further experiments revealed that miR-155 was induced by activation of the STAT3/NK-κB pathway upon SOCS3 deficiency, which consequently activated the Wnt/β-catenin pathway via targeting C/EBPβ. Furthermore, applying a specific miR-155 antagonist or the autophagy agonist rapamycin efficiently suppressed tumor growth and eMDSC infiltration in vivo. Overall, these findings indicated that SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of e-MDSCs via the miR-155/C/EBPβ/Wnt axis, and thus targeted therapy against this pathway could be a potential therapeutic target in breast cancer. Citation Format: Wenwen Zhang, Mengmeng Jiang, Rui Zhang, Pengpeng Liu, Yingnan Ye, Xiubao Ren, Wenwen Yu, Jinpu Yu. SOCS3 deficiency blocked autophagy-dependent myeloid differentiation of early-stage myeloid-derived suppressor cells via the miR-155/C/EBPβ/Wnt axis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 985.

Expression profiling and pathway analysis of iron oxide nanoparticles toxicity on human lung alveolar epithelial cell line using microarray analysis

Toxicogenomics is a developing area searching for cellular pathways and mechanisms including cancer, immunological diseases, environmental responses, gene-gene interactions and drug toxicity. Nanoparticles (NPs) become important candidates for analyzing in toxicogenomic experiments because of their unusual properties in various biological activities. Therefore, we examined the nanotoxicity of iron oxide (Fe2O3) on gene expression profiling of human alveolar epithelial cells (HPAEpiC) in the study. For this aim, iron oxide nanoparticles were synthesized by zone melting method and characterized via using X-ray crystallography (XRD) and transmission electron microscope (TEM) techniques. Cell viability and cytotoxicity were determined by 3-(4,5-dimethyl-thiazol-2-yl) 2,5-diphenyltetrazolium bromide (MTT), neutral red (NR) and lactate dehydrogenase (LDH) release tests. Whole-genome microarray expression analysis was performed to explore the effects of iron oxide nanoparticles on gene expression in cultured human alveolar epithelial cells. For further analyses, these genes were functionally classified by using DAVID (The Database for Annotation, Visualization and Integrated Discovery) with gene ontology (GO) analysis. The results from this study indicated that iron oxide-mediated toxicity directly or indirectly affecting the regulation of cell proliferation, response to hormone stimulus, estrogen stimulus, cytokine activity and blood circulation by stimulating diverse genes.

Copy number evolution with weighted aberrations in cancer.

MotivationCopy number aberrations (CNAs), which delete or amplify large contiguous segments of the genome, are a common type of somatic mutation in cancer. Copy number profiles, representing the number of copies of each region of a genome, are readily obtained from whole-genome sequencing or microarrays. However, modeling copy number evolution is a substantial challenge, because different CNAs may overlap with one another on the genome. A recent popular model for copy number evolution is the copy number distance (CND), defined as the length of a shortest sequence of deletions and amplifications of contiguous segments that transforms one profile into the other. In the CND, all events contribute equally; however, it is well known that rates of CNAs vary by length, genomic position and type (amplification versus deletion).ResultsWe introduce a weighted CND that allows events to have varying weights, or probabilities, based on their length, position and type. We derive an efficient algorithm to compute the weighted CND as well as the associated transformation. This algorithm is based on the observation that the constraint matrix of the underlying optimization problem is totally unimodular. We show that the weighted CND improves phylogenetic reconstruction on simulated data where CNAs occur with varying probabilities, aids in the derivation of phylogenies from ultra-low-coverage single-cell DNA sequencing data and helps estimate CNA rates in a large pan-cancer dataset.Availability and implementationCode is available at https://github.com/raphael-group/WCND.Supplementary information Supplementary data are available at Bioinformatics online.

DNA methylation status of imprinted H19 and KvDMR1 genes in human placentas after conception using assisted reproductive technology.

Assisted reproductive technologies (ARTs), such as in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI), are thought to destabilize genomic imprints. Previous studies examining the association between ART and aberrant DNA methylation have been inconclusive. The DNA methylation status of H19 and KvDMR1was compared between newborns conceived through ART and those conceived naturally to evaluate the safety of ART. Placental tissues from 6 full-term, naturally conceived pregnancies (no gestational comorbidities) and six full-term ART pregnancies (no gestational complication) were collected. Genomic DNA (gDNA) and RNA were extracted from both groups. Real-time PCR was used to analyze the mRNA expression levels of H19 and KvDMR1 in the placenta for both groups. A whole-genome DNA methylation microarray was used to examine three placentas from full-term, naturally conceived pregnancies and three placentas from full-term IVF pregnancies. The expression level of H19 in the IVF group was significantly higher than that in the natural pregnancy group, whereas the expression level of KvDMR1 was significantly lower in the ART group than in the natural pregnancy group. Also, human ART manipulation resulted in placental gDNA methylation modifications. Conclusion: Abnormal methylation patterns were detected in phenotypically normal phenotype conceived by ART, which may occur due to imprinting errors in sperm/oocyte cells or side effects of in vitro embryo culture procedures. Further investigation is necessary to determine whether imprinted gene expression and DNA methylation can be regulated through other mechanisms. Assisted reproductive technology (ART); placenta; methylation; H19; KvDMR1.

MTOR up-regulation of SNRPA1 contributes to hepatocellular carcinoma development.

Hepatocellular carcinoma (HCC) is the second leading cause of cancer-related death worldwide. Recent studies showed that snRNPs were implicated in human cancer development. The role of SNRPA1, which is a member of U2 snRNPs, in HCC, remains undocumented. Here, we found that SNRPA1 was highly expressed in HCC tissue compared with normal adjacent liver tissues. Up-regulation of SNRPA1 was correlated with the clinical stage of HCC and the overall survival of HCC patients. In vitro and in vivo results showed that knockdown of SNPRA1 inhibited the cell proliferation, colony formation and xenografted tumorigenesis of HCC cells. Apoptosis was induced by SNPRA1 down-regulation. Mechanistically, SNPRA1 was stimulated by mTOR activation. In addition, whole-genome microarray analysis identified that 262 genes were up-regulated and 462 genes were down-regulated by SNPRA1 knockdown in HCC cells. qPCR analysis suggested that the fibroblast growth factor-2 (FGF2), Alpha-fetoprotein (AFP), β-catenin, Ki-67 and cyclin B1 were down-regulated and caspase 3, p53 as well as p21 were up-regulated after SNRPA1 knockdown. Taken together, our findings implicate that SNPRA1 functions as an oncogene in HCC.

Oxaliplatin-induced neuropathic pain involves HOXA6 via a TET1-dependent demethylation of the SOX10 promoter.

Chemotherapy-induced neuropathic pain is a common dose-limiting side effect of cancer treatment but the underlying mechanisms are largely unknown. Here, we used a whole-genome expression microarray and gene ontology analysis to identify the upregulation of a sequence-specific DNA-binding protein, HOXA6, in the spinal dorsal horn on Day 10 after injection of rats with oxaliplatin. Genetic disruption of HOXA6 with siRNAs alleviated mechanical allodynia after oxaliplatin administration. Reduced representation bisulfite sequencing assays indicated that oxaliplatin decreased the methylation levels of the SOX10 promoter but not of HOXA6. TET1 was also upregulated by oxaliplatin. Genetic disruption of TET1 with siRNA blocked the promoter demethylation of SOX10 and the upregulation of HOXA6 and SOX10. Importantly, inhibition of SOX10 by intrathecal application of SOX10 siRNA ameliorated the mechanical allodynia induced by oxaliplatin and downregulated the expression of HOXA6. Consistently, overexpression of SOX10 through intraspinal injection of AAV-SOX10-EGFP produced mechanical allodynia and upregulated the expression of spinal dorsal horn HOXA6. Moreover, chromatin immunoprecipitation assays demonstrated that oxaliplatin increased the binding of SOX10 to the promoter region of HOXA6. Taken together, our data suggest that HOXA6 upregulation through the TET1-mediated promoter demethylation of SOX10 may contribute to oxaliplatin-induced neuropathic pain.

Pro-angiogenic Activity Discriminates Human Adipose-Derived Stromal Cells From Retinal Pericytes: Considerations for Cell-Based Therapy of Diabetic Retinopathy.

Diabetic retinopathy (DR) is a frequent diabetes-associated complication. Pericyte dropout can cause increased vascular permeability and contribute to vascular occlusion. Adipose-derived stromal cells (ASC) have been suggested to replace pericytes and restore microvascular support as potential therapy of DR. In models of DR, ASC not only generated a cytoprotective and reparative environment by the secretion of trophic factors but also engrafted and integrated into the retina in a pericyte-like fashion. The aim of this study was to compare the pro-angiogenic features of human ASC and human retinal microvascular pericytes (HRMVPC) in vitro. The proliferation and the expression of ASC and HRMVPC markers were compared. Adhesion to high glucose-conditioned endothelial extracellular matrix, mimicking the diabetic microenvironment, was measured. The angiogenesis-promoting features of both cell types and their conditioned media on human retinal endothelial cells (EC) were assessed. To identify a molecular basis for the observed differences, gene expression profiling was performed using whole-genome microarrays, and data were validated using PCR arrays and flow cytometry. Based on multiplex cytokine results, functional studies on selected growth factors were performed to assess their role in angiogenic support. Despite a distinct heterogeneity in ASC and HRMVPC cultures with an overlap of expressed markers, ASC differed functionally from HRMVPC. Most importantly, the pro-angiogenic activity was solely featured by ASC, whereas HRMVPC actively suppressed vascular network formation. HRMVPC, in contrast to ASC, showed impaired adhesion and proliferation on the high glucose-conditioned endothelial extracellular matrix. These data were supported by gene expression profiles with differentially expressed genes. The vessel-stabilizing factors were more highly expressed in HRMVPC, and the angiogenesis-promoting factors were more highly expressed in ASC. The vascular endothelial growth factor receptor-2 inhibition efficiently abolished the ASC angiogenic supportive capacities, whereas the addition of angiopoietin-1 and angiopoietin-2 did not alter these effects. Our results clearly show that ASC are pro-angiogenic, whereas HRMVPC are marked by anti-angiogenic/EC-stabilizing features. These data support ASC as pericyte replacement in DR but also suggest a careful risk-to-benefit analysis to take full advantage of the ASC therapeutic features.

The effect of maternal antibiotic use in sows on intestinal development in offspring.

The objective of this study is to investigate the effect of a maternal antibiotic administration during the last week of gestation on the early life intestinal development in neonatal piglets. Colonization of the gut with bacteria starts during birth and plays a major role in the intestinal and immunological development of the intestine. We demonstrate that maternal interventions induced changes in the sows (n = 6 to 8 per treatment) fecal microbiota diversity around birth (P < 0.001, day 1). Whole-genome microarray analysis in small intestinal samples of 1-d old piglets (n = 6 to 8 per treatment) showed significantly expressed genes (Padj < 0.05) which were involved in processes of tight junction formation and immunoglobulin production. Furthermore, when performing morphometry analysis, the number of goblet cells in jejunum was significantly (P < 0.001) lower in piglets from amoxicillin administered sows compared with the respective control piglets. Both significantly expressed genes (Padj < 0.05) and significant morphometry data (jejunum P < 0.05 and ileum P < 0.01) indicate that the crypts of piglets from amoxicillin administered sows deepen around weaning (day 26) as an effect of the amoxicillin administration in sows. The latter might imply that the intestinal development of piglets was delayed by maternal antibiotic administration. Taken together, these results show that maternally oral antibiotic administration changes in early life can affect intestinal development of the offspring piglets for a period of at least 5 wk after the maternal antibiotic administration was finished. These results show that modulation of the neonatal intestine is possible by maternal interventions.

Immune infiltration and angiogenesis as markers of outcome in the post-nephrectomy setting: Transcriptomic data from patients receiving placebo on a randomized phase III trial (PROTECT).

5082 Background: Defined stromal and immune features of the tumor microenvironment (TME) have proven relevant for outcomes with systemic therapy in advanced clear cell renal cell carcinoma (ccRCC). We hypothesized that these may matter beyond therapeutic applications and could be relevant much earlier in the disease course. We sought to study the TME in high risk ccRCC patients undergoing definitive surgery. Methods: Clinical, pathologic, immunohistochemical, and whole-genome microarray data were acquired on 236 out of 769 patients in the Placebo arm of PROTECT trial (NCT01235962 - pazopanib vs placebo). Transcriptomic scores assessing angiogenesis and myeloid infiltration with individual annotations above/below median were used to categorize patients into four groups (angiogenesis high vs. low; myeloid high vs. low). We tested categorical association with disease free (DFS) and overall survival (OS) using logrank testing and assessed interdependence with relevant clinicopathologic variables, including the UCLA Integrated Staging System (UISS) in a cox regression model. Results: Tumors from236 patients were available for analysis. Overall, 37% developed metastatic recurrence and 81% were alive at last follow up. On univariate analysis increasing tumor stage, higher UISS score, and angiogenesis/myeloid subgroups (high – H and low – L) were associated with worse DFS and OS (all p values &lt;0.05). On multivariate analysis TME subgroups remained significant for worse DFS and OS (Table). Conclusions: Microenvironmental subgroups stratified into angiogenic and myeloid expression profiles carry independent prognostic significance and should be further explored to guide future biomarker-directed adjuvant trials. Clinical trial information: NCT01235962 . [Table: see text]

Early Response to the Plant Toxin Stenodactylin in Acute Myeloid Leukemia Cells Involves Inflammatory and Apoptotic Signaling.

Stenodactylin, a highly toxic type 2 ribosome-inactivating protein purified from the caudex of Adenia stenodactyla Harms, is a potential anticancer drug candidate. Previous studies demonstrated that stenodactylin induces apoptosis and necroptosis in treated cells, involving the production of reactive oxygen species. We analyzed the effect of stenodactylin on Raji and Ramos (Human Burkitt’s lymphoma cells) and MOLM-13 (acute myeloid leukemia cells). Moreover, we focused on the early events in MOLM-13 cells that characterize the cellular response to the toxin by whole-genome microarray analysis of gene expression. Treatment with stenodactylin induced the depurination of 28S rRNA within 4 h and increased the phosphorylation of p38 and JNK. A time-dependent activation of caspase 1, 2, 8, 9, 3/7 was also observed. Genome-wide gene expression microarray analysis revealed early changes in the expression of genes involved in the regulation of cell death, inflammation and stress response. After 4 h, a significant increase of transcript level was detectable for ATF3, BTG2, DUSP1, EGR1, and JUN. Increased upstream JUN signaling was also confirmed at protein level. The early response to stenodactylin treatment involves inflammatory and apoptotic signaling compatible with the activation of multiple cell death pathways. Because of the above described properties toward acute myeloid leukemia cells, stenodactylin may be a promising candidate for the design of new immunoconjugates for experimental cancer treatment.

Hepatotoxic pyrrolizidine alkaloids induce DNA damage response in rat liver in a 28-day feeding study

Pyrrolizidine alkaloids (PA) are secondary plant metabolites that occur as food and feed contaminants. Acute and subacute PA poisoning can lead to severe liver damage in humans and animals, comprising liver pain, hepatomegaly and the development of ascites due to occlusion of the hepatic sinusoids (veno-occlusive disease). Chronic exposure to low levels of PA can induce liver cirrhosis and liver cancer. However, it is not well understood which transcriptional changes are induced by PA and whether all hepatotoxic PA, regardless of their structure, induce similar responses. Therefore, a 28-day subacute rat feeding study was performed with six structurally different PA heliotrine, echimidine, lasiocarpine, senecionine, senkirkine, and platyphylline, administered at not acutely toxic doses from 0.1 to 3.3 mg/kg body weight. This dose range is relevant for humans, since consumption of contaminated tea may result in doses of ~ 8 µg/kg in adults and cases of PA ingestion by contaminated food was reported for infants with doses up to 3 mg/kg body weight. ALT and AST were not increased in all treatment groups. Whole-genome microarray analyses revealed pronounced effects on gene expression in the high-dose treatment groups resulting in a set of 36 commonly regulated genes. However, platyphylline, the only 1,2-saturated and, therefore, presumably non-hepatotoxic PA, did not induce significant expression changes. Biological functions identified to be affected by high-dose treatments (3.3 mg/kg body weight) comprise cell-cycle regulation associated with DNA damage response. These functions were found to be affected by all analyzed 1,2-unsaturated PA.In conclusion, 1,2-unsaturated hepatotoxic PA induced cell cycle regulation processes associated with DNA damage response. Similar effects were observed for all hepatotoxic PA. Effects were observed in a dose range inducing no histopathological alterations and no increase in liver enzymes. Therefore, transcriptomics studies identified changes in expression of genes known to be involved in response to genotoxic compounds at PA doses relevant to humans under worst case exposure scenarios.

Identification of a Gene-Related Risk Signature in Melanoma Patients Using Bioinformatic Profiling.

Introduction Gene signature has been used to predict prognosis in melanoma patients. Meanwhile, the efficacy of immunotherapy was correlated with particular genes expression or mutation. In this study, we systematically explored the gene expression pattern in the melanoma-immune microenvironment and its relationship with prognosis. Methods A cohort of 122 melanoma cases with whole-genome microarray expression data were enrolled from the Gene Expression Omnibus (GEO) database. The findings were validated using The Cancer Genome Atlas (TCGA) database. A principal component analysis (PCA), gene set enrichment analysis (GSEA), and gene oncology (GO) analysis were performed to explore the bioinformatic implications. Results Different gene expression patterns were identified according to the clinical stage. All eligible gene sets were analyzed, and the 8 genes (GPR87, KIT, SH3GL3, PVRL1, ATP1B1, CDAN1, FAU, and TNFSF14) with the greatest prognostic impact on melanoma. A gene-related risk signature was developed to distinguish patients with a high or low risk of an unfavorable outcome, and this signature was validated using the TCGA database. Furthermore, the prognostic significance of the signature between the classified subgroups was verified as an independent prognostic predictor of melanoma. Additionally, the low-risk melanoma patients presented an enhanced immune phenotype compared to that of the high-risk gene signature patients. Conclusions The gene pattern differences in melanoma were profiled, and a gene signature that could independently predict melanoma patients with a high risk of poor survival was established, highlighting the relationship between prognosis and the local immune response.

Reliable Analysis of Clinical Tumor-Only Whole-Exome Sequencing Data.

PURPOSEAllele-specific copy number alteration (CNA) analysis is essential to study the functional impact of single-nucleotide variants (SNVs) and the process of tumorigenesis. However, controversy over whether it can be performed with sufficient accuracy in data without matched normal profiles and a lack of open-source implementations have limited its application in clinical research and diagnosis.METHODSWe benchmark allele-specific CNA analysis performance of whole-exome sequencing (WES) data against gold standard whole-genome SNP6 microarray data and against WES data sets with matched normal samples. We provide a workflow based on the open-source PureCN R/Bioconductor package in conjunction with widely used variant-calling and copy number segmentation algorithms for allele-specific CNA analysis from WES without matched normals. This workflow further classifies SNVs by somatic status and then uses this information to infer somatic mutational signatures and tumor mutational burden (TMB).RESULTSApplication of our workflow to tumor-only WES data produces tumor purity and ploidy estimates that are highly concordant with estimates from SNP6 microarray data and matched normal WES data. The presence of cancer type–specific somatic mutational signatures was inferred with high accuracy. We also demonstrate high concordance of TMB between our tumor-only workflow and matched normal pipelines.CONCLUSIONThe proposed workflow provides, to our knowledge, the only open-source option with demonstrated high accuracy for comprehensive allele-specific CNA analysis and SNV classification of tumor-only WES. An implementation of the workflow is available on the Terra Cloud platform of the Broad Institute (Cambridge, MA).