Gene Transcription Data Research Articles

Human-specific epigenomic states in spermatogenesis

Infertility is becoming increasingly common, affecting one in six people globally. Half of these cases can be attributed to male factors, many driven by abnormalities in the process of sperm development. Emerging evidence from genome-wide association studies, genetic screening of patient cohorts, and animal models highlights an important genetic contribution to spermatogenic defects, but comprehensive identification and characterization of the genes critical for male fertility remain lacking. High divergence of gene regulation in spermatogenic cells across species poses challenges for delineating the genetic pathways required for human spermatogenesis using common model organisms. In this study, we leveraged post-translational histone modification and gene transcription data for 15,491 genes in four mammalian species (human, rhesus macaque, mouse, and opossum), to identify human-specific patterns of gene regulation during spermatogenesis. We combined H3K27me3 ChIP-seq, H3K4me3 ChIP-seq, and RNA-seq data to define epigenetic states for each gene at two stages of spermatogenesis, pachytene spermatocytes and round spermatids, in each species. We identified 239 genes that are uniquely active, poised, or dynamically regulated in human spermatogenic cells distinct from the other three species. While some of these genes have been implicated in reproductive functions, many more have not yet been associated with human infertility and may be candidates for further molecular and epidemiologic studies. Our analysis offers an example of the opportunities provided by evolutionary and epigenomic data for broadly screening candidate genes implicated in reproduction, which might lead to discoveries of novel genetic targets for diagnosis and management of male infertility and male contraception.

CITMIC: Comprehensive Estimation of Cell Infiltration in Tumor Microenvironment based on Individualized Intercellular Crosstalk.

The tumor microenvironment (TME) cells interact with each other and play a pivotal role in tumor progression and treatment response. A comprehensive characterization of cell and intercellular crosstalk in the TME is essential for understanding tumor biology and developing effective therapies. However, current cell infiltration analysis methods only partially describe the TME's cellular landscape and overlook cell-cell crosstalk. Here, this approach, CITMIC, can infer the cell infiltration of TME by simultaneously measuring 86 different cell types, constructing an individualized cell-cell crosstalk network based on functional similarities between cells, and using only gene transcription data. This is a novel approach to estimating the relative cell infiltration levels, which are shown to be superior to the current methods. The TME cell-based features generated by analyzing melanoma data are effective in predicting prognosis and treatment response. Interestingly, these features are found to be particularly effective in assessing the prognosis of high-stage patients, and this method is applied to multiple high-stage adenocarcinomas, where more significant prognostic performance is also observed. In conclusion, CITMIC offers a more comprehensive description of TME cell composition by considering cell-cell crosstalk, providing an important reference for the discovery of predictive biomarkers and the development of new therapeutic strategies.

Therapeutic JAK1 Inhibition Reverses Lupus Nephritis in a Mouse Model and Demonstrates Transcriptional Changes Consistent With Human Disease.

Janus kinase family members are essential for signaling by multiple cytokines, including many implicated in systemic lupus erythematosus (SLE) pathogenesis. To test whether inhibition of JAK1 signaling can be efficacious in SLE, we used a JAK1-selective inhibitor (ABT-317) and evaluated its ability to ameliorate disease in murine SLE. Efficacy of ABT-317 was evaluated using NZB/W-F1 mice treated prophylactically and therapeutically. Primary endpoints were proteinuria, survival, and saliva production. Other endpoints included histological analysis of kidneys and salivary glands, flow cytometric analysis of splenic cell populations, and gene expression analysis by RNA sequencing in the kidneys, salivary glands, and blood. Publicly available human kidney gene transcription data were used to assess the translatability of the mouse findings. ABT-317 was efficacious when dosed prophylactically and prevented disease for up to two months after treatment cessation. When dosed therapeutically, ABT-317 quickly reversed severe proteinuria and restored saliva production, as well as diminished kidney and salivary gland inflammation. ABT-317-induced changes in glomerular morphology coincided with normalization of a human nephrotic gene signature, suggesting translatability to human lupus nephritis (LN). JAK1 inhibition prevented and reversed kidney and salivary gland manifestations of murine lupus with long-lasting effects after treatment cessation. These data, along with the presence of JAK1 and nephrotic gene signatures in human LN glomeruli, suggest that a JAK1-selective inhibitor may be an effective therapeutic in the treatment of human SLE and LN.

180 Dietary milk fat globule membrane alters intestinal microbiota of neonatal piglet following lipopolysaccharide challenge and marker of neuroinflammation

Abstract Early life stress exposure can increase morbidity and reduce animal wellbeing throughout life. Dysregulated gut function has been associated with neurological changes, and neurological changes affect gut homeostasis. Maternal milk is the gold-standard for yielding developmental benefits to offspring. Identification of milk components has led to an understanding of the bioactive nutrients important in neonatal development. Components of the milk fat globule membrane (MFGM) contribute immensely to neonatal gut health and neurodevelopment. However, mechanisms of actions are still being defined. The objective is to understand how dietary MFGM modulates the gut microbiota, blood-brain barrier tight junctions, and brain inflammatory markers following lipopolysaccharide (LPS) challenge. Piglets (n = 24, one-d-old) were assigned to soy (CON) or MFGM phospholipid supplemented diet (0.75% wt/wt) ± LPS (100 µg/kg body weight) in a 2 × 2 factorial design (n = 6 animals•diet-1•challenge-1.). Piglets were fed for 21 d and 6 animals/diet received saline or LPS injections 4 h before euthanasia. Colonic microbiota was characterized through 16s rRNA gene sequencing, while hypothalamus samples were collected to evaluate brain barrier function and inflammation-related genes using RT-qPCR. Microbiota data were processed using QIIME2 with DADA2 plugin and analyzed for alpha-diversity, beta-diversity, and differential abundance in R software. Gene transcription data were analyzed by a 2 × 2 experimental factorial design using the PROC MIXED procedure of SAS. Principal coordinates analysis indicated that MFGM-fed groups exhibited an observable separation from CON-fed groups in terms of microbial community structures (P < 0.05). Firmicutes_A, Bacteroidota, and Firmicutes_D were the predominant phyla across all treatments, with average proportions to be 52.21 %, 24.36 %, and 18.46 %, respectively. At genus level, MFGM + Saline group down-regulated Corynebacterium by 88.00 % (q < 0.05) and Unclassified Muribaculaceae by 73.33 % (0.050.05). However, there was an MFGM × LPS interaction (P < 0.05) observed for nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor alpha, in which MFGM attenuated the LPS-induced increase in mRNA relative abundance (3.62 and 2.39 ± 0.38). In conclusion, dietary MFGM altered neonatal piglet intestinal microbiota and an important signaling molecule of the neuroinflammatory response following LPS challenge.

Gene Transcription Data Are Correlated with Pain-Related Phenotypic Traits in Individuals with Sickle Cell Disease

Background: Sickle cell disease (SCD) pain is acute and chronic and associated with significant morbidity across the lifespan. Interindividual variability in SCD pain exists, despite the same hemoglobin gene defect. The biology that explains this variability is not well understood, which is a barrier to novel analgesics and disease modifying therapy. Our prior work used plasma-based gene transcription via a peripheral blood mononuclear cell (PBMC) reporter assay to identify differentially expressed transcripts between individuals with SCD and healthy Black controls. In this study, we sought to provide clinical meaning to gene transcription data in the context of SCD pain. We hypothesized that we could identify gene modules using Weighted Gene Co-expression Network Analysis (WGCNA) that are significantly associated with pain-related phenotypic traits in individuals with SCD. Methods: We leveraged our prior data from 149 individuals with SCD (median age 13.6, IQR 9.3-17.1) during baseline health and 45 healthy Black controls to discover differentially expressed transcripts between the two groups. Using our well-developed assay, PBMCs from a healthy blood donor functioned as reporter cells to plasma-born factors present in individuals with SCD during baseline health and controls. Gene expression in PBMCs was measured with Affymetrix array. Using our pre-defined filtering criteria of retaining transcripts differentially expressed between individuals with SCD and controls that exhibit a fold change >1.4, ANOVA p-value of <0.05 and an FDR <10%, we identified 3028 genes differentially expressed between individuals with SCD and controls. We then applied this gene list to an independent cohort of newly recruited individuals with SCD during baseline health. Plasma-based transcription using our PBMC reporter cell assay was conducted in the new independent cohort using identical methods to our derivation cohort. The gene list (n=3028) generated from our derivation cohort was used as the input list for WGCNA to investigate the association of gene modules with pain-related phenotypic traits in our new cohort. Clinical pain-related data included: patient-reported outcomes (PROs) (i.e., PROMIS Pain Interference, Pain Behavior, Pain Quality-Neuropathic, PedsQL Pain Impact), acute care pain visits, chronic pain phenotyping. PROs were scored as per developers' guidelines; means and final PRO scores were used as WGCNA traits. Descriptive statistics summarized clinical variables. WGCNA was used to assess the correlation of co-expressed gene modules with SCD pain-related phenotypic data. The correlation coefficient generated by WGCNA is between the module eigengene (i.e., first principal component of a principal component analysis which is representative of expression profiles of all genes within that module) and the phenotypic trait. We retained modules with significant correlations of ≥0.3 between these modules and SCD pain-related data. Database for Annotation, Visualization and Integrated Discovery (DAVID) was used to identify biological processes represented by the differentially expressed transcripts within each module. Results: A total of 87 individuals with SCD at baseline health were included in our current cohort. Mean age was 14.5 (SD 14.2) years, 51.7% were female and 70.1% had HbSS/HbSβ 0thal. Our WGCNA analyses identified 11 modules (Figure 1). Of these, 10 exhibited significant correlations with pain-related phenotypic traits that met our module inclusion criteria of ≥0.3. Modules with the highest number of significant pain-related trait correlations included: 1) pink, black, blue (4 each) and 2) lime, brown, red, turquoise (3 each). DAVID analyses of the biological processes contained within the modules significantly associated with pain-related traits are displayed in Figure 1. A strong immune-mediated component was found across the modules significantly associated with pain-related traits. Conclusions: We identified gene modules significantly associated with pain-related phenotypic data. These analyses give clinical meaning to gene expression data which is imperative for translational SCD pain research. A strong immune-mediated component was identified in the modules significantly associated with pain. The transcriptional data and pathways identified can be leveraged to investigate SCD pain biology and discover targets for novel pain therapeutics.

Epigenetic Reprogramming By Histone Deacetylase Inhibitors Improves CAR-T Cell Functions

Background: The exhaustion and diminished persistence of CAR-T cells primarily affect clinical efficacy and long-term outcomes. Epigenetic is an innate mechanism in regulating the biological function CAR-T cells. We previously dissected comprehensive dynamic changes of chromatin accessibility and transcription factors during CAR-T cell exhaustion (Jiang P, et al. Leukemia. 2022 Nov;36(11):2656-2668). Therefore, combined application with epigenetic drugs have much therapeutic potential. Methods: We initially performed an extensive screening among 370 epigenetic drugs to select candidates that could resist exhaustion and maintain phenotype of memory subsets, and thoroughly verify their effects on CAR-T cells in vitro. Next, we conducted multi-omics analysis comprising RNA-seq, ATAC-seq, and CUT&Tag-seq to explore underlying regulative mechanisms of epigenetic manipulation by epi-drugs. In the end, we used preclinical B-ALL NSG mouse model to verify anti-tumor activities of drug-treated CAR-T cells in vivo. Results: Among those screened epigenetic drugs, we identified two histone deacetylase inhibitors (HDACi), Chidamide and M344, could best promote maintenance of central memory subsets and resistance to exhaustion of CAR-T cells, with enhanced anti-leukemia activities in vitro (Figure 1a-c). Chidamide and M344 inhibited enzymatic activity and expression of HDAC1, which elevated the acetylation level of histone H3, particularly at H3K27 in CD19 CAR-T cells. Genetic knock-down of HDAC1 could partially reflect the phenotypic mimicry by Chidamide and M344 on CAR-T cells. By combining multi-omics data from bulk RNA-seq, ATAC-seq, and H3K27ac-targeted CUT&Tag-seq in Chidamide-or M344-treated CAR-T cells, we identified distinct patterns of epigenetic reprogramming, and enrichment of genes associated with exhaustion, as well as regulation of cellular differentiation and formation of memory subsets. In addition, analysis of motif and interaction network among transcription factors revealed altered regulation of gene transcription (data not shown). Finally, either Chidamide-pretreated CAR-T cells or in vivo administration of Chidamide after CAR-T cells infusion had exhibited enhanced and persistent tumor clearance in preclinical B-ALL NSG mouse model (Figure 1d). Conclusion: we conducted comprehensive screening of 370 epigenetic drugs and identified that administration of HDACi, particularly Chidamide and M344, could induce significantly enhanced anti-tumor function with maintenance of memory phenotype and reduced exhaustion in CAR-T cells through epigenetic reprogramming and altered regulation of gene transcription, which provided with basis and therapeutic targets for the rational combination of CAR-T cell therapy and epigenetic drugs in patients with hematological malignancies. Figure legends: Figure1. Screening of 370 epigenetic drugs identified M344 and Chidamide that enhanced maintenance of memory phenotype, resistance to exhaustion, and ability of tumor clearance in CAR-T cells. (a) The scatterplot of screening 370 compounds based on relative CD45RO +CD62L + proportion of central memory in treated CAR-T cell, showing of compared with DMSO control group by flow cytometry. Red indicates an increase in the proportion of memory CAR-T cells, while blue represents a decrease. (b) Frequency of co-expression of exhaustion-related inhibitory receptors (LAG-3, TIM-3, and PD-1) in DMSO or HDACi-treated groups. Data presented as mean ± SEM of independent donors (n=3 or more). (c) Flow cytometric analysis of central memory and effector memory subsets in CAR-T cells in each group. Data presented as mean ± SEM of independent donors (n=3 or more). (d) Bioluminescent imaging of tumor clearance in B-ALL NSG mouse. ‘X’ represents dead subject (end point). Survival of B-ALL NSG mice i.v. infused with T cells or treated CAR-T cells. Statistical analysis by Mantel-Cox test. Data presented as pooled from independent experiments (n=2).

Glycine betaine uptake and metabolism in marine microbial communities.

SummaryGlycine betaine (GBT) is a compatible solute in high concentrations in marine microorganisms. As a component of labile organic matter, GBT has complex biochemical potential as a substrate for microbial use that is unconstrained in the environment. Here we determine the uptake kinetics and metabolic fate of GBT in two natural microbial communities in the North Pacific characterized by different nitrate concentrations. Dissolved GBT had maximum uptake rates of 0.36 and 0.56 nM h−1 with half‐saturation constants of 79 and 11 nM in the high nitrate and low nitrate stations respectively. During multiday incubations, most GBT taken into cells was retained as a compatible solute. Stable isotopes derived from the added GBT were also observed in other metabolites, including choline, carnitine and sarcosine, suggesting that GBT was used for biosynthesis and for catabolism to pyruvate and ammonium. Where nitrate was scarce, GBT was primarily metabolized via demethylation to glycine. Gene transcript data were consistent with SAR11 using GBT as a source of methyl groups to fuel the methionine cycle. Where nitrate concentrations were higher, more GBT was partitioned for lipid biosynthesis by both bacteria and eukaryotic phytoplankton. Our data highlight unexpected metabolic pathways and potential routes of microbial metabolite exchange.

Evaluation of Optimal Reference Genes for qRT-PCR Analysis in Hyphantria cunea (Drury).

Simple SummaryReverse transcription-quantitative polymerase chain reaction (qRT-PCR) has rapidly become the most accessible and widely-applied method for quantifying gene transcription. Stable reference genes need to be normalized to facilitate gene expression studies and obtain more accurate qRT-PCR data. In this research, eight reference genes from different functional categories and gene families were used to assess the stability level of gene expression under different conditions in H. cunea. Finally, the expression pattern of the target gene serine proteases (HcSP1) was evaluated under different conditions to verify the precision and reliability of the results. This study provides a comprehensive list of suitable reference genes for analyses and optional reference genes for other gene function research in H. cunea.The relative quantification of gene expression is mainly achieved through reverse transcription-quantitative PCR (qRT-PCR); however, its reliability and precision rely on proper data normalization using one or more optimal reference genes. Hyphantria cunea (Drury) has been an invasive pest of forest trees, ornamental plants, and fruit trees in China for many years. Currently, the molecular physiological role of reference genes in H. cunea is unclear, which hinders functional gene study. Therefore, eight common reference genes, RPS26, RPL13, UBI, AK, RPS15, EIF4A, β-actin, α-tub, were selected to evaluate levels of gene expression stability when subjected to varied experimental conditions, including developmental stage and gender, different tissues, larvae reared on different hosts and different larval density. The geNorm, BestKeeper, ΔCt method, and NormFinder statistical algorithms were used to normalize gene transcription data. Furthermore, the stability/suitability of these candidates was ranked overall by RefFinder. This study provides a comprehensive evaluation of reference genes in H. cunea and could help select reference genes for other Lepidoptera species.

N-acetyl cysteine alters the genotoxic and estrogenic properties of Alternaria toxins in naturally occurring mixtures

It is unclear if complex mycotoxin mixtures produced by Alternaria spp. act estrogenic and/or genotoxic under physiological conditions, particularly considering the co-occurrence with antioxidants in food. Thus, this study focused on enlightening the impact of N-acetyl cysteine (NAC), as a representative anti-oxidative SH-donor, on the mentioned toxicological endpoints of the signature Alternaria toxins alternariol (AOH), altertoxin-II (ATX-II) and a complex extract (CE) of an Alternaria alternata culture.Using Ishikawa cells as an in vitro model, we monitored alterations in toxin concentrations by LC-MS/MS, estrogenicity by alkaline phosphatase assays, cytotoxicity by sulforhodamine B assays, genotoxicity by single-cell gel electrophoresis and the transcription of selected genes of interest by quantitative real-time PCR.The results indicate that the strong genotoxic effects of epoxide-carrying perylene quinones such as ATX-II are erased in the presence of NAC. The cellular effects of ATX-II/AOH mixtures are dominated by the genotoxicity of the perylene chinone. In this mixture, AOH regained its estrogenicity when co-incubated with NAC. In contrast, NAC treatment of an AOH/CE mixture did not result in a recovery of estrogenicity, but in potentiated anti-estrogenic effects. These findings were in line with gene transcription data, that indicated the aryl hydrocarbon receptor (AhR) to be a prime mediator of Alternaria toxin – induced antagonistic effects towards estrogen receptor signaling.Taken together, further studies on potential endocrine-disruptive properties of non-genotoxic perylene quinones should be a future research priority in the field of these emerging contaminants.

Multispecies Populations of Methanotrophic Methyloprofundus and Cultivation of a Likely Dominant Species from the Iheya North Deep-Sea Hydrothermal Field.

ABSTRACTThe Methyloprofundus clade is represented by uncultivated methanotrophic bacterial endosymbionts of deep-sea bathymodiolin mussels, but only a single free-living species has been cultivated to date. This study reveals the existence of free-living Methyloprofundus variants in the Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough. A clade-targeted amplicon analysis of the particulate methane monooxygenase gene (pmoA) detected 647 amplicon sequence variants (ASVs) of the Methyloprofundus clade in microbial communities newly formed in in situ colonization systems. Such systems were deployed at colonies of bathymodiolin mussels and a galatheoid crab in diffuse-flow areas. These ASVs were classified into 161 species-like groups. The proportion of the species-like groups representing endosymbionts of mussels was unexpectedly low. A methanotrophic bacterium designated INp10, a likely dominant species in the Methyloprofundus population in this field, was enriched in a biofilm formed in a methane-fed cultivation system operated at 10°C. Genomic characterization with the gene transcription data set of INp10 from the biofilm suggested traits advantageous to niche competition in environments, such as mobility, chemotaxis, biofilm formation, offensive and defensive systems, and hypoxia tolerance. The notable metabolic traits that INp10 shares with some Methyloprofundus members are the use of lanthanide-dependent XoxF as the sole methanol dehydrogenase due to the absence of the canonical MxaFI, the glycolytic pathway using fructose-6-phosphate aldolase instead of fructose-1,6-bisphosphate aldolase, and the potential to perform partial denitrification from nitrate under oxygen-limited conditions. These findings help us better understand the ecological strategies of this possibly widespread marine-specific methanotrophic clade.IMPORTANCE The Iheya North deep-sea hydrothermal field in the mid-Okinawa Trough is characterized by abundant methane derived from organic-rich sediments and diverse chemosynthetic animal species, including those harboring methanotrophic bacterial symbionts, such as bathymodiolin mussels Bathymodiolus japonicus and “Bathymodiolus” platifrons and a galatheoid crab, Shinkaia crosnieri. Symbiotic methanotrophs have attracted significant attention, and yet free-living methanotrophs in this environment have not been studied in detail. We focused on the free-living Methyloprofundus spp. that thrive in this hydrothermal field and identified an unexpectedly large number of species-like groups in this clade. Moreover, we enriched and characterized a methanotroph whose genome sequence indicated that it corresponds to a new species in the genus Methyloprofundus. This species might be a dominant member of the indigenous Methyloprofundus population. New information on free-living Methyloprofundus populations suggests that the hydrothermal field is a promising locale at which to investigate the adaptive capacity and associated genetic diversity of Methyloprofundus spp.

Use of RNAi With OsMYB76R as a Reporter for Candidate Genes Can Efficiently Create and Verify Gametophytic Male Sterility in Rice.

Gametophytic male sterility (GMS) plays an important role in the study of pollen development and seed propagation of recessive nuclear male sterile lines insensitive to the environmental conditions in hybrid rice breeding. Since the inherent phenotypic and genetic characteristics of GMS, it is very difficult to find and identify the GMS mutants. However, due to the abundance of gene transcription data, a large number of pollen-specific genes have been found, and most of them may be associated with GMS. To promote the study of these genes in pollen development and heterosis utilization, in this study, an easy and efficient method of creating and identifying GMS was established using RNAi and OsMYB76R as a reporter. First, the OsC1/OsMYB76 gene involved in anthocyanin synthesis was modified, and we have validated that the modified OsMYB76R is workable as the same as the pre-modified OsMYB76 gene. Then, the ascorbic acid oxidase gene OsPTD1 was downregulated using RNAi, driven by its own promoter that resulted in abnormal pollen tube growth. Finally, the RNAi elements were linked with OsMYB76R and transformed into an osmyb76 mutant, and the distortion of purple color segregation was found in T1 and F1 generations. This indicates that the OsPTD1 GMS was prepared successfully. Compared to current methods, there are several advantages to this method. First, time is saved in material preparation, as one generation less needs to be compared than in the conventional method, and mutation screening can be avoided. In addition, for identification, the cost is lower; PCR, electrophoresis, and other processes are not needed; and no expensive chemicals or instruments are required. Finally, the results are more accurate, with much lower background effects, and no damage to the plant. The result is an easy, efficient, low-cost, and accurate method of preparing and identifying GMS genes.

The Leloir Cycle in Glioblastoma: Galactose Scavenging and Metabolic Remodeling.

Simple SummaryProliferation of glioblastoma (GBM) depends on access to extracellular nutrients. Inadequate tumor perfusion creates a short supply of the most common nutrients, such as glucose (Glc) and glutamine. As a result, GBMs undergo metabolic remodeling and scavenge alternative nutrients from the tumor microenvironment for their growth and proliferation. GBM expresses sugar transporters, such as Glut3 and Glut14. Galactose (Gal) is a good substrate for Glut3/Glut14, and GBM cells can scavenge Gal from the circulation/extracellular space. The Leloir pathway allows GBM to transport and metabolize Gal at physiologic Glc concentrations, providing GBM cells with an alternate energy source. GBM cultures proliferated when grown solely on Gal. GBMs metabolize Gal via the Leloir pathway, glycolysis, and pentose phosphate pathway (PPP) to optimize ATP production, while the mitochondrial metabolism of Gal in GBM is limited. The selective targeting of the Leloir/PPP pathway may provide new treatment strategies for GBM.Background: Glioblastoma (GBM) can use metabolic fuels other than glucose (Glc). The ability of GBM to use galactose (Gal) as a fuel via the Leloir pathway is investigated. Methods: Gene transcript data were accessed to determine the association between expression of genes of the Leloir pathway and patient outcomes. Growth studies were performed on five primary patient-derived GBM cultures using Glc-free media supplemented with Gal. The role of Glut3/Glut14 in sugar import was investigated using antibody inhibition of hexose transport. A specific inhibitor of GALK1 (Cpd36) was used to inhibit Gal catabolism. Gal metabolism was examined using proton, carbon and phosphorous NMR spectroscopy, with 13C-labeled Glc and Gal as tracers. Results: Data analysis from published databases revealed that elevated levels of mRNA transcripts of SLC2A3 (Glut3), SLC2A14 (Glut14) and key Leloir pathway enzymes correlate with poor patient outcomes. GBM cultures proliferated when grown solely on Gal in Glc-free media and switching Glc-grown GBM cells into Gal-enriched/Glc-free media produced elevated levels of Glut3 and/or Glut14 enzymes. The 13C NMR-based metabolic flux analysis demonstrated a fully functional Leloir pathway and elevated pentose phosphate pathway activity for efficient Gal metabolism in GBM cells. Conclusion: Expression of Glut3 and/or Glut14 together with the enzymes of the Leloir pathway allows GBM to transport and metabolize Gal at physiological glucose concentrations, providing GBM cells with an alternate energy source. The presence of this pathway in GBM and its selective targeting may provide new treatment strategies.

Molecular profiling of long-term responders to immune checkpoint inhibitors in advanced non-small cell lung cancer.

Immunotherapy has transformed advanced non-small cell lung cancer (NSCLC) treatment strategies and has led to unprecedented long-lasting responses in some patients. However, the molecular determinants driving these long-term responses remain elusive. To address this issue, we performed an integrative analysis of genomic and transcriptomic features of long-term immune checkpoint inhibitors (ICIs)-associated responders. We assembled a cohort of 47 patients with NSCLC receiving ICIs that was enriched in long-term responders [>18months of progression-free survival (PFS)]. We performed whole-exome sequencing from tumor samples, estimated the tumor mutational burden (TMB), and inferred the somatic copy number alterations (SCNAs). We also obtained gene transcription data for a subset of patients using Nanostring, which we used to assess the tumor immune infiltration status and PD-L1 expression. Our results indicate that there is an association between TMB and benefit to ICIs, which is driven by those patients with long-term response. Additionally, high SCNAs burden is associated with poor response and negatively correlates with the presence of several immune cell types (B cells, natural killers, regulatory T cells or effector CD8 T cells). Also, CD274 (PD-L1) expression is increased in patients with benefit, mainly in those with long-term response. In our cohort, combined assessment of TMB and SCNAs burden enabled identification of long-term responders (considering PFS and overall survival). Notably, the association between TMB, SCNAs burden, and PD-L1 expression with the outcomes of ICIs treatment was validated in two public datasets of ICI-treated patients with NSCLC. Thus, our data indicate that TMB is associated with long-term benefit following ICIs treatment in NSCLC and that TMB, SCNAs burden, and PD-L1 are complementary determinants of response to ICIs.

Tissue-Specific Transcription Footprinting Using RNA PoI DamID (RAPID) in Caenorhabditis elegans.

Differential gene expression across cell types underlies development and cell physiology in multicellular organisms. Caenorhabditis elegans is a powerful, extensively used model to address these biological questions. A remaining bottleneck relates to the difficulty to obtain comprehensive tissue-specific gene transcription data, since available methods are still challenging to execute and/or require large worm populations. Here, we introduce the RNA Polymerase DamID (RAPID) approach, in which the Dam methyltransferase is fused to a ubiquitous RNA polymerase subunit to create transcriptional footprints via methyl marks on the DNA of transcribed genes. To validate the method, we determined the polymerase footprints in whole animals, in sorted embryonic blastomeres and in different tissues from intact young adults by driving tissue-specific Dam fusion expression. We obtained meaningful transcriptional footprints in line with RNA-sequencing (RNA-seq) studies in whole animals or specific tissues. To challenge the sensitivity of RAPID and demonstrate its utility to determine novel tissue-specific transcriptional profiles, we determined the transcriptional footprints of the pair of XXX neuroendocrine cells, representing 0.2% of the somatic cell content of the animals. We identified 3901 candidate genes with putatively active transcription in XXX cells, including the few previously known markers for these cells. Using transcriptional reporters for a subset of new hits, we confirmed that the majority of them were expressed in XXX cells and identified novel XXX-specific markers. Taken together, our work establishes RAPID as a valid method for the determination of RNA polymerase footprints in specific tissues of C. elegans without the need for cell sorting or RNA tagging.

Proteomic signatures of clear cell renal cell carcinoma.

In recent years, the molecular view of clear cell renal cell carcinoma (ccRCC) has been based primarily on gene transcription data with limited information on protein features. A new study led by the Clinical Proteomic Tumor Analysis Consortium now offers a comprehensive view of the ccRCC proteome.

A Model of Glucocorticoid Receptor Interaction With Coregulators Predicts Transcriptional Regulation of Target Genes.

Regulatory factors that control gene transcription in multicellular organisms are assembled in multicomponent complexes by combinatorial interactions. In this context, nuclear receptors provide well-characterized and physiologically relevant systems to study ligand-induced transcription resulting from the integration of cellular and genomic information in a cell- and gene-specific manner. Here, we developed a mathematical model describing the interactions between the glucocorticoid receptor (GR) and other components of a multifactorial regulatory complex controlling the transcription of GR-target genes, such as coregulator peptides. We support the validity of the model in relation to gene-specific GR transactivation with gene transcription data from A549 cells and in vitro real time quantification of coregulator-GR interactions. The model accurately describes and helps to interpret ligand-specific and gene-specific transcriptional regulation by the GR. The comprehensive character of the model allows future insight into the function and relative contribution of the molecular species proposed in ligand- and gene-specific transcriptional regulation.

Differential gene transcription across the life cycle in Daphnia magna using a new all genome custom-made microarray

BackgroundUnravelling the link between genes and environment across the life cycle is a challenging goal that requires model organisms with well-characterized life-cycles, ecological interactions in nature, tractability in the laboratory, and available genomic tools. Very few well-studied invertebrate model species meet these requirements, being the waterflea Daphnia magna one of them. Here we report a full genome transcription profiling of D. magna during its life-cycle. The study was performed using a new microarray platform designed from the complete set of gene models representing the whole transcribed genome of D. magna.ResultsUp to 93% of the existing 41,317 D. magna gene models showed differential transcription patterns across the developmental stages of D. magna, 59% of which were functionally annotated. Embryos showed the highest number of unique transcribed genes, mainly related to DNA, RNA, and ribosome biogenesis, likely related to cellular proliferation and morphogenesis of the several body organs. Adult females showed an enrichment of transcripts for genes involved in reproductive processes. These female-specific transcripts were essentially absent in males, whose transcriptome was enriched in specific genes of male sexual differentiation genes, like doublesex.ConclusionOur results define major characteristics of transcriptional programs involved in the life-cycle, differentiate males and females, and show that large scale gene-transcription data collected in whole animals can be used to identify genes involved in specific biological and biochemical processes.

In the hunt for genomic markers of metabolic resistance to pyrethroids in the mosquito Aedes aegypti: An integrated next-generation sequencing approach.

BackgroundThe capacity of Aedes mosquitoes to resist chemical insecticides threatens the control of major arbovirus diseases worldwide. Until alternative control tools are widely deployed, monitoring insecticide resistance levels and identifying resistance mechanisms in field mosquito populations is crucial for implementing appropriate management strategies. Metabolic resistance to pyrethroids is common in Aedes aegypti but the monitoring of the dynamics of resistant alleles is impeded by the lack of robust genomic markers.Methodology/Principal findingsIn an attempt to identify the genomic bases of metabolic resistance to deltamethrin, multiple resistant and susceptible populations originating from various continents were compared using both RNA-seq and a targeted DNA-seq approach focused on the upstream regions of detoxification genes. Multiple detoxification enzymes were over transcribed in resistant populations, frequently associated with an increase in their gene copy number. Targeted sequencing identified potential promoter variations associated with their over transcription. Non-synonymous variations affecting detoxification enzymes were also identified in resistant populations.Conclusion /SignificanceThis study not only confirmed the role of gene copy number variations as a frequent cause of the over expression of detoxification enzymes associated with insecticide resistance in Aedes aegypti but also identified novel genomic resistance markers potentially associated with their cis-regulation and modifications of their protein structure conformation. As for gene transcription data, polymorphism patterns were frequently conserved within regions but differed among continents confirming the selection of different resistance factors worldwide. Overall, this study paves the way of the identification of a comprehensive set of genomic markers for monitoring the spatio-temporal dynamics of the variety of insecticide resistance mechanisms in Aedes aegypti.

Morphological and functional development of the interbranchial lymphoid tissue (ILT) in Atlantic salmon (Salmo salar L)

The interbranchial lymphoid tissue (ILT) of Atlantic salmon originates from an embryological location that in higher vertebrates gives rise to both primary and secondary lymphoid tissues. Still much is unknown about the morphological and functional development of the ILT. In the present work a standardized method of organ volume determination was established to study its development in relation to its containing gill and the thymus. Based on morphological findings and gene transcription data, the ILT shows no signs of primary lymphoid function. In contrast to the thymus, an ILT-complex first became discernible after the yolk-sac period. After its appearance, the ILT-complex constitutes 3–7% of the total volume of the gill (excluding the gill arch) with the newly described distal ILT constituting a major part, and in adult fish it is approximately 13 times larger than the thymus. Confined regions of T-cell proliferation are present within the ILT. Communication with systemic circulation through the distal ILT is also highly plausible thus offering both internal and external recruitment of immune cells in the growing ILT.

Cancer therapeutic target genes identified on chromosome 20q

Cancer therapeutic target genes identified on chromosome 20q