Changes In Transcriptional Profile Research Articles

Characterization of the Immunologic Phenotype of Dendritic Cells Infected With Herpes Simplex Virus 1.

Due to viral envelope glycoprotein D binding to cellular membrane HVEM receptor, HSV-1 can infect certain dendritic cells, which becomes an event in the viral strategy to interfere with the host’s immune system. We previously generated the HSV-1 mutant strain M6, which produced an attenuated phenotype in mice and rhesus monkeys. The attenuated M6 strain was used to investigate how HSV-1 infection of dendritic cells interferes with both innate and adaptive immunity. Our study showed that dendritic cells membrane HVEM receptors could mediate infection of the wild-type strain and attenuated M6 strain and that dendritic cells infected by both viruses in local tissues of animals exhibited changes in transcriptional profiles associated with innate immune and inflammatory responses. The infection of pDCs and cDCs by the two strains promoted cell differentiation to the CD103+ phenotype, but varied transcriptional profiles were observed, implying a strategy that the HSV-1 wild-type strain interferes with antiviral immunity, probably due to viral modification of the immunological phenotype of dendritic cells during processing and presentation of antigen to T cells, leading to a series of deviations in immune responses, ultimately generating the deficient immune phenotype observed in infected individuals in the clinical.

Abstract 288: An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics

Abstract Background: Molecular changes underlying immune cell signaling in response to new therapy development are crucial to validate the clinical efficacy of immunotherapeutics. There are several preclinical tools including patient derived cell lines, organoid and xenograft (PDX) models to assess the efficacy of drugs. However, it is important to recapitulate the complexity of human malignancy and immune contexture within the tumor microenvironment. Here we report, an ex vivo platform (3D-EXplore) using fresh patient tumor samples with intact stromal and immune cell components to assess treatment-mediated changes in molecular and transcriptional profiles of tumor resident immune cells using a Cellular Indexing of Transcriptomes and Epitopes by sequencing (CITE-seq) platform. Methods: All tumor samples were obtained with patient consent and relevant IRB approval. Unpropagated 3D tumoroids measuring 150 µm in size with intact tumor immune microenvironment were prepared from fresh tumor samples of colon and lung tissues using proprietary technology developed at Nilogen Oncosystems. No enzymatic digestion, propagation or reassembly was used during the preparation of the tumoroids. Hundreds of tumoroids originated from different parts of each patient’s tumor sample were pooled to represent the tumor heterogeneity and treated ex vivo with or without immunostimulatory agents for 48h. Here, we applied multi-modal CITE-seq profiling using the 10X Genomics platform to interrogate cellular responses to ex vivo treatment. Culture supernatants were collected for multiplex analysis of cytokine release in media. Additionally, flow cytometry was used to assess the activation profile of resident immune cells. Results: Following sequencing of FACS sorted viable CD45 populations, the FAST-Q output files were uploaded into Cell Ranger for analysis with Loupe Browser. Multimodal analysis of transcriptomes or proteomics at the single-cell level revealed significant changes in the top 100 gene expression levels specific to CD4 and CD8+ T cells, NK cells and B cells signaling and activation upon stimulation with immunostimulatory agents. In addition, several pro and anti-inflammatory chemokines and cytokines were upregulated in response to ex vivo treatment in both colon and lung tumoroids, which coincided with marked changes in the activation status of tumor resident immune cells, as detected by multiparameter flow cytometry analysis. Conclusions: These results demonstrate Nilogen’s 3D fresh tumoroid model reflects the significant impact on activation and proliferation of tumor resident immune cells within the intact tumor microenvironment at the transcriptional level. We believe this model system can serve as a clinically relevant tool for accelerating immuno-oncology drug development. Citation Format: Jonathan P. Waterman-Smith, Jared Ehrhart, Brittany Bunch, Krithika Kodumudi, Matt Weitzman, Olivia MacIntosh, Kelly Sussman, Soner Altiok. An ex-vivo 3D tumoroid model of fresh patient tissue (3D-EXplore) to assess transcriptional and compositional changes of the immune landscape in intact tumor microenvironment using single-cell proteogenomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 288.

Transcriptional profile changes after treatment of ischemia reperfusion injury-induced kidney fibrosis with 18β-glycyrrhetinic acid

Introduction Chronic kidney disease (CKD) is characterized by renal fibrosis without effective therapy. 18β-Glycyrrhetinic acid (GA) is reported to have detoxification and anti-inflammatory functions and promotes tissue repair. However, the role of GA in CKD remains unclear. In this study, we investigated whether GA has a potential therapeutic effect in kidney fibrosis. Methods A renal fibrosis mouse model was established by ischemia/reperfusion (I/R) injury via clamping unilateral left renal pedicle for 45 min; then, the mice were treated with vehicle or GA. Kidney tissues and blood samples were extracted 14 days after reperfusion and renal function, histopathological staining, quantitative PCR, and western blotting were performed. RNA-seq was performed to explore the changes in the transcriptional profile after GA treatment. Results Renal function, pathological and molecular analysis displayed that fibrosis was successfully induced in the I/R model. In the GA treatment group, the severity of fibrosis gradually reduced with the best effect seen at a concentration of 25 mg kg −1. A total of 970 differentially expressed genes were identified. Pathway enrichment showed that reduced activation and migration of inflammatory cells and decreased chemokine interaction in significant pathways. Protein–protein interaction networks were constructed and 15 hub genes were selected by degree rank, including chemokines, such as C3, Ccl6, Ccr2, Ptafr, Timp1, and Pf4. Conclusions GA may alleviate renal fibrosis by inhibiting the inflammatory response. GA is a promising therapy that may perhaps be used in treating renal fibrosis and CKD.

The carbohydrate elicitor Riclinoctaose facilitates defense and growth of potato roots by inducing changes in transcriptional and metabolic profiles

Promoting both root growth and defense is conducive to the production of potatoes (Solanum tuberosum L.), while the role of elicitors in this topic hasn’t been fully understood. To investigate the effect of Riclinoctaose (RiOc) on root growth and defense, potato tissue cuttings were cultivated with different concentration of RiOc (0, 50, 200 mg/L) for 5 weeks and changes in root morphology, transcription, enzymatic and metabolomic profiles were monitored over time. The results indicated that RiOc triggered the salicylic acid (SA)-mediated defense response and facilitated the growth of adventitious and lateral roots in a dose- and time-dependent manner. MPK3/MPK6, SA- and auxin-signaling pathways and transcription factors such as WUS, SCR and GRAS4/GRAS9 participated in this process. Moreover, the 1H NMR based metabolome profiling demonstrated that potato roots altered the primary metabolism to respond to the RiOc elicitation and efficiency in production and allocation of defense and growth-related metabolites was improved. After 5-week treatment, the level of glucose, N-acetylglucosamine, glutamine, asparagine, isoleucine, valine, 3-hydroxyisovalerate and ferulate increased, while acetate, acetoacetate, fucose, and 2-hydroxyphenylacetate declined. In conclusion, RiOc played dual roles in activating the SA-mediated defense response and in promoting growth of potato roots by inducing changes in root transcription and metabolism.

Microglia and macrophages contribute to the development and maintenance of sciatica in lumbar disc herniation.

Lumbar disc herniation (LDH) is a major cause of sciatica. Emerging evidence indicated that inflammation induced by the herniated nucleus pulposus (NP) tissues plays a major role in the pathogenesis of sciatica. However, the underlying mechanisms are still elusive. Although microglia and macrophages have been implicated in nerve injury-induced neuropathic pain, their roles in LDH-induced sciatica largely remain unknown. This study successfully established and modified a mouse model of LDH. We found that nerve root compression using degenerated NP tissues can initiate remarkable and persistent sciatica, with increased and prolonged macrophage infiltration in dorsal root ganglia (DRG) and significant activation of microglia in the spinal dorsal horn. Instead, compression of the nerve root with nondegenerated NP tissues only led to transient sciatica, with transient infiltration and activation of macrophages and microglia. Moreover, continuous treatment of PLX5622, a specific colony-stimulating factor 1 receptor antagonist, ablated both macrophages and microglia, which effectively alleviated LDH-induced sciatica. However, mechanical allodynia reoccurred along with the repopulation of macrophages and microglia after the withdrawal of PLX5622. Using RNA sequencing analysis, the current study depicted transcriptional profile changes of DRG after LDH and identified several macrophage-related potential target candidates. Our results suggested that microglia and macrophages may play an essential role in the development and maintenance of LDH-induced sciatica. Targeting microglia and macrophages may be a promising treatment for chronic LDH-induced sciatica.

RBBP4 dysfunction reshapes the genomic landscape of H3K27 methylation and acetylation and disrupts gene expression.

RBBP4 is a subunit of the chromatin remodeling complexes known as Polycomb repressive complex 2 and histone deacetylase 1/2-containing complexes. These complexes are responsible for histone H3 lysine 27 methylation and deacetylation, respectively. How RBBP4 modulates the functions of these complexes remains largely unknown. We generated viable Rbbp4 mutant alleles in mouse embryonic stem cell lines by CRISPR-Cas9. The mutations disrupted Polycomb repressive complex 2 assembly and H3K27me3 establishment on target chromatin and altered histone H3 lysine 27 acetylation genome wide. Moreover, Rbbp4 mutant cells underwent dramatic changes in transcriptional profiles closely tied to the deregulation of H3K27ac. The alteration of H3K27ac due to RBBP4 dysfunction occurred on numerous cis-regulatory elements, especially putative enhancers. These data suggest that RBBP4 plays a central role in regulating histone H3 lysine 27 methylation and acetylation to modulate gene expression.

Spermatozoal Mitochondrial Dynamics Markers and Other Functionality-Related Signaling Molecules Exert Circadian-like Response to Repeated Stress of Whole Organism.

In the search for the possible role of the mitochondrial dynamics markers in spermatozoa adaptation, an in vivo approach was designed to mimic situations in which human populations are exposed to 3 h of repeated psychological stress (the most common stress in human society) at different time points during the day (24 h). The hormones (stress hormone corticosterone and testosterone), the number and the functionality of spermatozoa (response to acrosome-reaction-inducer progesterone), as well as the transcriptional profiles of 22 mitochondrial dynamics and function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality were followed at three time points (ZT3, ZT11, and ZT23). The results show that repeated stress significantly decreased the number and functionality of spermatozoa at all time points. In the same samples, the transcriptional profiles of 91% (20/22) of mitochondrial dynamics and functionality markers and 86% (19/22) of signaling molecules were disturbed after repeated stress. It is important to point out that similar molecular changes in transcriptional profiles were observed at ZT3 and ZT23, but the opposite was observed at ZT11, suggesting the circadian nature of the adaptive response. The results of PCA analysis show the significant separation of repeated stress effects during the inactive/light and active/dark phases of the day, suggesting the circadian timing of molecular adaptations.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Guardians of the eye: new tales about retinal microglia and other ocular macrophages.

Dynamic molecular choreography induced by traffic exposure: A randomized, crossover trial using multi-omics profiling

The biological mechanism of adverse health outcomes related to exposure to traffic-related air pollution (TRAP) needs elucidation. We conducted a randomized, crossover trial among healthy young students in Shanghai, China. Participants wore earplugs and were randomly assigned to a 4-hour walking treatment either along a traffic-polluted road or through a traffic-free park. We conducted untargeted analyses of plasma exosome transcriptomics, serum mass spectrometry-based proteomics, and serum metabolomics to evaluate changes in genome-wide transcription, protein, and metabolite profiles in 35 randomly selected participants. Mean personal exposure levels of ultrafine particles, black carbon, nitrogen dioxide, and carbon monoxide in the road were 2–3 times higher than that in the park. We observed 3449 exosome mRNAs, 58 serum proteins, and 128 serum metabolites that were significantly associated with TRAP. The multi-omics analysis showed dozens of regulatory pathways altered in response to TRAP, such as inflammation, oxidative stress, coagulation, endothelin-1 signaling, and renin-angiotensin signaling. We found that several novel pathways activated in response to TRAP exposure: growth hormone signaling, adrenomedullin signaling, and arachidonic acid metabolism. Our study served as a demonstration and proof of concept on the evidence that associated TRAP exposure with global molecular changes based on the multi-omics level.

Ammonia exposure causes the imbalance of the gut-brain axis by altering gene networks associated with oxidative metabolism, inflammation and apoptosis

Ammonia is an acknowledged environment pollutant in atmosphere with irritating smell. Previous studies have shown that excessive ammonia has toxic effects on farm animals and humans. However, the detail toxicity mechanism of ammonia to pigs is still unknown so far. In order to clarify the mechanism of ammonia toxicity, we established a porcine exogenous ammonia poisoning model and assessed the effects of ammonia on the gut-brain axis by transcriptome sequencing, histological observation and chemical analysis. Our results showed that after 30 d of ammonia exposure, 578 differentially expressed genes (DEGs) and 407 DEGs were obtained in the hypothalamus and jejunum, respectively. These DEGs were enriched into Gene Ontology terms associated with inflammation, oxidative metabolism, apoptosis, and the highly expressed genes among these DEGs were verified by real-time quantitative PCR. The content of glutathione and the activities of glutathione peroxidase and superoxide dismutase were significantly decreased, while malondialdehyde content was increased after ammonia exposure. Corticotropin releasing factor, substance P, 5-hydroxytryptamine and ghrelin contents in serum elevated significantly. Furthermore, pathologic observation in the ammonia group revealed infiltration of lymphocytes in the hypothalamus and significant decrease of jejunal epithelial cells. Our results indicated that ammonia exposure mediated changes in transcriptional profiles, pathological damage, oxidative stress and brain-gut peptide of the pig jejunum and hypothalamus, and induced the imbalance of the brain-gut axis through the “oxidative stress-inflammation-apoptosis” interaction network. Our study not only provides a new perspective for the toxicity assessment of ammonia, but also enriches the toxicology mechanism of ammonia.

Immunosuppression, oxidative stress, and apoptosis in pig kidney caused by ammonia: Application of transcriptome analysis in risk assessment of ammonia exposure

Ammonia (NH3) is a recognized environmental contaminant around the world and has adverse effects on animal and human health. However, the mechanism of the renal toxicity of NH3 is not well understood. Pigs are considered an ideal model for biomedical and toxicological research because of the similarity to humans in physiological and biochemical basis. Therefore, in this study, twelve pigs were selected as research objects and randomly divided into two groups, namely the control group and the NH3 group. The formal experiment lasted 30 days. The effects of excessive NH3 inhalation on the kidney of fattening pig were evaluated by chemical analysis, ELISA, transcriptome analysis and real-time quantitative PCR (qRT-PCR) from the renal antioxidant level, renal function, blood ammonia content and gene level. Our results showed that excessive NH3 exposure could cause an increase in blood NH3 content, a reduction in renal GSH-Px, SOD and GSH, as well as an increase in MDA levels and an increase in serum creatinine, urea and uric acid levels. In addition, transcriptome analysis showed that NH3 exposure caused changes in 335 differentially expressed genes (DEGs) (including 126 up-regulated DEGs and 109 down-regulated DEGs). Some highly expressed DEGs were enriched into GO terms associated with immune function, oxidative stress, and apoptosis and were verified by qRT-PCR. The qRT-PCR results were comsistent with the transcriptome results. Our results indicated that NH3 exposure could cause changes in renal transcriptional profiles and kidney function, and induce kidney damage in the fattening pigs through oxidative stress, immune dysfunction and apoptosis. Our present study provides novel insights into the immunotoxicity mechanism of NH3 on kidney.

Transcriptional profile changes and quality maintenance of fresh-cut produce

Transcriptional profile changes and quality maintenance of fresh-cut produce

Characterization of Causal Variants at the GATA3 Loci Associated with Susceptibility to Ph-like Acute Lymphoblastic Leukemia

Acute lymphoblastic leukemia (ALL) is the most common cancer in children and there is growing evidence of inherited susceptibility to this type of leukemia. Recent genome-wide association studies have definitively identified at least 6 genomic loci with common variants that influence ALL risk, often in an age- and/or subtype-dependent fashion. In particular, we had previously reported germline intronic variants at the GATA3 locus that specifically associated with the risk of developing Philadelphia chromosome-like ALL (Ph-like ALL) and also inferior outcomes of ALL therapy (Nature Genetics 45:1494). However, the identity of the causal variant(s) that functionally alters GATA3 activity and drives the association with the susceptibility to Ph-like ALL remained unknown.To comprehensively identify ALL risk variants at the GATA3 locus, we first performed targeted sequencing of a ~27 Kb genomic region encompassing GATA3 exons and introns in 5,008 children with ALL enrolled on Children's Oncology Group and St. Jude Children's Research Hospital frontline trials. Of 1048 variants with sufficient sequencing quality, rs3824662 showed the strongest association with susceptibility to Ph-like ALL (P=1.2x10-8) and another 2 variants also reached statistical significance after corrected for multiple testing (P <1x10-5). However, multivariate analysis conditioning on rs3824662 revealed no independent signals, thus pointing to a single causal variant responsible for the association signal at this locus. This linkage disequilibrium block also overlapped with a chromatin segment epigenetically active in the hematopoietic cells, as inferred from histone modification marks.Next, we set out to systemically characterize potential functions of this regulatory DNA element and how its activity is influenced by rs3824662 genotype. The ALL risk variant A allele at rs3824662 was associated with higher enhancer activity in the reporter gene assay, and elevated levels of H3K4Me1 and H3K27Ac marks (for active chromatin) relative to the wildtype C allele in lymphoblastoid cells. ATAC-seq data from a representative panel of B-ALL cell lines confirmed a more open chromatin conformation linked to the A allele at this locus, e.g., in Ph-like ALL. By CRISPR-cas9 genome editing, we introduced the A allele at rs3824662 into the wildtype lymphoblastoid cell line GM12878. Engineered GM12878 cells with variant genotype (AA or CA) showed 3.7- and 3.8-fold increase in GATA3 expression as compared to isogenic cells with wildtype genotype, with a concomitant increase in H3K27Ac and H3K4Me1 modification at this locus, indicating that the risk allele at rs3824662 positively regulates GATA3 transcription.Ectopic expression of GATA3 in a non-Ph-like ALL cell line (Nalm6), induced a global change in gene transcription profile that resembles the Ph-like expression signature (P=0.0004), with particularly notable upregulation of CRLF2 . Chromosomal rearrangement-mediated CRLF2 overexpression is a defining molecular abnormality in up to 50% of Ph-like ALL cases. In fact, using GATA3 ChIP-seq, we identified 5 regions upstream of CRLF2 enriched for binding to this transcription factor in ALL cell line models, with binding sites located proximal to CRLF2 rearrangement hotspots observed in patients with Ph-like ALL. Inducible overexpression of GATA3 led to upregulation of CRLF2 in a time-dependent manner; conversely, downregulation of GATA3 suppressed CRLF2 transcription, further suggesting that GATA3 may function as transcriptional regulator of CRLF2 . In mouse hematopoietic cell BaF3, GATA3 overexpression resulted in elevated phosphorylation of JAK2 and STAT5, downstream of CRLF2 . Co-expression of GATA3 and JAK2R683G were sufficient to induce cytokine-independent growth and leukemic transformation of BaF3 cells, in a fashion similar to co-expression of CRLF2 and JAK2R683G. Interestingly, the addition of CRLF2 ligand, TSLP, potentiated transformation of BaF7 cells expressing GATA3 and JAK2R683G . Taken together, these data suggested that the GATA3 -mediated activation of CRLF2 - JAK signaling is a plausible mechanism by which it contributes to the pathogenesis of Ph-like ALL.In conclusion, our results indicate that rs3824662 causally regulates a cis-acting enhancer for GATA3 transcription and influences Ph-like ALL leukemogenesis by regulating CRLF2-JAK signaling. DisclosuresHunger:Erytech Pharmaceuticals: Consultancy; Novartis: Consultancy; Jazz Pharmaceuticals: Honoraria; Amgen: Consultancy, Equity Ownership. Mullighan:Amgen: Consultancy; Loxo Oncology: Research Funding.

Prediction of drug efficacy from transcriptional profiles with deep learning.

Drug discovery focused on target proteins has been a successful strategy, but many diseases and biological processes lack obvious targets to enable such approaches. Here, to overcome this challenge, we describe a deep learning-based efficacy prediction system (DLEPS) that identifies drug candidates using a change in the gene expression profile in the diseased state as input. DLEPS was trained using chemically induced changes in transcriptional profiles from the L1000 project. We found that the changes in transcriptional profiles for previously unexamined molecules were predicted with a Pearson correlation coefficient of 0.74. We examined three disorders and experimentally tested the top drug candidates in mouse disease models. Validation showed that perillen, chikusetsusaponin IV and trametinib confer disease-relevant impacts against obesity, hyperuricemia and nonalcoholic steatohepatitis, respectively. DLEPS can generate insights into pathogenic mechanisms, and we demonstrate that the MEK-ERK signaling pathway is a target for developing agents against nonalcoholic steatohepatitis. Our findings suggest that DLEPS is an effective tool for drug repurposing and discovery.

Emamectin benzoate induced enzymatic and transcriptional alternation in detoxification mechanism of predatory beetle Paederus fuscipes (Coleoptera: Staphylinidae) at the sublethal concentration.

In this study, the detoxification enzyme activity and the transcriptional profile changes in the second instar through RNA-sequencing technology due to emamectin benzoate (EB) were assessed. The cytochrome P450 monooxygenases (P450) enzyme activity was not altered by EB due to the change in concentration and exposure time in all treatments. The glutathione S-transferase (GST) enzyme was not considerably varying in all treatments, while exposure time significantly changed the enzyme activity. Results showed that the esterase (Ests) activity was elevated with the increasing concentrations and exposure time. Two libraries were generated, containing 107,767,542 and 108,142,289 clean reads for the samples treated with LC30 of EB and control. These reads were grouped into 218,070 transcripts and 38,097 unigenes. A total of 2257 differentially expressed genes (DEGs) were identified from these unigenes, of which 599 up-regulated and 1658 were down-regulated. The majority of these DEGs related to pesticides resistance were identified in numerous Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, e.g., steroid hormone biosynthesis, glutathione metabolism, drug metabolism-other enzymes, chemical carcinogenesis, pathways of cancer, metabolism of xenobiotics by cytochrome P450, drug metabolism of cytochrome P450, linoleic acid metabolism, retinol metabolism, and insect hormone biosynthesis. These pathways also shared the same genes as cytochrome P450 monooxygenases (P450s), glutathione S-transferases (GSTs), Esterase (Ests), UDP-glucosyltransferases (UGTs), and ATP-binding cassettes (ABCs). A heatmap analysis also showed that regulation of genes in a pathway causes a series of gene expression regulation in subsequent pathways. Our quantitative reverse transcription-PCR (qRT-PCR) results were consistent with the DEG's data of transcriptome analysis. The comprehensive transcriptome sequence resource attained through this study evidence that the EB induces significant modification in enzyme activity and transcriptome profile of Paederus fuscipes, which may enable more significant molecular underpinnings behind the insecticide-resistance mechanism for further investigations.

EMBR-01. CLASS I HDAC INHIBITORS AND PLK1 INHIBITORS SYNERGIZE IN MYC-AMPLIFIED MEDULLOBLASTOMA

BackgroundMedulloblastoma (MB) is one of the most common malignant pediatric CNS tumors. Patients with Group 3 MBs harboring MYC amplification exhibit low survival rates. Surviving patients suffer from therapy-induced sequelae, which calls for new targeted therapy strategies. We and others have previously shown the sensitivity of MYC-amplified MB to class I histone deacetylase (HDAC) inhibition. After demonstrating that the MYC target gene PLK1 is significantly downregulated upon class I HDACi treatment, we hypothesized that inhibition of both HDACs and PLK1 could have synergistic effects. MethodsCell metabolic activity changes upon HDAC and PLK1 inhibitor treatment were measured in MYC-amplified and non-amplified MB cell lines, as well as in an additional MYC-inducible cell line. The interaction effect of both inhibitors was determined by computation of the combination index (CI) using the Chou-Talalay method. Results were validated assessing cell viability, cell cycle, and apoptosis induction. Transcription profile changes after combination treatment were evaluated. Results MYC-amplified MB cell lines were more sensitive than non-amplified cell lines to PLK1i treatment, showing IC50 in clinically achievable concentration ranges. Inhibition of class I HDACs and PLK1 synergistically reduced cell metabolic activity in lower concentrations in MYC-amplified compared to non-amplified MB cell lines. We also observed a significant loss of viability and cells in G1 phase, as well as induction of apoptosis after combination treatment in MYC-amplified cells. MYC target gene sets were significantly downregulated in the MYC-amplified cell line HD-MB03 after treatment with combination. We demonstrated reduction of MYC protein levels upon PLK1i treatment. In vivo evaluation of combination treatment using orthotopic Group 3 MYC-amplified MB PDX models is ongoing. ConclusionOur data suggest that MYC-amplification is a predictive marker for PLK1i treatment in MB. The combination of HDACi and PLKi could be a candidate therapy for future clinical trials for MYC-amplified group 3 MB.

Transcriptional Analysis of Coccidioides immitis Mycelia and Spherules by RNA Sequencing.

Coccidioides immitis and C. posadasii are dimorphic fungi that transform from mycelia with internal arthroconidia in the soil to a tissue form known as a spherule in mammals. This process can be recapitulated in vitro by increasing the temperature, CO2 and changing other culture conditions. In this study, we have analyzed changes in gene expression in mycelia and young and mature spherules. Genes that were highly upregulated in young spherules include a spherule surface protein and iron and copper membrane transporters. Genes that are unique to Coccidioides spp. are also overrepresented in this group, suggesting that they may be important for spherule differentiation. Enriched GO terms in young spherule upregulated genes include oxidation-reduction, response to stress and membrane proteins. Downregulated genes are enriched for transcription factors, especially helix–loop–helix and C2H2 type zinc finger domain-containing proteins, which is consistent with the dramatic change in transcriptional profile. Almost all genes that are upregulated in young spherules remain upregulated in mature spherules, but a small number of genes are differentially expressed in those two stages of spherule development. Mature spherules express more Hsp31 and amylase and less tyrosinase than young spherules. Some expression of transposons was detected and most of the differentially expressed transposons were upregulated in spherules.

Acquisition of optimal TFH cell function is defined by specific molecular, positional, and TCR dynamic signatures

The development of follicular helper CD4 T (TFH) cells is a dynamic process resulting in a heterogenous pool of TFH subsets. However, the cellular and molecular determinants of this heterogeneity and the possible mechanistic links between them is not clear. We found that human TFH differentiation is associated with significant changes in phenotypic, chemokine, functional, metabolic and transcriptional profile. Furthermore, this differentiation was associated with distinct positioning to follicular proliferating B cells. Single-cell T cell receptor (TCR) clonotype analysis indicated the transitioning toward PD-1hiCD57hi phenotype. Furthermore, the differentiation of TFH cells was associated with significant reduction in TCR level and drastic changes in immunological synapse formation. TFH synapse lacks a tight cSMAC (central supra molecular activation Cluster) but displays the TCR in peripheral microclusters, which are potentially advantageous in the ability of germinal center (GC) B cells to receive necessary help. Our data reveal significant aspects of human TFH heterogeneity and suggest that the PD-1hiCD57hi TFH cells, in particular, are endowed with distinctive programming and spatial positioning for optimal GC B cell help.

Excessive ammonia inhalation causes liver damage and dysfunction by altering gene networks associated with oxidative stress and immune function

Ammonia (NH3) is a major gaseous pollutant in livestock production and has adverse effects on production, health and welfare of animals. The liver is one of the target organs of NH3, and excessive NH3 inhalation can induce liver damage. However, the toxicity assessment of NH3 on pig liver and its mechanism have not been reported yet. Recently, transcriptome analysis has become a major method to study the toxic mechanism of pollutants in environmental toxicology. Therefore, in the present study, we examined the effects of excessive NH3 inhalation on the liver of fattening pig through chemical analysis, ELISA, transcriptome analysis and real-time quantitative PCR (qRT-PCR). Our results showed that the transcriptome analysis database of fattening pig liver under excessive NH3 exposure, and 449 differentially expressed genes (DEGs) (including 181 up-regulated DEGs and 168 down-regulated DEGs) were found. Some genes associated with the 3 Gene Ontology (GO) terms (liver function, immune, antioxidant defense) were validated by quantitative real-time PCR. In addition, the activities of GPT and GOT in NH3 group were significantly increased by 63.5% and 37.4% (P < 0.05), respectively. Our results indicated that NH3 exposure could cause changes in transcriptional profiles and liver function, and induce liver damage in fattening pigs through oxidative stress and immune dysfunction. Our study results not only provide a new perspective for the toxicity assessment of NH3, but also enrich the toxicological mechanism of NH3.

Time-Resolved Transcriptional Profiling of Epithelial Cells Infected by Intracellular Acinetobacter baumannii

The rise in the number of antibiotic-resistant bacteria has become a serious threat to health, making it important to identify, characterize and optimize new molecules to help us to overcome the infections they cause. It is well known that Acinetobacter baumannii has a significant capacity to evade the actions of antibacterial drugs, leading to its emergence as one of the bacteria responsible for hospital and community-acquired infections. Nonetheless, how this pathogen infects and survives inside the host cell is unclear. In this study, we analyze the time-resolved transcriptional profile changes observed in human epithelial HeLa cells after infection by A. baumannii, demonstrating how it survives in host cells and starts to replicate 4 h post infection. These findings were achieved by sequencing RNA to obtain a set of Differentially Expressed Genes (DEGs) to understand how bacteria alter the host cells’ environment for their own benefit. We also determine common features observed in this set of genes and identify the protein–protein networks that reveal highly-interacted proteins. The combination of these findings paves the way for the discovery of new antimicrobial candidates for the treatment of multidrug-resistant bacteria.