Use of Microalgae-Derived Astaxanthin to Improve Cytoprotective Capacity in the Ileum of Heat-Induced Oxidative Stressed Broilers.

The gastrointestinal tract has a pivotal role in nutrient absorption, immune function, and overall homeostasis. The ileum segment of the small intestine plays respective roles in nutrient breakdown and absorption. The purpose of this study was to investigate the impact of heat-induced oxidative stress and the potential mitigating effects of an astaxanthin antioxidant treatment on the ileum of broilers. By comparing the growth performance and gene expression profiles among three groups-thermal neutral, heat stress, and heat stress with astaxanthin-thermal neutral temperature conditions of 21-22 °C and heat stress temperature of 32-35 °C, this research aims to elucidate the role of astaxanthin in supporting homeostasis and cellular protection in the ileum. Results showed both treatments under heat stress experienced reduced growth performance, while the group treated with astaxanthin showed a slightly lesser decline. Results further showed the astaxanthin treatment group significantly upregulated in the cytoprotective gene expression for HSF2, SOD2, GPX3, and TXN, as well as the upregulation of epithelial integrity genes LOX, CLDN1, and MUC2. In conclusion, our experimental findings demonstrate upregulation of cytoprotective and epithelial integrity genes, suggesting astaxanthin may effectively enhance the cellular response to heat stress to mitigate oxidative damage and contribute to cytoprotective capacity.

Circulating microRNA Biomarker for Detecting Breast Cancer in High-Risk Benign Breast Tumors.

High-risk benign breast tumors are known to develop breast cancer at high rates. However, it is still controversial whether they should be removed during diagnosis or followed up until cancer development becomes evident. Therefore, this study sought to identify circulating microRNAs (miRNAs) that could serve as detection markers of cancers arising from high-risk benign tumors. Small RNA-seq was performed using plasma samples collected from patients with early-stage breast cancer (CA) and high-risk (HB), moderate-risk (MB), and no-risk (Be) benign breast tumors. Proteomic profiling of CA and HB plasma was performed to investigate the underlying functions of the identified miRNAs. Our findings revealed that four miRNAs, hsa-mir-128-3p, hsa-mir-421, hsa-mir-130b-5p, and hsa-mir-28-5p, were differentially expressed in CA vs. HB and had diagnostic power to discriminate CA from HB with AUC scores greater than 0.7. Enriched pathways based on the target genes of these miRNAs indicated their association with IGF-1. Furthermore, the Ingenuity Pathway Analysis performed on the proteomic data revealed that the IGF-1 signaling pathway was significantly enriched in CA vs. HB. In conclusion, these findings suggest that these miRNAs could potentially serve as biomarkers for detecting early-stage breast cancer from high-risk benign tumors by monitoring IGF signaling-induced malignant transformation.

Single-cell RNA sequencing reveals characteristics of myeloid cells in post-acute sequelae of SARS-CoV-2 patients with persistent respiratory symptoms.

Background: Although our understanding of the immunopathology and subsequent risk and severity of COVID-19 disease is evolving, a detailed account of immune responses that contribute to the long-term consequences of pulmonary complications in COVID-19 infection remains unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequelae of SARS-CoV-2 infection (PASC) with persistent pulmonary symptoms. The dysregulation of the immune system that drives pulmonary sequelae in COVID-19 survivors and PASC sufferers remains largely unknown. Results: To characterize the immunological features of pulmonary PASC (PPASC), we performed droplet-based single-cell RNA sequencing (scRNA-seq) to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from a participant naïve to SARS-CoV-2 (Control) (n=1) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC) (n=2). After integrating scRNA-seq data with a naïve participant from a published dataset, 11 distinct cell populations were identified based on the expression of canonical markers. The proportion of myeloid-lineage cells ([MLCs]; CD14+/CD16+monocytes, and dendritic cells) was increased in PPASC (n=2) compared to controls (n=2). MLCs from PPASC displayed up-regulation of genes associated with pulmonary symptoms/fibrosis, while glycolysis metabolism-related genes were downregulated. Similarly, pathway analysis showed that fibrosis-related (VEGF, WNT, and SMAD) and cell death pathways were up-regulated, but immune pathways were down-regulated in PPASC. Further comparison of PPASC with scRNA-seq data with Severe COVID-19 (n=4) data demonstrated enrichment of fibrotic transcriptional signatures. In PPASC, we observed interactive VEGF ligand-receptor pairs among MLCs, and network modules in CD14+ (cluster 4) and CD16+ (Cluster 5) monocytes displayed a significant enrichment for biological pathways linked to adverse COVID-19 outcomes, fibrosis, and angiogenesis. Further analysis revealed a distinct metabolic alteration in MLCs with a down-regulation of glycolysis/gluconeogenesis in PPASC compared to SARS-CoV-2 naïve samples. Conclusion: Analysis of a small scRNA-seq dataset demonstrated alterations in the immune response and cellular landscape in PPASC. The presence of elevated MLC levels and their corresponding gene signatures associated with fibrosis, immune response suppression, and altered metabolic states suggests a potential role in PPASC development.

Exploring Optimal Biomarker Sources: A Comparative Analysis of Exosomes and Whole Plasma in Fasting and Non-Fasting Conditions for Liquid Biopsy Applications.

The study of liquid biopsy with plasma samples is being conducted to identify biomarkers for clinical use. Exosomes, containing nucleic acids and metabolites, have emerged as possible sources for biomarkers. To evaluate the effectiveness of exosomes over plasma, we analyzed the small non-coding RNAs (sncRNAs) and metabolites extracted from exosomes in comparison to those directly extracted from whole plasma under both fasting and non-fasting conditions. We found that sncRNA profiles were not affected by fasting in either exosome or plasma samples. Our results showed that exosomal sncRNAs were found to have more consistent profiles. The plasma miRNA profiles contained high concentrations of cell-derived miRNAs that were likely due to hemolysis. We determined that certain metabolites in whole plasma exhibited noteworthy concentration shifts in relation to fasting status, while others did not. Here, we propose that (1) fasting is not required for a liquid biopsy study that involves both sncRNA and metabolomic profiling, as long as metabolites that are not influenced by fasting status are selected, and (2) the utilization of exosomal RNAs promotes robust and consistent findings in plasma samples, mitigating the impact of batch effects derived from hemolysis. These findings advance the optimization of liquid biopsy methodologies for clinical applications.

RNA-Sequencing based analysis of bovine endometrium during the maternal recognition of pregnancy.

BACKGROUND: Maternal recognition is the crucial step for establishing pregnancy in cattle. This study aims to identify endometrial genes and biological pathways involved in the maternal recognition of pregnancy. Caruncular endometrial tissues were collected from Day 15-17 of gestation (pregnant), non-pregnant (absence of conceptus), and cyclic (non-bred) heifers. RESULTS: Total RNAs were isolated from the caruncular endometrial tissues of pregnant, non-pregnant, and cyclic heifers, and were subjected to high-throughput RNA-sequencing. The genes with at least two-fold change and Benjamini and Hochberg p-value ≤ 0.05 were considered differentially expressed genes and further confirmed with quantitative real-time PCR. A total of 107 genes (pregnant vs cyclic) and 98 genes (pregnant vs non-pregnant) were differentially expressed in the pregnant endometrium. The most highly up-regulated genes in the pregnant endometrium were MRS2, CST6, FOS, VLDLR, ISG15, IFI6, MX2, C15H11ORF34, EIF3M, PRSS22, MS4A8, and TINAGL1. Interferon signaling, immune response, nutrient transporter, synthesis, and secretion of proteins are crucial pathways during the maternal recognition of pregnancy. CONCLUSIONS: The study demonstrated that the presence of conceptus at Day 15-17 of gestation affects the endometrial gene expression related to endometrial remodeling, immune response, nutrients and ion transporters, and relevant signaling pathways in the caruncular region of bovine endometrium during the maternal recognition of pregnancy.

T-cell activation decreases miRNA-15a/16 levels to promote MEK1-ERK1/2-Elk1 signaling and proliferative capacity.

While miRs have been extensively studied in the context of malignancy and tumor progression, their functions in regulating T-cell activation are less clear. In initial studies, we found reduced levels of miR-15a/16 at 3 to 18 h post-T-cell receptor (TCR) stimulation, suggesting a role for decreased levels of this miR pair in shaping T-cell activation. To further explore this, we developed an inducible miR15a/16 transgenic mouse model to determine how elevating miR-15a/16 levels during early stages of activation would affect T-cell proliferation and to identify TCR signaling pathways regulated by this miR pair. Doxycycline (DOX)-induced expression of miR-15a/16 from 0 to 18 h post-TCR stimulation decreased ex vivo T-cell proliferation as well as in vivo antigen-specific T-cell proliferation. We also combined bioinformatics and proteomics approaches to identify the mitogen-activated protein kinase kinase 1 (MEK1) (Map2k1) as a target of miR-15a/16. MEK1 targeting by miR-15a/16 was confirmed using miR mimics that decreased Map2k1 mRNA containing the 3'-UTR target nucleotide sequence (UGCUGCUA) but did not decrease Map2k1 containing a mutated control sequence (AAAAAAAA). Phosphorylation of downstream signaling molecules, extracellular signal-regulated protein kinase 1/2 (ERK1/2) and Elk1, was also decreased by DOX-induced miR-15a/16 expression. In addition to MEK1, ERK1 was subsequently found to be targeted by miR-15a/16, with DOX-induced miR-15a/16 reducing total ERK1 levels in T cells. These findings show that TCR stimulation reduces miR-15a/16 levels at early stages of T-cell activation to facilitate increased MEK1 and ERK1, which promotes the sustained MEK1-ERK1/2-Elk1 signaling required for optimal proliferation.

RSK1 and RSK2 serine/threonine kinases regulate different transcription programs in cancer.

The 90 kDa ribosomal S6 kinases (RSKs) are serine threonine kinases comprising four isoforms. The isoforms can have overlapping functions in regulation of migration, invasion, proliferation, survival, and transcription in various cancer types. However, isoform specific differences in RSK1 versus RSK2 functions in gene regulation are not yet defined. Here, we delineate ribosomal S6 kinases isoform-specific transcriptional gene regulation by comparing transcription programs in RSK1 and RSK2 knockout cells using microarray analysis. Microarray analysis revealed significantly different mRNA expression patterns between RSK1 knockout and RSK2 knockout cell lines. Importantly some of these functions have not been previously recognized. Our analysis revealed RSK1 has specific roles in cell adhesion, cell cycle regulation and DNA replication and repair pathways, while RSK2 has specific roles in the immune response and interferon signaling pathways. We further validated that the identified gene sets significantly correlated with mRNA datasets from cancer patients. We examined the functional significance of the identified transcriptional programs using cell assays. In alignment with the microarray analysis, we found that RSK1 modulates the mRNA and protein expression of Fibronectin1, affecting cell adhesion and CDK2, affecting S-phase arrest in the cell cycle, and impairing DNA replication and repair. Under similar conditions, RSK2 showed increased ISG15 transcriptional expression, affecting the immune response pathway and cytokine expression. Collectively, our findings revealed the occurrence of RSK1 and RSK2 specific transcriptional regulation, defining separate functions of these closely related isoforms.

The miRNA Profile of Inflammatory Colorectal Tumors Identify TGF-ß as a Companion Target for Checkpoint Blockade Immunotherapy.

Extrinsic factors such as expression of PD-L1 (programmed dealth-ligand 1) in the tumor microenvironment (TME) have been shown to correlate with responses to checkpoint blockade therapy. More recently two intrinsic factors related to tumor genetics, microsatellite instability (MSI), and tumor mutation burden (TMB), have been linked to high response rates to checkpoint blockade drugs. These response rates led to the first tissue-agnostic approval of any cancer therapy by the FDA for the treatment of metastatic, MSI-H tumors with anti-PD-1 immunotherapy. But there are still very few studies focusing on the association of miRNAs with immune therapy through checkpoint inhibitors. Our team sought to explore the biology of such tumors further and suggest potential companion therapeutics to current checkpoint inhibitors. Analysis by Pearson Correlation revealed 41 total miRNAs correlated with mutation burden, 62 miRNAs correlated with MSI, and 17 miRNAs correlated with PD-L1 expression. Three miRNAs were correlated with all three of these tumor features as well as M1 macrophage polarization. No miRNAs in any group were associated with overall survival. TGF-ß was predicted to be influenced by these three miRNAs (p = 0.008). Exploring miRNA targets as companions to treatment by immune checkpoint blockade revealed three potential miRNA targets predicted to impact TGF-ß. M1 macrophage polarization state was also associated with tumors predicted to respond to therapy by immune checkpoint blockade.

Bacterial Diversity Correlates with Overall Survival in Cancers of the Head and Neck, Liver, and Stomach.

One in five cancers is attributed to infectious agents, and the extent of the impact on the initiation, progression, and disease outcomes may be underestimated. Infection-associated cancers are commonly attributed to viral, and to a lesser extent, parasitic and bacterial etiologies. There is growing evidence that microbial community variation rather than a single agent can influence cancer development, progression, response to therapy, and outcome. We evaluated microbial sequences from a subset of infection-associated cancers-namely, head and neck squamous cell carcinoma (HNSC), liver hepatocellular carcinoma (LIHC), and stomach adenocarcinoma (STAD) from The Cancer Genome Atlas (TCGA). A total of 470 paired tumor and adjacent normal samples were analyzed. In STAD, concurrent presence of EBV and Selemonas sputigena with a high diversity index were associated with poorer survival (HR: 2.23, 95% CI 1.26-3.94, p = 0.006 and HR: 2.31, 95% CI 1.1-4.9, p = 0.03, respectively). In LIHC, lower microbial diversity was associated with poorer overall survival (HR: 2.57, 95% CI: 1.2, 5.5, p = 0.14). Bacterial within-sample diversity correlates with overall survival in infection-associated cancers in a subset of TCGA cohorts.

Ischemic heart injury leads to HIF1-dependent differential splicing of CaMK2γ.

Ischemic heart disease is a leading cause of heart failure and hypoxia inducible factor 1 (HIF1) is a key transcription factor in the response to hypoxic injury. Our lab has developed a mouse model in which a mutated, oxygen-stable form of HIF1α (HIF-PPN) can be inducibly expressed in cardiomyocytes. We observed rapid cardiac dilation and loss of contractility in these mice due to lower expression of excitation-contraction coupling genes and reduced calcium flux. As alternative splicing plays an underappreciated role in transcriptional regulation, we used RNA sequencing to search for splicing changes in calcium-handling genes of HIF-PPN hearts and compared them to previous sequencing data from a model of myocardial infarction (MI) to select for transcripts that are modified in a pathological setting. We found overlap between genes differentially expressed in HIF-PPN and post-MI mice (54/131 genes upregulated in HIF-PPN hearts at 1 day and/or 3 days post-MI, and 45/78 downregulated), as well as changes in alternative splicing. Interestingly, calcium/calmodulin dependent protein kinase II, gamma (CAMK2G) was alternatively spliced in both settings, with variant 1 (v1) substantially decreased compared to variants 2 (v2) and 3 (v3). These findings were also replicated in vitro when cells were transfected with HIF-PPN or exposed to hypoxia. Further analysis of CAMK2γ protein abundance revealed only v1 was detectable and substantially decreased up to 7 days post-MI. Rbfox1, a splicing factor of CAMK2G, was also decreased in HIF-PPN and post-MI hearts. Subcellular fractionation showed CAMK2γ v1 was found in the nuclear and cytoplasmic fractions, and abundance decreased in both fractions post-MI. Chromatin immunoprecipitation analysis of HIF1 in post-MI hearts also demonstrated direct HIF1 binding to CAMK2G. CaMK2 is a key transducer of calcium signals in both physiological and pathological settings. The predominantly expressed isoform in the heart, CaMK2δ, has been extensively studied in cardiac injury, but the specific role of CaMK2γ is not well defined. Our data suggest that loss of CaMK2γ after MI is HIF1-dependent and may play an important role in the heart's calcium signaling and transcriptional response to hypoxia.

Dysregulated KRAS gene-signaling axis and abnormal chromatin remodeling drive therapeutic resistance in heterogeneous-sized circulating tumor cells in gastric cancer patients.

The mechanism by which heterogeneous-sized circulating tumor cells (CTCs) in gastric cancer (GC) patients are resistant to the targeted therapy and/or chemotherapy remains unclear. This study investigated prognostic value and genomic variations of size-heterogenous CTCs, in an attempt to unravel the molecular mechanisms underlying the therapeutic resistance, which is relevant to poor prognosis in GC. Aneuploid CTCs, detected in 111 advanced GC patients, were categorized into small (≤white blood cell [WBC], 25.54%) and large (>WBC, 74.46%) cells. Pre-treatment patients possessing ≥3 baseline small CTCs with trisomy 8 (SCTCstri) or ≥6 large multiploid CTCs (LCTCsmulti) showed an inferior median progression-free survival. Moreover, the cut-off value of ≥6 LCTCsmulti was also an effective prognosticator for poor median overall survival. Single cell-based DNA sequencing of 50 targeted CTCs indicated that SCTCstri and LCTCsmulti harbored distinct gene variations respectively. Mutations in the KRAS and Rap1 pathway were remarkably abundant in SCTCstri, whereas several unique mutations in the MET/PI3K/AKT pathway and SMARCB1 gene were identified in LCTCsmulti. Obtained results suggested that SCTCstri and LCTCsmulti exhibited different mechanisms to therapy resistance and correlated with patients' poor outcome.

Upregulated ethanolamine phospholipid synthesis via selenoprotein I is required for effective metabolic reprogramming during T cell activation.

OBJECTIVE: T cell activation triggers metabolic reprogramming to meet increased demands for energy and metabolites required for cellular proliferation. Ethanolamine phospholipid synthesis has emerged as a regulator of metabolic shifts in stem cells and cancer cells, which led us to investigate its potential role during T cell activation. METHODS: As selenoprotein I (SELENOI) is an enzyme participating in two metabolic pathways for the synthesis of phosphatidylethanolamine (PE) and plasmenyl PE, we generated SELENOI-deficient mouse models to determine loss-of-function effects on metabolic reprogramming during T cell activation. Ex vivo and in vivo assays were carried out along with metabolomic, transcriptomic, and protein analyses to determine the role of SELENOI and the ethanolamine phospholipids synthesized by this enzyme in cell signaling and metabolic pathways that promote T cell activation and proliferation. RESULTS: SELENOI knockout (KO) in mouse T cells led to reduced de novo synthesis of PE and plasmenyl PE during activation and impaired proliferation. SELENOI KO did not affect T cell receptor signaling, but reduced activation of the metabolic sensor AMPK. AMPK was inhibited by high [ATP], consistent with results showing SELENOI KO causing ATP accumulation, along with disrupted metabolic pathways and reduced glycosylphosphatidylinositol (GPI) anchor synthesis/attachment CONCLUSIONS: T cell activation upregulates SELENOI-dependent PE and plasmenyl PE synthesis as a key component of metabolic reprogramming and proliferation.

Adaptive Thermogenesis in a Mouse Model Lacking Selenoprotein Biosynthesis in Brown Adipocytes.

Selenoproteins are a class of proteins with the selenium-containing amino acid selenocysteine (Sec) in their primary structure. Sec is incorporated into selenoproteins via recoding of the stop codon UGA, with specific cis and trans factors required during translation to avoid UGA recognition as a stop codon, including a Sec-specific tRNA, tRNA[Ser]Sec, encoded in mice by the gene Trsp. Whole-body deletion of Trsp in mouse is embryonically lethal, while targeted deletion of Trsp in mice has been used to understand the role of selenoproteins in the health and physiology of various tissues. We developed a mouse model with the targeted deletion of Trsp in brown adipocytes (Trspf/f-Ucp1-Cre+/-), a cell type predominant in brown adipose tissue (BAT) controlling energy expenditure via activation of adaptive thermogenesis, mostly using uncoupling protein 1 (Ucp1). At room temperature, Trspf/f-Ucp1-Cre+/- mice maintain oxygen consumption and Ucp1 expression, with male Trspf/f-Ucp1-Cre+/- mice accumulating more triglycerides in BAT than both female Trspf/f-Ucp1-Cre+/- mice or Trspf/f controls. Acute cold exposure neither reduced core body temperature nor changed the expression of selenoprotein iodothyronine deiodinase type II (Dio2), a marker of adaptive thermogenesis, in Trspf/f-Ucp1-Cre+/- mice. Microarray analysis of BAT from Trspf/f-Ucp1-Cre+/- mice revealed glutathione S-transferase alpha 3 (Gsta3) and ELMO domain containing 2 (Elmod2) as the transcripts most affected by the loss of Trsp. Male Trspf/f-Ucp1-Cre+/- mice showed mild hypothyroidism while downregulating thyroid hormone-responsive genes Thrsp and Tshr in their BATs. In summary, modest changes in the BAT of Trspf/f-Ucp1-Cre +/- mice implicate a mild thyroid hormone dysfunction in brown adipocytes.

Selenoprotein M Promotes Hypothalamic Leptin Signaling and Thioredoxin Antioxidant Activity.

Aims: Selenoproteins are an essential class of proteins involved in redox signaling and energy metabolism. However, the functions of many selenoproteins are not clearly established. Selenoprotein M (SELENOM), an endoplasmic reticulum (ER)-resident oxidoreductase bearing structural similarity to thioredoxin (TXN), is among those yet to be fully characterized. This protein is highly expressed in hypothalamic regions involved in leptin signaling and has been previously linked to energy metabolism. Herein, we performed a series of studies using in vivo and in vitro models to probe the specific influence of SELENOM on hypothalamic leptin signaling and assess SELENOM-regulated pathways. Innovation and Results: Our initial experiment in vivo demonstrated that (i) leptin promotes hypothalamic expression of SELENOM and (ii) leptin-induced STAT3 phosphorylation is impeded by SELENOM deficiency. Additional in vitro studies using mHypoE-44 immortalized hypothalamic neurons corroborated these findings, as SELENOM deficiency obstructed downstream STAT3 phosphorylation and cytosolic calcium responses evoked by leptin treatment. Correspondingly, SELENOM overexpression enhanced leptin sensitivity. Microarray analysis conducted in parallel on hypothalamic tissue and mHypoE-44 cells revealed multiple genes significantly affected by SELENOM deficiency, including thioredoxin interacting protein, a negative regulator of the TXN system. Further analysis determined that (i) SELENOM itself possesses intrinsic TXN activity and (ii) SELENOM deficiency leads to a reduction in overall TXN activity. Finally, mHypoE-44 cells lacking SELENOM displayed diminished activation of the nuclear factor kappa-light-chain enhancer of activated B-cells (NF-κB) signaling pathway and increased susceptibility to ER stress-mediated cell death. Conclusion: In sum, these findings establish SELENOM as a positive regulator of leptin signaling and TXN antioxidant activity in the hypothalamus. Antioxid. Redox Signal. 35, 775-787.

Tissue-associated microbial detection in cancer using human sequencing data.

Cancer is one of the leading causes of morbidity and mortality in the globe. Microbiological infections account for up to 20% of the total global cancer burden. The human microbiota within each organ system is distinct, and their compositional variation and interactions with the human host have been known to attribute detrimental and beneficial effects on tumor progression. With the advent of next generation sequencing (NGS) technologies, data generated from NGS is being used for pathogen detection in cancer. Numerous bioinformatics computational frameworks have been developed to study viral information from host-sequencing data and can be adapted to bacterial studies. This review highlights existing popular computational frameworks that utilize NGS data as input to decipher microbial composition, which output can predict functional compositional differences with clinically relevant applicability in the development of treatment and prevention strategies.

miR-125 family regulates XIRP1 and FIH in response to myocardial infarction.

MicroRNAs (miRNAs) are powerful regulators of protein expression. Many play important roles in cardiac development and disease. While several miRNAs and targets have been well characterized, the abundance of miRNAs and the numerous potential targets for each suggest that the vast majority of these interactions have yet to be described. The goal of this study was to characterize miRNA expression in the mouse heart after coronary artery ligation (LIG) and identify novel mRNA targets altered during the initial response to ischemic stress. We performed small RNA sequencing (RNA-Seq) of ischemic heart tissue 1 day and 3 days after ligation and identified 182 differentially expressed miRNAs. We then selected relevant mRNA targets from all potential targets by correlating miRNA and mRNA expression from a corresponding RNA-Seq data set. From this analysis we chose to focus, as proof of principle, on two miRNAs from the miR-125 family, miR-125a and miR-351, and two of their potential mRNA targets, Xin actin-binding repeat-containing protein 1 (XIRP1) and factor inhibiting hypoxia-inducible factor (FIH). We found miR-125a to be less abundant and XIRP1 more abundant after ligation. In contrast, the related murine miRNA miR-351 was substantially upregulated in response to ischemic injury, and FIH expression correspondingly decreased. Luciferase reporter assays confirmed direct interactions between these miRNAs and targets. In summary, we utilized a correlative analysis strategy combining miRNA and mRNA expression data to identify functional miRNA-mRNA relationships in the heart after ligation. These findings provide insight into the response to ischemic injury and suggest future therapeutic targets.

Changes in gastrointestinal microbial communities influence HIV-specific CD8+ T-cell responsiveness to immune checkpoint blockade.

OBJECTIVES: The aim of this study was to examine the relationship between gut microbial communities in HIV-infected individuals on suppressive antiretroviral therapy (cART), and the peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo immune checkpoint blockade (ICB). DESIGN: Thirty-four HIV-seropositive, 10 HIV-seronegative and 12 HIV-seropositive receiving faecal microbiota transplant (FMT) participants were included. Gut microbial communities, peripheral and gut associated negative checkpoint receptors (NCRs) and peripheral effector functions were assessed. METHODS: Bacterial 16s rRNA sequencing for gut microbiome study and flow-based assays for peripheral and gut NCR and their cognate ligand expression, including peripheral HIV-Gag-specific CD8 T-cell responses before and after ex-vivo anti-PD-L1 and anti-TIGIT ICB were performed. RESULTS: Fusobacteria abundance was significantly higher in HIV-infected donors compared to uninfected controls. In HIV-infected participants receiving Fusobacteria-free FMT, Fusobacteria persisted up to 24 weeks in stool post FMT. PD-1 TIGIT and their ligands were expanded in mucosal vs. peripheral T cells and dendritic cells, respectively. PD-L1 and TIGIT blockade significantly increased the magnitude of peripheral anti-HIV-Gag-specific CD8 T-cell responses. Higher gut Fusobacteria abundance was associated with lower magnitude of peripheral IFN-γ+ HIV-Gag-specific CD8 T-cell responses following ICB. CONCLUSION: The gut colonization of Fusobacteria in HIV infection is persistent and may influence anti-HIV T-cell immunity to PD-1 or TIGIT blockade. Strategies modulating Fusobacteria colonization may elicit a favourable mucosal immune landscape to enhance the efficacy of ICB for HIV cure.

The landscape of bacterial presence in tumor and adjacent normal tissue across 9 major cancer types using TCGA exome sequencing.

Identification of microbial composition directly from tumor tissue permits studying the relationship between microbial changes and cancer pathogenesis. We interrogated bacterial presence in tumor and adjacent normal tissue strictly in pairs utilizing human whole exome sequencing to generate microbial profiles. Profiles were generated for 813 cases from stomach, liver, colon, rectal, lung, head & neck, cervical and bladder TCGA cohorts. Core microbiota examination revealed twelve taxa to be common across the nine cancer types at all classification levels. Paired analyses demonstrated significant differences in bacterial shifts between tumor and adjacent normal tissue across stomach, colon, lung squamous cell, and head & neck cohorts, whereas little or no differences were evident in liver, rectal, lung adenocarcinoma, cervical and bladder cancer cohorts in adjusted models. Helicobacter pylori in stomach and Bacteroides vulgatus in colon were found to be significantly higher in adjacent normal compared to tumor tissue after false discovery rate correction. Computational results were validated with tissue from an independent population by species-specific qPCR showing similar patterns of co-occurrence among Fusobacterium nucleatum and Selenomonas sputigena in gastric samples. This study demonstrates the ability to identify bacteria differential composition derived from human tissue whole exome sequences. Taken together our results suggest the microbial profiles shift with advanced disease and that the microbial composition of the adjacent tissue can be indicative of cancer stage disease progression.

Disabled Homolog 2 (DAB2) Protein in Tumor Microenvironment Correlates with Aggressive Phenotype in Human Urothelial Carcinoma of the Bladder.

Disabled homolog-2 (DAB2) has been reported to be a tumor suppressor gene. However, a number of contrary studies suggested that DAB2 promotes tumor invasion in urothelial carcinoma of the bladder (UCB). Here, we investigated the clinical role and biological function of DAB2 in human UCB. Immunohistochemical staining analysis for DAB2 was carried out on UCB tissue specimens. DAB2 expression levels were compared with clinicopathological factors. DAB2 was knocked-down by small interfering RNA (siRNA) transfection, and then its effects on cell proliferation, invasion, and migration, and changes to epithelial-mesenchymal transition (EMT)-related proteins were evaluated. In our in vivo assays, tumor-bearing athymic nude mice subcutaneously inoculated with human UCB cells (MGH-U-3 or UM-UC-3) were treated by DAB2-targeting siRNA. Higher expression of DAB2 was associated with higher clinical T category, high tumor grade, and poor oncological outcome. The knock-down of DAB2 decreased both invasion and migration ability and expression of EMT-related proteins. Significant inhibitory effects on tumor growth and invasion were observed in xenograft tumors of UM-UC-3 treated by DAB2-targeting siRNA. Our findings suggested that DAB2 expression was associated with poor prognosis through increased oncogenic properties including tumor proliferation, migration, invasion, and enhancement of EMT in human UCB.

Lower Interferon Regulatory Factor-8 Expression in Peripheral Myeloid Cells Tracks With Adverse Central Nervous System Outcomes in Treated HIV Infection.

Cognitive dysfunction persists in 30-50% of chronically HIV-infected individuals despite combination antiretroviral therapy (ART). Although monocytes are implicated in poor cognitive performance, distinct biological mechanisms associated with cognitive dysfunction in HIV infection are unclear. We previously showed that a regulatory region of the interferon regulatory factor-8 (IRF8) gene is hyper-methylated in HIV-infected individuals with cognitive impairment compared to those with normal cognition. Here, we investigated IRF-8 protein expression and assessed relationships with multiple parameters associated with brain health. Intracellular IRF-8 expression was measured in cryopreserved peripheral blood mononuclear cells from chronically HIV-infected individuals on ART using flow cytometry. Neuropsychological performance was assessed by generating domain-specific standardized (NPZ) scores, with a global score defined by aggregating individual domain scores. Regional brain volumes were obtained by magnetic resonance imaging and soluble inflammatory factors were assessed by immunosorbent assays. Non-parametric analyses were conducted and statistical significance was defined as p < 0.05. Twenty aviremic (HIV RNA<50 copies/ml) participants, 84% male, median age 51 [interquartile range (IQR) 46, 55], median CD4 count 548 [439, 700] were evaluated. IRF-8 expression was highest in plasmacytoid dendritic cells (pDCs). Assessing cognitive function, lower IRF-8 density in classical monocytes significantly correlated with worse NPZ_learning memory (LM; rho = 0.556) and NPZ_working memory (WM; rho = 0.612) scores, in intermediate monocytes with worse NPZ_LM (rho = 0.532) scores, and in non-classical monocytes, lower IRF-8 correlated with worse global NPZ (rho = 0.646), NPZ_LM (rho = 0.536), NPZ_WM (rho = 0.647), and NPZ_executive function (rho = 0.605) scores. In myeloid DCs (mDCs) lower IRF-8 correlated with worse NPZ_WM (rho = 0.48) scores and in pDCs with worse NPZ_WM (rho = 0.561) scores. Declines in IRF-8 in classical monocytes significantly correlated with smaller hippocampal volume (rho = 0.573) and in intermediate and non-classical monocytes with smaller cerebral white matter volume (rho = 0.509 and rho = 0.473, respectively). IRF-8 density in DCs did not significantly correlate with brain volumes. Among biomarkers tested, higher soluble ICAM-1 levels significantly correlated with higher IRF-8 in all monocyte and DC subsets. These data may implicate IRF-8 as a novel transcription factor in the neuropathophysiology of brain abnormalities in treated HIV and serve as a potential therapeutic target to decrease the burden of cognitive dysfunction in this population.