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.

sRNAfrag: A pipeline and suite of tools to analyze fragmentation in small RNA sequencing data.

Fragments derived from small RNAs such as small nucleolar RNAs hold biological relevance. However, they remain poorly understood, calling for more comprehensive methods for analysis. We developed sRNAfrag, a standardized workflow and set of scripts to quantify and analyze sRNA fragmentation of any biotype. In a benchmark, it is able to detect loci of mature microRNAs fragmented from precursors and, utilizing multi-mapping events, the conserved 5' seed sequence of miRNAs which we believe may extraoplate to other small RNA fragments. The tool detected 1411 snoRNA fragment conservation events between 2/4 eukaryotic species, providing the opportunity to explore motifs and fragmentation patterns not only within species, but between. Availability: https://github.com/kenminsoo/sRNAfrag.

Single-cell RNA sequencing reveals characteristics of myeloid cells in pulmonary post-acute sequelae of SARS-CoV-2.

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 complication in COVID-19 infection remain unclear. Few studies have detailed the immune and cytokine profiles associated with post-acute sequalae of SARS-CoV-2 infection with persistent pulmonary symptoms (PPASC). However, 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 to study the transcriptomic profiles of peripheral blood mononuclear cells (PBMCs) from participants naïve to SARS-CoV-2 (Control) and infected with SARS-CoV-2 with chronic pulmonary symptoms (PPASC). We analyzed more than 34,139 PBMCs by integrating our dataset with previously reported control datasets (GSM4509024) cell distribution. In total, 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 compared to controls. 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. 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: This study offers valuable insights into 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 their potential role as a driver of PPASC.

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.

sRNAfrag: a pipeline and suite of tools to analyze fragmentation in small RNA sequencing data.

Fragments derived from small RNAs such as small nucleolar RNAs are biologically relevant but remain poorly understood. To address this gap, we developed sRNAfrag, a modular and interoperable tool designed to standardize the quantification and analysis of small RNA fragmentation across various biotypes. The tool outputs a set of tables forming a relational database, allowing for an in-depth exploration of biologically complex events such as multi-mapping and RNA fragment stability across different cell types. In a benchmark test, sRNAfrag was able to identify established loci of mature microRNAs solely based on sequencing data. Furthermore, the 5' seed sequence could be rediscovered by utilizing a visualization approach primarily applied in multi-sequence-alignments. Utilizing the relational database outputs, we detected 1411 snoRNA fragment conservation events between two out of four eukaryotic species, providing an opportunity to explore motifs through evolutionary time and conserved fragmentation patterns. Additionally, the tool's interoperability with other bioinformatics tools like ViennaRNA amplifies its utility for customized analyses. We also introduce a novel loci-level variance-score which provides insights into the noise around peaks and demonstrates biological relevance by distinctly separating breast cancer and neuroblastoma cell lines after dimension reduction when applied to small nucleolar RNAs. Overall, sRNAfrag serves as a versatile foundation for advancing our understanding of small RNA fragments and offers a functional foundation to further small RNA research. Availability: https://github.com/kenminsoo/sRNAfrag.

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.

Adherence to Mediterranean diet impacts gastrointestinal microbial diversity throughout pregnancy.

BACKGROUND: Consumption of a diet with high adherence to a Mediterranean diet pattern (MDP) has been associated with a favorable gastrointestinal tract (GIT) microbiome. A healthy GIT microbiome in pregnancy, as defined by increased alpha diversity, is associated with lower chance of adverse perinatal outcomes. This study aimed to evaluate the impact of adherence to an MDP on GIT microbial diversity longitudinally throughout pregnancy. METHODS: Adherence to MDP was scored by the Alternate Mediterranean (aMED) Diet Quality Score, after being applied to a validated Food Frequency Questionnaire. Association of aMED Scores with GIT alpha diversity profiles were compared linearly and across time using a linear mixed model, including covariates of age, body mass index (BMI), ethnicity, and parity. RESULTS: Forty-one participants of Filipino, Japanese, Native Hawaiian, and Non-Hispanic White descent provided dietary information and microbiome samples during each trimester of pregnancy. Alpha diversity profiles changed over gestation, with decreased microbial diversity in the third trimester. aMED scores positively correlated with Chao1 Index and Observed Species Number (r = 0.244, p = 0.017, and r = 0.233, p = 0.023, respectively). The strongest association was detected in the third trimester (Chao 1: r = 0.43, p = 0.020, Observed Species Number: r = 0.41, p = 0.026). Participants with higher aMED scores had higher relative abundance of Acidaminoacaeae at the family level (p = 0.0169), as well as higher abundance of several species known to increase production of short chain fatty acids within the GIT. CONCLUSIONS: Adherence to MDP pattern is associated with increased maternal GIT microbial diversity, and promotes the abundance of bacteria that produce short chain fatty acids. Increased consumption of fruits, vegetables and legumes with low red meat consumption were key components driving this association. The effect of nutrition however, was less of an effect than pregnancy itself. Further studies are needed to determine if adherence to a Mediterranean diet translates not only into microbial health, but also into reduced risk of adverse pregnancy outcomes.

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.

RNA sequencing-based analysis of the magnum tissues revealed the novel genes and biological pathways involved in the egg-white formation in the laying hen.

BACKGROUND: The mechanism of egg formation in the oviduct of laying hens is tightly controlled; each segment of the oviduct contributes a unique component of the egg. Several genes/proteins are involved in the synthesis of a completely healthy egg. This implies a time- and tissue-specific expression of genes and proteins in the different oviductal segments. We used hens at different physiological stages and time points to understand the transcriptional regulation of egg-white (albumen) synthesis and secretion onto the eggs in the magnum of laying hens. This study used Next-Generation Sequencing and quantitative real-time PCR (qPCR) to detect the novel genes and the cognate biological pathways that regulate the major events during the albumen formation. RESULTS: Magnum tissues collected from laying (n = 5 each at 3 h post-ovulation, p.o. and 15-20 h p.o.), non-laying (n = 4), and molting (n = 5) hens were used for differential gene expression analyses. A total of 540 genes (152 upregulated and 388 down-regulated) were differentially expressed at 3 h p.o. in the magnum of laying hens. Kyoto Encyclopedia of Genes and Genomes pathways analysis of the 152 upregulated genes revealed that glycine, serine, and threonine metabolism was the most-enriched biological pathway. Furthermore, the top two most enriched keywords for the upregulated genes were amino-acid biosynthesis and proteases. Nine candidate genes associated with albumen formation were validated with qPCR to have differential expression in laying, non-laying, and molting hens. Proteases such as TMPRSS9, CAPN2, MMP1, and MMP9 (protein maturation, ECM degradation, and angiogenesis); enzymes such as PSPH, PHGDH, and PSAT1 (amino-acid biosynthesis); RLN3, ACE, and REN (albumen synthesis, secretion and egg transport); and AVD, AvBD11, and GPX3 (antimicrobial and antioxidants) were recognized as essential molecules linked to albumen deposition in the magnum. CONCLUSIONS: This study revealed some novel genes that participate in the signaling pathways for egg-white synthesis and secretion along with some well-known functional genes. These findings help to understand the mechanisms involved in albumen biosynthesis.

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.