Dual RNA-Seq of Trunk Kidneys Extracted From Channel Catfish Infected With Yersinia ruckeri Reveals Novel Insights Into Host-Pathogen Interactions

Host-pathogen intectarions are complex, involving large dynamic changes in gene expression through the process of infection. These interactions are essential for understanding anti-infective immunity as well as pathogenesis. In this study, the host-pathogen interaction was analyzed using a model of acute infection where channel catfish were infected with Yersinia ruckeri. The infected fish showed signs of body surface hyperemia as well as hyperemia and swelling in the trunk kidney. Double RNA sequencing was performed on trunk kidneys extracted from infected channel catfish and transcriptome data was compared with data from uninfected trunk kidneys. Results revealed that the host-pathogen interaction was dynamically regulated and that the host-pathogen transcriptome fluctuated during infection. More specifically, these data revealed that the expression levels of immune genes involved in Cytokine-cytokine receptor interactions, the NF-kappa B signaling pathway, the JAK-STAT signaling pathway, Toll-like receptor signaling and other immune-related pathways were significantly upregulated. Y. ruckeri mainly promote pathogenesis through the flagellum gene fliC in channel catfish. The weighted gene co-expression network analysis (WGCNA) R package was used to reveal that the infection of catfish is closely related to metabolic pathways. This study contributes to the understanding of the host-pathogen interaction between channel catfish and Y. ruckeri, more specifically how catfish respond to infection through a transcriptional perspective and how this infection leads to enteric red mouth disease (ERM) in these fish.

Novel Immune Subtypes Identification of HER2 Positive Breast Cancer Based On Immunogenomic Landscape

Background: HER2 positive BC is heterogeneous. But few studies discussed the classification of HER2 positive BC based on immune-related signatures.Methods: Using two publicly BC genomics datasets, we classified HER2 positive BC based on 33 immune-related signatures and used unsupervised machine learning methods to predict and perform the classification.Results: We grouped three HER2 positive BC subtypes that we called Immune-High (IM-H), Immune-Medium (IM-M), and Immune-Low (IM-L), and manifested this categorization was predictable, duplicable and reliable by analyzing another dataset. Compared to other subtypes, IM-H had a higher immune cell infiltration level and stronger anti-tumor immune activities, as well as better clinical survival outcome. Besides these signatures, there were some cancer-related pathways which were hyperactivated in IM-H, including cytokine-cytokine receptor interactions, antigen processing and presentation pathways, natural killer cell-mediated cytotoxicity, Th1 and Th2 cell differentiation, chemokine signaling pathway, Th17 cell differentiation, B and T cell receptor signaling, NF-kappa B signaling, PD-L1 expression and PD-1 checkpoint pathway in cancer, TNF signaling, IL-17 signaling, NOD-like receptor signaling and Toll-like receptor signaling. By contrast, IM-L showed depressed immune-related signatures and enhanced activation of lycosylphosphatidylinositol-anchor biosynthesis and mismatch repair. Moreover, we discovered a gene co-expression network focused on eight transcription factor genes (EOMES, TBX21, GFI1, IRF4, POU2AF1, CIITA, FOXP3 and TOX) and one tumor suppress gene (PRF1), which were closely related with tumor immune.Conclusion: We identified three HER2 positive BC subtypes based on immune-related signatures, which had potential clinical implications and promoted the optimal stratification of HER2 positive BC responsive to immunotherapy.

Associations between DNA methylation and BMI vary by metabolic health status: a potential link to disparate cardiovascular outcomes

Background Body mass index (BMI), a well-known risk factor for poor cardiovascular outcomes, is associated with differential DNA methylation (DNAm). Similarly, metabolic health has also been associated with changes in DNAm. It is unclear how overall metabolic health outside of BMI may modify the relationship between BMI and methylation profiles, and what consequences this may have on downstream cardiovascular disease. The purpose of this study was to identify cytosine-phosphate-guanine (CpG) sites at which the association between BMI and DNAm could be modified by overall metabolic health. Results The discovery study population was derived from three Women’s Health Initiative (WHI) ancillary studies (n = 3977) and two Atherosclerosis Risk in Communities (ARIC) ancillary studies (n = 3520). Findings were validated in the Multi-Ethnic Study of Atherosclerosis (MESA) cohort (n = 1200). Generalized linear models regressed methylation β values on the interaction between BMI and metabolic health Z score (BMI × MHZ) adjusted for BMI, MHZ, cell composition, chip number and location, study characteristics, top three ancestry principal components, smoking, age, ethnicity (WHI), and sex (ARIC). Among the 429,566 sites examined, differential associations between BMI × MHZ and DNAm were identified at 22 CpG sites (FDR q < 0.05), with one site replicated in MESA (cg18989722, in the TRAPPC9 gene). Three of the 22 sites were associated with incident coronary heart disease (CHD) in WHI. For each 0.01 unit increase in DNAm β value, the risk of incident CHD increased by 9% in one site and decreased by 6–10% in two sites over 25 years. Conclusions Differential associations between DNAm and BMI by MHZ were identified at 22 sites, one of which was validated (cg18989722) and three of which were predictive of incident CHD. These sites are located in several genes related to NF-kappa-B signaling, suggesting a potential role for inflammation between DNA methylation and BMI-associated metabolic health.

Exploring the Mechanisms of Arsenic Trioxide (Pishuang) in Hepatocellular Carcinoma Based on Network Pharmacology

Objective. Arsenic trioxide (Pishuang, Pishi, arsenolite, As2O3, and CAS 1327-53-3), a naturally occurring and toxic mineral as a drug for more than 2000 years in China, has been found to have a valuable function in hepatocellular carcinoma (HCC) in recent years. However, its exact mechanism remains to be elucidated. Therefore, this study was intended to explore the potential anti-HCC mechanism of arsenic trioxide through network pharmacology. Methods. The potential targets of arsenic trioxide were collected from PubChem and TargetNet. HCC targets were obtained from the GeneCards database. Then, a protein-protein interaction (PPI) network of arsenic trioxide and HCC common targets was established using STRING. GO and KEGG pathway enrichment analyses were performed by the Database for Annotation, Visualization, and Integrated Discovery (DAVID). Finally, an arsenic trioxide-target-pathway-HCC network was built by Cytoscape 3.2.1, and network topological analysis was carried out to screen the key candidate targets. Results. A total of 346 corresponding targets of arsenic trioxide and 521 HCC-related targets were collected. After target mapping, a total of 52 common targets were obtained. GO analysis showed that the biological process was mainly involved in the negative regulation of cellular senescence, response to tumor necrosis factor, and cellular response to hypoxia. Molecular functions included NF-kappa B binding, enzyme binding, p53 binding, and transcription factor binding. Cellular components mainly were replication fork, ESC/E(Z) complex, RNA polymerase II transcription factor complex, and organelle membrane. KEGG pathways were mainly enriched in the PI3K-Akt signaling pathway, VEGF signaling pathway, p53 signaling pathway, HIF-1 signaling pathway, TNF signaling pathway, AMPK signaling pathway, NF-kappa B signaling pathway, FoxO signaling pathway, ErbB signaling pathway, and MAPK signaling pathway. In the arsenic trioxide-target-pathway-HCC network, targets such as AKT1, RAF1, RELA, TP53, and PTEN had a higher degree. Conclusions. Our study showed that key targets of arsenic trioxide were mainly involved in multiple biological processes and pathways. It provided a theoretical basis for the screening of drug targets.

Genome-wide characterization of SARS-CoV-2 cytopathogenic proteins in the search of antiviral targets

Therapeutic inhibition of critical viral functions is important for curtailing coronavirus disease-2019 (COVID-19). We sought to identify antiviral targets through genome-wide characterization of SARS-CoV-2 proteins that are crucial for viral pathogenesis and that cause harmful cytopathic effects. All twenty-nine viral proteins were tested in a fission yeast cell-based system using inducible gene expression. Twelve proteins including eight non-structural proteins (NSP1, NSP3, NSP4, NSP5, NSP6, NSP13, NSP14 and NSP15) and four accessory proteins (ORF3a, ORF6, ORF7a and ORF7b) were identified that altered cellular proliferation and integrity, and induced cell death. Cell death correlated with the activation of cellular oxidative stress. Of the twelve proteins, ORF3a was chosen for further study in mammalian cells. In human pulmonary and kidney epithelial cells, ORF3a induced cellular oxidative stress associated with apoptosis and necrosis, and caused activation of pro-inflammatory response with production of the cytokines TNF-alpha, IL-6, and IFN-beta1, possibly through the activation of NF-kappa B. To further characterize the mechanism, we tested a natural ORF3a Beta variant, Q57H, and a mutant with deletion of the highly conserved residue, deltaG188. Compared to wild type ORF3a, the delataG188 variant yielded more robust activation of cellular oxidative stress, cell death, and innate immune response. Since cellular oxidative stress and inflammation contribute to cell death and tissue damage linked to the severity of COVID-19, our findings suggest that ORF3a is a promising, novel therapeutic target against COVID-19.

EFFECT OF AMMONIUM PYRROLIDINE DITHIOCARBAMBATE ON THE FORMATION OF RACTIVE OXYGEN AND NITROGEN SPECIES IN LIVER OF RATS KEPT ON CARBOHYDRATE-LIPID DIET AND EXPOSED TO ROUND-THE-CLOCK LIGHTING

This aim of the experiment performed on 21 white rats is to clear up the effect of the nuclear factor kappa B (NF-kappa B) inhibitor on the production of reactive oxygen and nitrogen species in the liver of animals kept on carbohydrate-lipid diet and exposed to round-the-clock lighting. The study has demonstrated the administration of ammonium pyrrolidine diothiocarbamate (76 mg/kg three times a week, starting on the 30th day of the experiment) under the conditions of high-calorie carbohydrate-lipid diet (60 days) and exposure of the test animals to round-the-clock light (1500 lux for the last 30 days of the experiment) reduces the production of superoxide anion radical in the liver tissues by NADPH- and NADH-dependent electron transport chains by 45.2% and 43.5%. The production of this radical by leukocytes by NADPH oxidase was 41.6% lower than in the comparison group. The activity of NO-synthase (total and its inducible isoenzyme) decreased by 34.4 and 29.8%, the activity of the constitutive isoform increased threefold. The coupling index of the latter elevated by 5.16 times that indicates the restoration of the coupling state of this isoenzyme. The decrease in the generation of superoxide anion radical and nitric oxide was accompanied by lowering in the peroxynitrites concentration by 36.4%. We can conclude that the administration of ammonium pyrrolidine dithiocarbamate, an NF-kappa B inhibitor, during the metabolic syndrome simulated in rats by round-the-clock lightening exposure and diet rich in carbohydrates and fats is an effective means to restrict the production of reactive oxygen species.

Comprehensive Analysis of N6-Methyladenosine (m6A) Methylation in Neuromyelitis Optica Spectrum Disorders

Background: N6-Methyladenosine (m6A) methylation is the most prevalent internal posttranscriptional modification on mammalian mRNA. But its role in neuromyelitis optica spectrum disorders (NMOSD) is not known.Aims: To explore the mechanism of m6A in NMOSD patients.Methods: This study assessed the m6A methylation levels in blood from two groups: NMOSD patients and healthy controls. Methylated RNA immunoprecipitation Sequencing (MeRIP-seq) and RNA-seq were performed to assess differences in m6A methylation between NMOSD patients and healthy controls. Ultra-high performance liquid chromatography coupled with triple quadruple mass spectrometry (UPLC-QQQ-MS) method was performed to check m6A level. Differential m6A methylation genes were validated by MeRIP-qPCR.Results: Compared with that in the control group, the total m6A level was decreased in the NMOSD group. Genes with upregulated methylation were primarily enriched in processes associated with RNA splicing, mRNA processing, and innate immune response, while genes with downregulated methylation were enriched in processes associated with the regulation of transcription, DNA-templating, and the positive regulation of I-kappa B kinase/NF-kappa B signalling.Conclusion: These findings demonstrate that differential m6A methylation may act on functional genes to regulate immune homeostasis in NMOSD.

Proteasome Inhibition Attenuates Self-Renewal in Human Acute Myeloid Leukemia By Targeting NF-Kappa B in Leukemia Stem Cells

In acute myeloid leukemia (AML), relapse following standard chemotherapy is common, leading to 2-year survival rates of less than 30%. Relapse is caused by leukemia stem cells (LSCs), a rare population of mostly quiescent cells that are chemo-refractory and can recapitulate the disease. Our overall goal is to define mechanisms of self-renewal that could be targeted to cure AML and prevent relapse. Functionally, self-renewal is defined by the ability to propagate leukemia in vivo. We use transcriptional and protein profiling to define the functional states of LSCs. We previously demonstrated that NRAS G12V facilitates self-renewal in a mouse model of AML (Mll-AF9/NRAS G12V). Using single-cell RNA sequencing and in vivo leukemia assays, we showed that the stem cell compartment in this model has two distinct subpopulations which differ in their self-renewal and proliferative abilities. The subset of LSCs marked by CD36 LowCD69 High (CD69 High) expression can self-renew and are poorly proliferative. The CD36 HighCD69 Low (CD36 High) subset is unable to self-renew and is highly proliferative. These data demonstrated that self-renewal and rapid proliferation are mutually exclusive functions among LSCs. We demonstrated that the gene expression profiles associated with these functionally distinct LSC subsets are also similarly differentially expressed in primary human LSCs, at the single-cell level (Sachs Cancer Research 2020). Given the functional differences between the CD36 LowCD69 High and CD36 HighCD69 Low subsets in murine AML, we tested whether CD69 and CD36 likewise discriminate self-renewal and proliferation in human AML. We sorted primary human AML samples according to CD69 and CD36 expression and found that Lin -CD69 High cells formed more colonies than Lin -CD36 High cells in every sample tested (n=6). Notably, in three of the six samples, the Lin -CD36 High population was unable to form any colonies (Panel A). These data suggest that CD69 may identify a self-renewing subset of human AML as it does in murine AML. Next, we asked whether Lin -CD69 High and Lin -CD36 High subsets harbor unique signaling protein activation profiles that could potentially be targeted therapeutically. We used CyTOF (mass cytometry) to compare the levels of 12 intracellular signaling proteins between these subpopulations in 14 human AML samples with intermediate and poor risk genetics. CyTOF quantitatively measures a panel of proteins at the single-cell level and allows us to profile activated signaling pathways within well-defined immunophenotypic subpopulations. We found upregulation of both total and phosphorylated (t- and p-) NF-kappa B, pERK, p4EBP1, pMAPKAPKII, and its upstream activator, pP38 in the Lin -CD69 High subset relative to the Lin -CD36 High. Notably, CD69 is a NF-kappa B target gene. Indeed, our single-cell transcriptional data showed that NF-kappa B target genes were upregulated in the self-renewing, CD69 High subset of murine and human LSCs (Sachs Cancer Research 2020). NF-kappa B is a well-known mediator of AML-self-renewal. Next, we asked whether targeting NF-kappa B with proteasome inhibitors might reduce self-renewal capacity in human AML. We plated six primary human samples in colony-forming assays with pan-proteasome inhibitor, carfilzomib, immunoproteasome inhibitor, PR957, or vehicle and found that both inhibitors reduced colony formation in every sample tested. We harvested colonies from each of the inhibitor and vehicle groups and plated them in secondary colony-forming assays. Colonies harvested from vehicle-treated samples formed colonies but those harvested from either carfilzomib or PR957-treated samples were unable to form any secondary colonies (Panel B). Using CyTOF, we validated that both inhibitors reduce NF-kappa B levels in these samples. These data demonstrate that CD69 marks a self-renewing subset of human AML, as it does in our murine AML model, and suggest that the transcriptional mechanisms of self-renewal that we defined in our murine model at the single-cell level may be shared with human AML. Additionally, these data demonstrate enhanced NF-kappa B levels in this self-renewing subset and that targeting the NF-kappa B pathway with proteasome inhibitors attenuates self-renewal in primary human AML samples (Panel C). These findings suggest that proteasome inhibition may be an effective approach to treating and preventing relapse in AML. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Transcriptome Dynamics Reveal Stage-Specific and Melatonin-Triggered Gene Expression Patterns During The Cashmere Growth Cycle in Capra Hircus

Background Cashmere goat is famous for its high-quality fibers. The growth of cashmere in secondary hair follicles exhibits a seasonal pattern arising from circannual changes in the natural photoperiod. Although several studies have compared and analyzed the differences in gene expression between different cashmere growth stages, the selection of samples in these studies relies on research experience or morphological evidence. Distinguishing cashmere growth cycle according to gene expression patterns may help to explore the regulation mechanisms related to cashmere growth and the effect of melatonin from a molecular level more accurately. Results In this study, we applied RNA-sequencing to the hair follicles of three normal and three melatonin-treated Inner Mongolian cashmere goats sampled every month during a whole cashmere growth cycle. A total of 3559 and 988 genes were subjected as seasonal changing genes (SCGs) in the control and treated groups, respectively. The SCGs in the normal group are divided into three clusters, and their specific expression patterns help to group the cashmere growth cycle into anagen, catagen and telogen stages. Some canonical pathways such as Wnt, TGF-beta and Hippo signaling pathways are detected as promoting the cashmere growth, while Cell adhesion molecules (CAMs), Cytokine-cytokine receptor interaction, Jak-STAT, Fc epsilon RI, NOD-like receptor, Rap1, PI3K-Akt, cAMP, NF-kappa B and many immune-related pathways are detected in the catagen and telogen stages. The PI3K-Akt signaling, ECM-receptor interaction and Focal adhesion are found in the transition stage between telogen to anagen, which may serve as candidate biomarkers for telogen-anagen regeneration. Pairwise comparisons between the control and melatonin-treated groups also indicate 941 monthly differentially expressed genes (monthly DEGs). These monthly DEGs are mainly distributed from April and September, which reveal a potential signal pathway map regulating the anagen stage triggered by melatonin. Enrichment analysis shows that Wnt, Hedgehog, ECM, Chemokines and NF-kappa B signaling pathways may be involved in the regulation of non-quiescence and secondary shedding under the influence of melatonin. Conclusions Our study decodes the key regulators of the whole cashmere growth cycle, laying the foundation for the control of cashmere growth and improvement of cashmere yield.