Differential Expression Changes Research Articles

Abstract LB012: Evaluating computational approaches for CPTAC pan-cancer cross-cohort protein expression comparison

Abstract Recently, the National Cancer Institute’s Clinical Proteomic Tumor Analysis Consortium (CPTAC) has generated harmonized genomic, transcriptomic, proteomic, and clinical data for >1000 tumors in 10 cohorts to facilitate pan-cancer discovery research. However, protein expression comparison across CPTAC cohorts remains challenging due to non-uniform missing data and protein expression distribution patterns across tumor types. Here we present our efforts to evaluate various missing data handling and normalization strategies to generate a normalized pan-cancer protein expression dataset. First, we developed a novel algorithm to select robustly expressed proteins in tumors in any of the CPTAC cohorts, Second, we applied a cohort hybrid imputation approach to protein abundance values from FragPipe within each cohort based on protein expression distribution patterns. Third, we calculated iBAQ using protein abundance value and applied global quantile normalization or smooth quantile normalization methods. To assess if our missing data imputation and normalization strategy affected downstream analyses, we compared the fold change in differential protein expression between tumor and matched normal for each cohort using non-normalized, global quantile normalized and smooth quantile normalized protein iBAQ values. Our results demonstrate a strong correlation in fold change between global quantile normalized data and non-normalized data (Pearson r = 0.97 (ccRCC), r = 0.96 (COAD), r = 0.99 (LUAD) and r = 0.99 (LSCC)). Similar results were observed comparing smooth quantile normalized data to non-normalized data (Pearson r = 1.00 for ccRCC, COAD, LUAD, and LSCC), indicating both normalization methods retained biological differences between tumor and matched normal tissues within cohorts. Lastly, we identified several proteins (ERAP2, CA9, GSTM3, MX1, STAT1) whose protein and RNA expression were highly correlated across eight CPTAC cohorts (r > 0.7 for COAD, BRCA, LUAD, ccRCC, PDAC, UCEC, HNSCC, and LSCC). We then compared their protein expression rank across CPTAC cohorts with their RNA expression rank across corresponding TCGA cohorts. Specifically, median log2(iBAQ) of CPTAC and median log2(TPM) of TCGA are calculated for those proteins within indications, then indications are ranked by median log2(iBAQ) and median log2(TPM) in CPTAC and TCGA, respectively. Weighted rank correlation was used to measure rank agreement. Global quantile normalization has the highest rank correlation (weighted rank correlation between 0.597 to 0.931) compared to smooth quantile normalization or without normalization. These results suggest that combination of cohort hybrid imputation and global quantile normalization is a reasonable approach to generate a normalized CPTAC pan-cancer protein dataset that could be leveraged to interrogate protein expression across different cancer types. Citation Format: Jixin Wang, Xiaowen Tian, Wen Yu, John Bullen, Elaine Hurt, Wenyan Zhong. Evaluating computational approaches for CPTAC pan-cancer cross-cohort protein expression comparison [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB012.

Kidney toxicity and transcriptome analyses of male ICR mice acutely exposed to the mushroom toxin α-amanitin

Amatoxins are responsible for most fatal mushroom poisoning cases, as it causes both hepatotoxicity and nephrotoxicity. However, studies on amatoxin nephrotoxicity are limited. Here, we investigated nephrotoxicity over 4 days and nephrotoxicity/hepatotoxicity over 14 days in mice. The organ weight ratio, serological indices, and tissue histology results indicated that a nephrotoxicity mouse model was established with two stages: (1) no apparent effects within 24 h; and (2) the appearance of adverse effects, with gradual worsening within 2–14 days. For each stage, the kidney transcriptome revealed patterns of differential mRNA expression and significant pathway changes, and Western blot analysis verified the expression of key proteins. Amanitin-induced nephrotoxicity was directly related to RNA polymerase II because mRNA levels decreased, RNA polymerase II-related pathways were significantly enriched at the transcription level, and RNA polymerase II protein was degraded in the early poisoning stage. In the late stage, nephrotoxicity was more severe than hepatotoxicity. This is likely associated with inflammation because inflammation-related pathways were significantly enriched and NF-κB activation was increased in the kidney.

Single-cell RNA sequencing reveals roles of unique retinal microglia types in early diabetic retinopathy

BackgroundThe pathophysiological mechanisms of diabetic retinopathy (DR), a blinding disease, are intricate. DR was thought to be a microvascular disease previously. However, growing studies have indicated that the retinal microglia-induced inflammation precedes microangiopathy. The binary concept of microglial M1/M2 polarization paradigms during inflammatory activation has been debated. In this study, we confirmed microglia had the most significant changes in early DR using single-cell RNA sequencing.MethodsA total of five retinal specimens were collected from donor SD rats. Changes in various cells of the retina at the early stage of DR were analyzed using single-cell sequencing technology.ResultsWe defined three new microglial subtypes at cellular level, including two M1 types (Egr2+ M1 and Egr2− M1) and one M2 type. We also revealed the anatomical location between these subtypes, the dynamic changes of polarization phenotypes, and the possible activation sequence and mutual activation regulatory mechanism of different cells. Furthermore, we constructed an inflammatory network involving microglia, blood-derived macrophages and other retinal nonneuronal cells. The targeted study of new disease-specific microglial subtypes can shorten the time for drug screening and clinical application, which provided insight for the early control and reversal of DR.ConclusionsWe found that microglia show the most obvious differential expression changes in early DR and reveal the changes in microglia in a high-glucose microenvironment at the single-cell level. Our comprehensive analysis will help achieve early reversal and control the occurrence and progression of DR.

Leveraging baseline transcriptional features and information from single-cell data to power the prediction of influenza vaccine response.

Vaccination is still the primary means for preventing influenza virus infection, but the protective effects vary greatly among individuals. Identifying individuals at risk of low response to influenza vaccination is important. This study aimed to explore improved strategies for constructing predictive models of influenza vaccine response using gene expression data. We first used gene expression and immune response data from the Immune Signatures Data Resource (IS2) to define influenza vaccine response-related transcriptional expression and alteration features at different time points across vaccination via differential expression analysis. Then, we mapped these features to single-cell resolution using additional published single-cell data to investigate the possible mechanism. Finally, we explored the potential of these identified transcriptional features in predicting influenza vaccine response. We used several modeling strategies and also attempted to leverage the information from single-cell RNA sequencing (scRNA-seq) data to optimize the predictive models. The results showed that models based on genes showing differential expression (DEGs) or fold change (DFGs) at day 7 post-vaccination performed the best in internal validation, while models based on DFGs had a better performance in external validation than those based on DEGs. In addition, incorporating baseline predictors could improve the performance of models based on days 1-3, while the model based on the expression profile of plasma cells deconvoluted from the model that used DEGs at day 7 as predictors showed an improved performance in external validation. Our study emphasizes the value of using combination modeling strategy and leveraging information from single-cell levels in constructing influenza vaccine response predictive models.

Abscisic Acid Affects Phenolic Acid Content to Increase Tolerance to UV-B Stress in Rhododendron chrysanthum Pall.

The presence of the ozone hole increases the amount of UV radiation reaching a plant's surface, and UV-B radiation is an abiotic stress capable of affecting plant growth. Rhododendron chrysanthum Pall. (R. chrysanthum) grows in alpine regions, where strong UV-B radiation is present, and has been able to adapt to strong UV-B radiation over a long period of evolution. We investigated the response of R. chrysanthum leaves to UV-B radiation using widely targeted metabolomics and transcriptomics. Although phytohormones have been studied for many years in plant growth and development and adaptation to environmental stresses, this paper is innovative in terms of the species studied and the methods used. Using unique species and the latest research methods, this paper was able to add information to this topic for the species R. chrysanthum. We treated R. chrysanthum grown in a simulated alpine environment, with group M receiving no UV-B radiation and groups N and Q (externally applied abscisic acid treatment) receiving UV-B radiation for 2 days (8 h per day). The results of the MN group showed significant changes in phenolic acid accumulation and differential expression of genes related to phenolic acid synthesis in leaves of R. chrysanthum after UV-B radiation. We combined transcriptomics and metabolomics data to map the metabolic regulatory network of phenolic acids under UV-B stress in order to investigate the response of such secondary metabolites to stress. L-phenylalanine, L-tyrosine and phenylpyruvic acid contents in R. chrysanthum were significantly increased after UV-B radiation. Simultaneously, the levels of 3-hydroxyphenylacetic acid, 2-phenylethanol, anthranilate, 2-hydroxycinnamic acid, 3-hydroxycinnamic acid, α-hydroxycinnamic acid and 2-hydroxy-3-phenylpropanoic acid in this pathway were elevated in response to UV-B stress. In contrast, the study in the NQ group found that externally applied abscisic acid (ABA) in R. chrysanthum had greater tolerance to UV-B radiation, and phenolic acid accumulation under the influence of ABA also showed greater differences. The contents of 2-phenylethanol, 1-o-p-coumaroyl-β-d-glucose, 2-hydroxy-3-phenylpropanoic acid, 3-(4-hydroxyphenyl)-propionic acid and 3-o-feruloylquinic ac-id-o-glucoside were significantly elevated in R. chrysanthum after external application of ABA to protect against UV-B stress. Taken together, these studies of the three groups indicated that ABA can influence phenolic acid production to promote the response of R. chrysanthum to UV-B stress, which provided a theoretical reference for the study of its complex molecular regulatory mechanism.

Comparative Analysis of Differential Cellular Transcriptome and Proteome Regulation by HIV-1 and HIV-2 Pseudovirions in the Early Phase of Infection.

In spite of the similar structural and genomic organization of human immunodeficiency viruses type 1 and 2 (HIV-1 and HIV-2), striking differences exist between them in terms of replication dynamics and clinical manifestation of infection. Although the pathomechanism of HIV-1 infection is well characterized, relatively few data are available regarding HIV-2 viral replication and its interaction with host-cell proteins during the early phase of infection. We utilized proteo-transcriptomic analyses to determine differential genome expression and proteomic changes induced by transduction with HIV-1/2 pseudovirions during 8, 12 and 26 h time-points in HEK-293T cells. We show that alteration in the cellular milieu was indeed different between the two pseudovirions. The significantly higher number of genes altered by HIV-2 in the first two time-points suggests a more diverse yet subtle effect on the host cell, preparing the infected cell for integration and latency. On the other hand, GO analysis showed that, while HIV-1 induced cellular oxidative stress and had a greater effect on cellular metabolism, HIV-2 mostly affected genes involved in cell adhesion, extracellular matrix organization or cellular differentiation. Proteomics analysis revealed that HIV-2 significantly downregulated the expression of proteins involved in mRNA processing and translation. Meanwhile, HIV-1 influenced the cellular level of translation initiation factors and chaperones. Our study provides insight into the understudied replication cycle of HIV-2 and enriches our knowledge about the use of HIV-based lentiviral vectors in general.

Transcutaneous electrical acupoint stimulation attenuated neuroinflammation and oxidative stress by activating SIRT1-induced signaling pathway in MCAO/R rat models

BackgroundSilent information regulator 1 (SIRT1) plays a beneficial role in cerebral ischemic injury. Previous reports have demonstrated that transcutaneous electrical acupoint stimulation (TEAS) exerts a beneficial effect on ischemic stroke; however, whether SIRT1 participates in the underlying mechanism for the neuroprotective effects of TEAS against ischemic brain damage has not been confirmed. MethodsThe rat models of middle cerebral artery occlusion/reperfusion (MCAO/R) were utilized in the current experiment. After MCAO/R surgery, rats in TEAS, EC and EX group received TEAS intervention with or without the injection of EX527, the SIRT1 inhibitor. Neurological deficit scores, infarct volume, hematoxylin eosin (HE) staining and apoptotic cell number were measured. The results of RNA sequencing were analyzed to determine the differential expression changes of genes among sham, MCAO and TEAS groups, in order to investigate the possible pathological processes involved in cerebral ischemia and explore the protective mechanisms of TEAS. Moreover, oxidative stress markers including MDA, SOD, GSH and GSH-Px were measured with assay kits. The levels of the proinflammatory cytokines, such as IL-6, IL-1β and TNF-α, were detected by ELISA assay, and Iba-1 (the microglia marker protein) positive cells was measured by immunofluorescence (IF). Western blot and IF were utilized to examine the levels of key molecules in SIRT1/FOXO3a and SIRT1/BRCC3/NLRP3 signaling pathways. ResultsTEAS significantly decreased brain infarcted size and apoptotic neuronal number, and alleviated neurological deficit scores and morphological injury by activating SIRT1. The results of RNA-seq and bioinformatic analysis revealed that oxidative stress and inflammation were the key pathological mechanisms, and TEAS alleviated oxidative injury and inflammatory reactions following ischemic stroke. Then, further investigation indicated that TEAS notably attenuated neuronal apoptosis, neuroinflammation and oxidative stress damage in the hippocampus of rats with MCAO/R surgery. Moreover, TEAS intervention in the MCAO/R model significantly elevated the expressions of SIRT1, FOXO3a, CAT, BRCC3, NLRP3 in the hippocampus. Furthermore, EX527, as the inhibitor of SIRT1, obviously abolished the anti-oxidative stress and anti-neuroinflammatory roles of TEAS, as well as reversed the TEAS-mediated elevation of SIRT1, FOXO3a, CAT and reduction of BRCC3 and NLRP3 mediated by following MCAO/R surgery. ConclusionsIn summary, these findings clearly suggested that TEAS attenuated brain damage by suppressing apoptosis, oxidative stress and neuroinflammation through modulating SIRT1/FOXO3a and SIRT1/BRCC3/NLRP3 signaling pathways following ischemic stroke, which can be a promising treatment for stroke patients.

Single Cell RNASeq Analysis of AD and Control Post Mortem Brain Samples

Alzheimer’s disease (AD) is a devastative late life disease, molecularly characterized by biochemical lesions of amyloid β plaques and Tau tangles. the 2nd most frequent one worldwide with no early detection methods/cure. I have applied single cell sequencing and analysed post mortem brain samples (using a Chromium machine) from 2 AD and 2 control frontal cortex samples. All the samples had ethics approval, and the RNA was tested for quality. The samples were obtained from the Edenborough MRC brain bank. Very few genes have been linked to far to AD (mainly through GWAS studies [1]). Overall, I quantified about 24,000 nuclei. Using statistical, bioinformatics and computational analyses I have detected significant differential expression changes in astrocyte, neuronal and microglial marker genes. I also conducted network analyses. The percentage of reads passing filter was slightly low (68%). Overall output was a bit lower than optimal. There was still over 1.6Bn clusters generated though, which is within the specification for an S1 flow-cell, and base call quality (quality control) was excellent: 97% >Q30. The read counts mapping to genome occupancy rate was also fairy low (63-81%). The number of barcodes is as follows: sample 1 (AD, Braak I-II) - 6,223 sample 2 (control) 6,262 sample 3 (AD Braak I-II) 5,332 sample 4 (AD, Braak I-II) 5,332 as well. The data is available for download under the GEO (accession number GSE175814). to assess global and cell-type specific gene expression impacts in Alzheimer’s versus control brain samples, we performed a holistic comparison of matching cell populations in AD and controls with the software cell Harmony. To identify possible gene regulatory network underlying transcriptional regulation in these distinct populations, we further explored prior evidenced transcription factor targets relationships from cell Harmony. Importantly, we observe both common and cell-type specific core transcription factors regulators, with both enriched downstream targets and regulated transcription factors. Upregulated networks in Astrocytes were associated with broad regulation by HIF1A, SOX2, NRF1, and RB1. the proportion of endothelial cells was higher in the Alzheimer’s disease samples than the neurological control samples. results reveal that the cell type-specific transcriptomic changes in Alzheimer’s disease are associated with 4 molecular pathways: angiogenesis in endothelial cells, immune response in endothelial cells and microglia, myelination in oligodendrocytes, and synaptic signalling in astrocytes and neurons. I also compared the results with microarray data from large cohort studies that examined samples from the prefrontal cortex (Alzheimer’s disease: n = 310; Neurological controls: n = 157) or temporal cortex (Alzheimer’s disease: n = 106; Neurological controls: n = 135). Among the DEGs identified in our snRNA-seq analysis, 1,113 and 764 genes were significantly differentially expressed in the microarray data from the prefrontal cortex and temporal cortex, respectively. subcluster analysis of microglia identified 13 subpopulations; VGF signalling was also found as changed. In the future, imaging of post mortem Alzheimer’s patients may verify brain cellular morphology/cell volume of vascular changes in the Alzheimer’s compared to control samples. My study findings [2] may open new venues to our understanding of the disease underlying molecular processes and cellular signaling. Regarding the glial cell changes, our main conclusion is that the glial cells may be reactive in the disease and not causal.

Maternal separation modifies spontaneous synaptic activity in the infralimbic cortex of stress-resilient male rats.

Glutamate and GABA signaling systems are necessary to maintain proper function of the central nervous system through excitation/inhibition (E/I) balance. Alteration of this balance in the medial prefrontal cortex (mPFC), as an effect of early-life stress, may lead to the development of anxiety and depressive disorders. Few studies exist in the infralimbic division of the mPFC to understand the effect of early-life stress at different ages, which is the purpose of the present work. Newborn Sprague Dawley male rats were subjected to maternal separation (MS) for two weeks. First, tests measuring anxiety- and depression-like behaviors were performed on adolescent and adult rats subjected to MS (MS-rats). Then, to establish a relationship with behavioral results, electrophysiological recordings were performed in neurons of the infralimbic cortex in acute brain slices of infant, adolescent, and adult rats. In the behavioral tests, there were no significant differences in MS-rats compared to control rats at any age. Moreover, MS had no effect on the passive membrane properties nor neuronal excitability in the infralimbic cortex, whereas spontaneous synaptic activity in infralimbic neurons was altered. The frequency of spontaneous glutamatergic synaptic events increased in infant MS-rats, whereas in adolescent MS-rats both the frequency and the amplitude of spontaneous GABAergic events increased without any effect on glutamatergic synaptic responses. In adult MS-rats, these two parameters decreased in spontaneous GABAergic synaptic events, whereas only the frequency of glutamatergic events decreased. These data suggest that rats subjected to MS did not exhibit behavioral changes and presented an age-dependent E/I imbalance in the infralimbic cortex, possibly due to differential changes in neurotransmitter release and/or receptor expression.

Age-Dependent Regulation of Dendritic Spine Density and Protein Expression in Mir324 KO Mice.

Dendritic spines are small, dynamic protrusions along the dendrite that comprise more than 90% of excitatory connections in the brain, making them essential sites for neuronal communication. These synaptic sites change throughout the process of development, reducing in density and shifting morphology as synapses are refined. One important class of dendritic spine regulators is microRNA (miRNA), small-noncoding RNAs that post-transcriptionally regulate gene expression. Several studies suggest that miRNA-324-5p regulates dendritic spine formation. In addition, we have previously shown that miR-324-5p plays a role in seizure and long-term potentiation, both of which involve dendritic spine changes. In this study, we aimed to characterize the role of miRNA-324-5p in developmental spine regulation by assessing the effect of Mir324 knockout (KO) on dendritic spine density and expression of a subset of dendritic proteins at select developmental time points. We show that miR-324-5p expression is developmentally regulated and peaks at 4weeks of age. We demonstrate that loss of miR-324-5p expression leads to differential changes in both target protein expression and spine density at different time points during development, disrupting the pattern of spine density changes and leading to a premature loss of dendritic spines in KO mice, which is compensated later. Our findings indicate that miR-324-5p plays a role in synaptic refinement across development. Additionally, our data illustrate the importance of context in the study of miRNA, as regulation by and/or of miRNA can vary dramatically across development and in disease.

Decreased TAX1BP1 participates in systemic lupus erythematosus by regulating monocyte/macrophage function.

Systemic lupus erythematosus (SLE) involves disorders of innate and adaptive immune pathways. Tax1-binding protein 1 (TAX1BP1) modulates the production of antibodies in B cells and the T-cell cycle by regulating the NF-κB signaling pathway. However, the potential association of TAX1BP1 with SLE and its role in monocytes/macrophages have not been fully elucidated. In this study, we utilized whole-exome sequencing (WES) in combination with Sanger sequencing and identified 16 gene mutations, including in TAX1BP1, in an SLE family. TAX1BP1 protein expression with western blotting detection was reduced in SLE patients and correlated with disease activity negatively. Furthermore, RNA sequencing and 4D Label-Free Phosphoproteomic analysis were employed to characterize the transcriptome and phosphoproteome profiles in THP-1 and THP-1-differentiated M1 macrophages with TAX1BP1 knockdown. Silencing of TAX1BP1 in THP-1 and THP-1-differentiated M1 macrophages led to an increase in cluster of differentiation 80 (CD80) expression and differential changes in CD14 and CD16 expression, as assessed by flow cytometry. Additionally, western blot analysis showed that knockdown of TAX1BP1 led to a reduction in TRAF6 and p-p65 in THP-1-differentiated macrophages, with or without lipopolysaccharide (LPS) or tumor necrosis factor (TNF)-α stimulation. Taken together, our findings suggest that TAX1BP1 participates in SLE activity by regulating antigen presentation in monocytes and inflammatory responses in M1 macrophages.

Abstract PR006: Integrating noise into a signal: Luminal epithelial cells integrate variable responses to aging into stereotypical changes that underlie breast cancer susceptibility

Abstract Effects from aging in any single cell are unpredictable, whereas aging phenotypes at the organ and tissue levels tend to appear as stereotypical changes. The mammary epithelium, the origin of breast carcinomas, is a bilayer of two major phenotypically and functionally distinct cell lineages, the luminal epithelial and myoepithelial cells. We have shown that luminal epithelia exhibit substantial stereotypical changes with age, which merits attention as they are putative breast cancer cells of origin. Hallmark aging changes in mammary gland are loss of lineage specificity in luminal epithelia exemplified by acquiring expression of proteins normally reserved for myoepithelial cells, and progenitor cells that accumulate and attain a basal differentiation bias. We hypothesize that effects from aging that impinge upon maintenance of lineage specificity increases susceptibility to cancer initiation. Indeed, histologically normal breast tissue from young women, who were known to be highly susceptible to breast cancer because they harbor a germline mutation in BRCA1, BRCA2, or PALB2 genes, exhibited hallmarks of accelerated aging. These included increased proportions of luminal epithelial cells with acquired myoepithelial proteins, acceleration of a breast specific biological clock by 10 to 40 years compared to chronological age, and a basal differentiation bias or failure of differentiation of cKit+ progenitors. To gain insight into the underlying molecular changes we interrogated transcriptomes, proteomes, and methylomes of mammary epithelia at lineage resolution. Strikingly, age-dependent differential gene expression and DNA methylation occurred almost exclusively in luminal epithelia, whereas changes due to increased variance of gene and protein expression occurs in both luminal and myoepithelial lineages. Most gene changes that were previously associated with early breast cancer are detectable as age-driven changes in normal luminal epithelia. The genes with age-dependent differential and variant changes in expression distinguish normal tissue from breast cancers and are predictive of PAM50 breast cancer subtypes. Heterochronus, multilineage, co-cultures demonstrated that the age-dependent phenotype of luminal cells is at least partly dependent on the microenvironment created on the apical surfaces of neighboring myoepithelial cells. We show that increased variance is a substantial outcome of aging and is likely central to understanding increased susceptibility to breast cancer with age. We speculate that the luminal epithelium is a site of integration of the variant responses to aging in their surrounding tissue, and that their emergent phenotype of aging both endows cells with the ability to become a cancer cell of origin and embodies a biosensor that presages one’s cancer susceptibility. Citation Format: Masaru Miyano, Rosalyn W. Sayaman, Parijat Senapati, Stefan Hinz, Victoria E. Seewaldt, Dustin Schones, Mark A. LaBarge. Integrating noise into a signal: Luminal epithelial cells integrate variable responses to aging into stereotypical changes that underlie breast cancer susceptibility [abstract]. In: Proceedings of the AACR Special Conference: Aging and Cancer; 2022 Nov 17-20; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_1):Abstract nr PR006.

A potential role for SARS-CoV-2 small viral RNAs in targeting host microRNAs and modulating gene expression

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease (COVID-19) in humans, with symptoms ranging from mild to severe, including fatality. The molecular mechanisms surrounding the effects of viral infection on the host RNA machinery remain poorly characterized. We used a comparative transcriptomics approach to investigate the effects of SARS-CoV-2 infection on the host mRNA and sRNA expression machinery in a human lung epithelial cell line (Calu-3) and an African green monkey kidney cell line (Vero-E6). Upon infection, we observed global changes in host gene expression and differential expression of dozens of host miRNAs, many with known links to viral infection and immune response. Additionally, we discovered an expanded landscape of more than a hundred SARS-CoV-2-derived small viral RNAs (svRNAs) predicted to interact with differentially expressed host mRNAs and miRNAs. svRNAs are derived from distinct regions of the viral genome and sequence signatures suggest they are produced by a non-canonical biogenesis pathway. 52 of the 67 svRNAs identified in Calu-3 cells are predicted to interact with differentially expressed miRNAs, with many svRNAs having multiple targets. Accordingly, we speculate that these svRNAs may play a role in SARS-CoV-2 propagation by modulating post-transcriptional gene regulation, and that methods for antagonizing them may have therapeutic value.

Polycyclic aromatic hydrocarbons exposure was associated with microRNA differential expression and neurotransmitter changes: a cross-sectional study in coal miners.

Exposure to polycyclic aromatic hydrocarbons (PAHs) may cause neurobehavioral changes. This study aimed to explore the underlying mechanism of PAH neurotoxicity in coal miners. Urinary PAH metabolites, neurotransmitters, and oxidative stress biomarkers of 652 coal miners were examined. Subjects were divided into high and low-exposure groups based on the median of total urinary PAH metabolites. Differentially expressed miRNAs were screened from 5 samples in the low-exposure group (≤ 4.88 μmol/mol Cr) and 5 samples in the high-exposure group (> 4.88 μmol/mol Cr) using microarray technology, followed by bioinformatics analysis of the potential molecular functions of miRNA target genes. Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) was used to validate differentially expressed miRNAs. Restricted cubic splines (RCS) were applied to assess the possible dose-response relationships. Compared to the low PAH exposure group, the high-exposure group had higher levels of 5-hydroxytryptamine (5-HT), epinephrine (E), and acetylcholine (ACh), and lower levels of acetylcholinesterase (AChE). 1-OHP had a dose-response relationship with malondialdehyde (MDA), dopamine (DA), 5-HT, and AChE (P for overall associations < 0.05). There were 19 differentially expressed microRNAs in microarray analysis, significantly enriched in the cell membrane, molecular binding to regulate transcription, and several signaling pathways such as PI3K-Akt. And in the validation stage, miR-885-5p, miR-20a-5p, and let-7i-3p showed differences in the low and high-exposure groups (P < 0.05). Changes in neurotransmitters and microRNA expression levels among the coal miners were associated with PAH exposure. Their biological functions are mainly related to the transcriptional regulation of nervous system diseases or signaling pathways of disorders. These findings provide new insights for future research of PAH neurotoxicity.

Role of Neuregulin 3‐ErbB4 Signaling in a Murine Model of Co‐Morbid Nicotine Dependence and Schizophrenia

According to the NIH, rates of nicotine dependence among patients with schizophrenia are at nearly 90%, compared to smoking rates of approximately 12% in the general population. Current research shows that variations in the gene for Neuregulin 3 (NRG3), a member of the EGF superfamily and the cognate ligand for ErbB4 receptors, are considered some of the strongest genetic links contributing to schizophrenia risk. Additionally, NRG3 protein and mRNA have been found to be highly enriched following chronic nicotine exposure and withdrawal. However, specific mechanisms underpinning NRG3’s association with both increased schizophrenia risk and increased nicotine withdrawal symptomology are unknown. In this ongoing experiment, we are testing the overall hypothesis that NRG3‐ErbB4 signaling effects on NMDA receptor function may provide a discrete, mechanistic link between nicotine dependence and schizophrenia.We plan to utilize a novel genetic mouse model of both nicotine dependence and schizophrenia in a multidimensional approach to study how NRG3 mediates nicotine withdrawal‐induced alterations on behavioral and molecular levels and how the alterations relate to schizophrenic‐like dysfunction. To first begin these investigations, we examined the behavioral and molecular baseline effects of nicotine and aripiprazole, a long‐acting atypical antipsychotic used to treat schizophrenia, in wildtype littermates. Animals treated chronically for nicotine and then undergoing spontaneous nicotine withdrawal were examined in the alternating Y‐maze, social interaction test, and open field test for efficacy in reducing behavioral endophenotypes common to both nicotine withdrawal and schizophrenia in our model. Following behavioral testing, PFC tissues were analyzed to determine mRNA and protein expression of factors in the NRG3 signaling pathway. Preliminary results show differential changes in mRNA expression associated with glutamatergic and serotonergic transmission in the mPFC, OFC, and hippocampus. Ongoing experiments will extend these studies to include time‐ and site‐specific genetic deletion of NRG3 in the PFC and compare these results to those derived in wildtype littermates. Our findings will provide insight into the underlying mechanisms linking the NRG3 signaling pathway and nicotine dependence, and may provide a novel link between nicotine use and its highly co‐morbid psychiatric disorder, schizophrenia. Findings from these highly translational studies may lead to novel therapeutic development for both nicotine dependence and schizophrenia.

Single-cell transcriptomic and chromatin accessibility analyses of dairy cattle peripheral blood mononuclear cells and their responses to lipopolysaccharide

BackgroundGram-negative bacteria are important pathogens in cattle, causing severe infectious diseases, including mastitis. Lipopolysaccharides (LPS) are components of the outer membrane of Gram-negative bacteria and crucial mediators of chronic inflammation in cattle. LPS modulations of bovine immune responses have been studied before. However, the single-cell transcriptomic and chromatin accessibility analyses of bovine peripheral blood mononuclear cells (PBMCs) and their responses to LPS stimulation were never reported.ResultsWe performed single-cell RNA sequencing (scRNA-seq) and single-cell sequencing assay for transposase-accessible chromatin (scATAC-seq) in bovine PBMCs before and after LPS treatment and demonstrated that seven major cell types, which included CD4 T cells, CD8 T cells, and B cells, monocytes, natural killer cells, innate lymphoid cells, and dendritic cells. Bioinformatic analyses indicated that LPS could increase PBMC cell cycle progression, cellular differentiation, and chromatin accessibility. Gene analyses further showed significant changes in differential expression, transcription factor binding site, gene ontology, and regulatory interactions during the PBMC responses to LPS. Consistent with the findings of previous studies, LPS induced activation of monocytes and dendritic cells, likely through their upregulated TLR4 receptor. NF-κB was observed to be activated by LPS and an increased transcription of an array of pro-inflammatory cytokines, in agreement that NF-κB is an LPS-responsive regulator of innate immune responses. In addition, by integrating LPS-induced differentially expressed genes (DEGs) with large-scale GWAS of 45 complex traits in Holstein, we detected trait-relevant cell types. We found that selected DEGs were significantly associated with immune-relevant health, milk production, and body conformation traits.ConclusionThis study provided the first scRNAseq and scATAC-seq data for cattle PBMCs and their responses to the LPS stimulation to the best of our knowledge. These results should also serve as valuable resources for the future study of the bovine immune system and open the door for discoveries about immune cell roles in complex traits like mastitis at single-cell resolution.

Claudin-10a Deficiency Shifts Proximal Tubular Cl- Permeability to Cation Selectivity via Claudin-2 Redistribution.

The tight junction proteins claudin-2 and claudin-10a form paracellular cation and anion channels, respectively, and are expressed in the proximal tubule. However, the physiologic role of claudin-10a in the kidney has been unclear. To investigate the physiologic role of claudin-10a, we generated claudin-10a-deficient mice, confirmed successful knockout by Southern blot, Western blot, and immunofluorescence staining, and analyzed urine and serum of knockout and wild-type animals. We also used electrophysiologic studies to investigate the functionality of isolated proximal tubules, and studied compensatory regulation by pharmacologic intervention, RNA sequencing analysis, Western blot, immunofluorescence staining, and respirometry. Mice deficient in claudin-10a were fertile and without overt phenotypes. On knockout, claudin-10a was replaced by claudin-2 in all proximal tubule segments. Electrophysiology showed conversion from paracellular anion preference to cation preference and a loss of paracellular Cl- over HCO3- preference. As a result, there was tubular retention of calcium and magnesium, higher urine pH, and mild hypermagnesemia. A comparison with other urine and serum parameters under control conditions and sequential pharmacologic transport inhibition, and unchanged fractional lithium excretion, suggested compensative measures in proximal and distal tubular segments. Changes in proximal tubular oxygen handling and differential expression of genes regulating fatty acid metabolism indicated proximal tubular adaptation. Western blot and immunofluorescence revealed alterations in distal tubular transport. Claudin-10a is the major paracellular anion channel in the proximal tubule and its deletion causes calcium and magnesium hyper-reabsorption by claudin-2 redistribution. Transcellular transport in proximal and distal segments and proximal tubular metabolic adaptation compensate for loss of paracellular anion permeability.

Circulating miR-126-3p and miR-423-5p Expression in de novo Adult Acute Myeloid Leukemia: Correlations with Response to Induction Therapy and the 2-Year Overall Survival.

BackgroundAcute myeloid leukemia (AML) results from sequential genetic alterations in a normal hematopoietic stem cell or its progenitors giving rise to an autonomous clone that dominates the bone marrow leading to marrow failure. MicroRNAs are short non-coding nucleic acid sequences that regulate post-transcriptional gene expression by base-pairing with their target mRNAs. MiRNAs can be secreted into extracellular fluids and carried to target cells by vesicles or bound to proteins. Intracellular and circulating miRNAs are believed to be useful markers in the diagnosis, prognosis, and treatment of various cancers. Practically, circulating miRNAs are more stable at room temperatures and extreme conditions.PurposeThis study aimed to compare the expression of miR-126-3p and miR-423-5p in patients and normal subjects and correlate their expression with response to induction therapy and with their 2-year overall survival rate.Patients and MethodsCirculating miR-126-3p and miR-423-5p was measured in the plasma of 43 adult AML patients and 35 age- and sex-matched controls by quantitative reverse transcriptase PCR. The fold change in differential expression for each gene was calculated using the comparative cycle threshold method.ResultsThere was an increase in the expression of the studied miRNAs in patients compared to the control group. The average expression fold change of miR-126-3p was 3.02 (p= 0.010). The average expression fold change of miR-423-5p was 4.09 (p= 0.003). No significant correlation was found between the expression of miR-126-3p and miR-423-5p in the studied AML patients (r = 0.094, p = 0.22). Furthermore, no relationship was found between the expression of the studied miRNAs and response to induction therapy or the 2-year survival rate.ConclusionAlthough further studies are needed, our findings highlight the studied circulating miRNAs as possible diagnostic markers for AML.

CircRNA (circ_0008057) promotes uremic serum-mediated proliferation and migration of vascular smooth muscle cells via miR-370/PLk1 signaling pathway.

In order to explore the mechanism of gefitinib-acquired resistance in lung cancer, a new biomarker has been developed for early clinical diagnosis and intervention; human NSCLC (Non-Small Cell Lung Cancer) cell lines H292 (denoted as H292S) and PC9 (denoted as PC9S) were used to establish gefitinibresistant NSCLC cell lines H292 and PC9 models. CCK-8 (Cell Counting Kit-8) method was used to test the drug resistance of the cells. circRNAs (circular RNAs) that were differentially expressed before and after resistance were screened by RNA sequencing technology. The effects of circSETD3 overexpression and interference on the sensitivity of gefitinib was observed to analyze the nuclear localization of circSETD3 and verify the interaction between circSETD3-miR-520h-ABCG2. The results showed that the most significant change in differential expression of human NSCLC cell lines before and after drug resistance was hsa_circ_0000567, that is, circSETD3, which is mainly present in the cytoplasm. In H292S and PC9S, compared with the negative control group, the cell proliferation ability of the overexpression group was significantly increased, and the apoptosis ability was significantly decreased. In H292R and PC9R, compared with the negative control group, the proliferation ability of the interference group was significantly decreased, and the apoptosis ability was significantly increased. Overexpression of circSETD3 to H292S and PC9S, the expression of ABCG2 increased significantly. Also, the expression of ABCG2 decreased significantly after transfection with miR-520h mimics. H292R and PC9R interfered with circSETD3, the expression of ABCG2 decreased significantly. Moreover, the expression of ABCG2 increased significantly after transfection with miR-520h inhibitor. In conclusion, circSETD3 can be used as a novel biomarker for lung cancer. It relieves miR-520h degradation of the transporter ABCG2 by down-regulating the miR-520h expression, causing gefitinib to be pumped out of the cell.

A Comparative Study on Changes in Total and Differential Milk Cell Counts, Activity, and Expression of Milk Phagocytes of Healthy and Mastitic Indigenous Sahiwal Cows.

Milk somatic cell counts (SCCs) have been used as a gold standard to monitor mammary health as well as an indicator of raw milk quality. The present work was undertaken to compare the changes in the milk SCC, milk differential leukocyte counts (DLCs), phagocytic activity (PA) of milk neutrophils and macrophages (by nitroblue tetrazolium assay), extracellular trap formation (PicoGreen assay) and mRNA expression of various genes in milk neutrophils and macrophages (reverse transcription–polymerase chain reaction), and milk plasma cortisol concentration (enzyme-linked immunosorbent assay) in healthy, subclinical mastitis (SCM), and clinical mastitis (CM) cows. Milk was collected from healthy, SCM, and CM cows grouped based on their SCCs and California mastitis test with eight cows in each group. Milk SCC was estimated by SCC counter, and DLC was done after staining the milk slide under a microscope at 100×. Total SCCs in healthy, SCM, and CM cows were on an average of 128.30, 300.3, and 694.40 × 103 cells/mL, respectively. Milk DLCs indicated a lower percentage of macrophage and lymphocytes and a higher (p < 0.05) percentage of neutrophils in SCM and CM compared to healthy milk. The percentage of mature segmented neutrophils was lower, whereas immature band neutrophils were higher (p < 0.05) in the SCM and CM groups as compared to healthy cows. The viability, in vitro PA, and extracellular trap formation of neutrophils were lower (p < 0.05) in SCM and CM milk samples as compared to healthy samples. However, the PA of macrophage remained unchanged in all the studied groups. The relative mRNA expression of Toll-like receptors (TLR2, TLR4), myeloperoxidase, and interleukin 2α (IL-2α) receptor (CD25) were minimum in healthy samples and increased (p < 0.05) with the progress of mammary inflammation. However, CD44 decreased (p < 0.05), and CD62L remained unchanged in mastitis as compared to healthy cows. Plasma cortisol concentrations were higher (p < 0.05) in mastitis as compared to healthy cows and were negatively correlated with the number of milk macrophages and the functions of milk phagocytes. Estimation of total SCC, milk DLC, and activity of milk phagocytes is essential for effective control and prevention of incidence of mastitis in dairy cows.