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Abstract Background Despite wide scale assessments, it remains unclear how large-scale severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) vaccination affected the wastewater concentration of the virus or the overall disease burden as measured by hospitalization rates. Methods We used weekly SARS-CoV-2 wastewater concentration with a stratified random sampling of seroprevalence, and linked vaccination and hospitalization data, from April 2021–August 2021 in Jefferson County, Kentucky (USA). Our susceptible ( $$S$$ S ), vaccinated ( $$V$$ V ), variant-specific infected ( $${I}_{1}$$ I 1 and $${I}_{2}$$ I 2 ), recovered ( $$R$$ R ), and seropositive ( $$T$$ T ) model ( $${SV}{I}_{2}{RT}$$ S V I 2 R T ) tracked prevalence longitudinally. This was related to wastewater concentration. Results Here we show the 64% county vaccination rate translate into about a 61% decrease in SARS-CoV-2 incidence. The estimated effect of SARS-CoV-2 Delta variant emergence is a 24-fold increase of infection counts, which correspond to an over 9-fold increase in wastewater concentration. Hospitalization burden and wastewater concentration have the strongest correlation (r = 0.95) at 1 week lag. Conclusions Our study underscores the importance of continuing environmental surveillance post-vaccine and provides a proof-of-concept for environmental epidemiology monitoring of infectious disease for future pandemic preparedness.

Abstract Reducing the loss of oligodendrocytes (OLs) is a major goal for neuroprotection after spinal cord injury (SCI). Therefore, the OL translatome was determined in Ribotag:Plp1-CreERT2 mice at 2, 10, and 42 days after moderate contusive T9 SCI. At 2 and 42 days, mitochondrial respiration- or actin cytoskeleton/cell junction/cell adhesion mRNAs were upregulated or downregulated, respectively. The latter effect suggests myelin sheath loss/morphological simplification which is consistent with downregulation of cholesterol biosynthesis transcripts on days 10 and 42. Various regulators of pro-survival-, cell death-, and/or oxidative stress response pathways showed peak expression acutely, on day 2. Many acutely upregulated OL genes are part of the repressive SUZ12/PRC2 operon suggesting that epigenetic de-silencing contributes to SCI effects on OL gene expression. Acute OL upregulation of the iron oxidoreductase Steap3 was confirmed at the protein level and replicated in cultured OLs treated with the mitochondrial uncoupler FCCP. Hence, STEAP3 upregulation may mark mitochondrial dysfunction. Taken together, in SCI-challenged OLs, acute and subchronic enhancement of mitochondrial respiration may be driven by axonal loss and subsequent myelin sheath degeneration. Acutely, the OL switch to oxidative phosphorylation may lead to oxidative stress that is further amplified by upregulation of such enzymes as STEAP3.

Abstract Background Despite a clear appreciation of the impact of human pathogens on community health, efforts to understand pathogen dynamics within populations often follow a narrow-targeted approach and rely on the deployment of specific molecular probes for quantitative detection or rely on clinical detection and reporting. Main text Genomic analysis of wastewater samples for the broad detection of viruses, bacteria, fungi, and antibiotic resistance genes of interest/concern is inherently difficult, and while deep sequencing of wastewater provides a wealth of information, a robust and cooperative foundation is needed to support healthier communities. In addition to furthering the capacity of high-throughput sequencing wastewater-based epidemiology to detect human pathogens in an unbiased and agnostic manner, it is critical that collaborative networks among public health agencies, researchers, and community stakeholders be fostered to prepare communities for future public health emergencies or for the next pandemic. A more inclusive public health infrastructure must be built for better data reporting where there is a global human health risk burden. Conclusions As wastewater platforms continue to be developed and refined, high-throughput sequencing of human pathogens in wastewater samples will emerge as a gold standard for understanding community health.

Abstract Aims Macrophages play an essential role in cancer development. Tumor‐associated macrophages (TAMs) have predominantly M2‐like attributes that are associated with tumor progression and poor patient survival. Numerous methods have been reported for differentiating and polarizing macrophages in vitro, but there is no standardized and validated model for creating TAMs. Primary cells show varying cytokine responses depending on their origin and functional studies utilizing these cells may lack generalization and validity. A distinct cell line‐derived TAM‐like M2 subtype is required to investigate the mechanisms mediated by anti‐inflammatory TAMs in vitro. Our previous work demonstrated a standardized protocol for creating an M2 subtype derived from a human THP‐1 cell line. The cell expression profile, however, has not been validated. The aim of this study was to characterize and validate the TAM‐like M2 subtype macrophage created based on our protocol to introduce them as a standardized model for cancer research. Methods and results Using qRT‐PCR and ELISA, we demonstrated that proinflammatory, anti‐inflammatory, and tumor‐associated marker expression changed during THP‐1‐derived marcrophage development in vitro, mimicking a TAM‐related profile (e.g., TNFα, IL‐1β). The anti‐inflammatory marker IL‐8/CXCL8, however, is most highly expressed in young M0 macrophages. Flow cytometry showed increased expression of CD206 in the final TAM‐like M2 macrophage. Single‐cell RNA‐sequencing analysis of primary human monocytes and colon cancer tissue macrophages demonstrated that cell line‐derived M2 macrophages resembled a TAM‐related gene profile. Conclusions The THP‐1‐derived M2 macrophage based on a standardized cell line model represents a distinct anti‐inflammatory TAM‐like phenotype with an M2a subtype profile. This model may provide a basis for in vitro investigation of functional mechanisms in a variety of anti‐inflammatory settings, particularly colon cancer development.

BackgroundIt is well established that females are more susceptible to the toxic effects of alcohol, although the exact mechanisms are still poorly understood. Previous studies noted that alcohol reduces the expression of mitogen-activated protein kinase phosphatase 1 (MKP1), a negative regulator of mitogen-activated protein kinases (MAPK) in the liver. However, the role of hepatocyte- specific MKP1 in the pathogenesis of alcohol-associated liver disease (ALD) remains uncharacterized. This study aimed to evaluate the role of hepatocyte-specific MKP1 in the susceptibility and sexual dimorphism in alcohol-induced liver injury.MethodsC57Bl/6 mice were used in an intragastric ethanol feeding model of alcohol-associated steatohepatitis (ASH). Hepatocyte-specific Mkp1-/- knockout and (Mkp1+/+ “f/f” male and female mice were subjected to the NIAAA chronic plus binge model. Primary mouse hepatocytes were used for in vitro studies. Liver RNA sequencing was performed on an Illumina NextSeq 500. Liver injury was evaluated by plasma alanine transaminase (ALT), hepatic ER stress and inflammation markers. Statistical analysis was carried out using ANOVA and the unpaired Student’s t-test.ResultsASH was associated with the severe injury accompanied by increased endoplasmic reticulum (ER) stress and significant downregulation of Dusp1 mRNA expression. In vitro, ethanol treatment resulted in a time-dependent decrease in Dusp1 mRNA and protein expression in primary hepatocytes in both males and females; however, this effect was significantly more pronounced in hepatocytes from females. In vivo, female mice developed more liver injury in a chronic plus binge model which was accompanied by a significant decrease in liver Dusp1 mRNA expression. In comparison, liver Dusp1 was not changed in male mice, while they developed milder injury to alcohol. Mkp1 deletion in hepatocytes led to increased alcohol induced liver injury, ER stress and inflammation in both sexes.ConclusionHepatocyte Mkp1 plays a significant role in alcohol induced liver injury. Alcohol downregulates Mkp1 expression in hepatocytes in a sex dependent manner and could play a role in sexual dimorphism in increased female susceptibility to alcohol.

Abstract The integrated stress response (ISR)-activated transcription factors ATF4 and CHOP/DDIT3 may regulate oligodendrocyte (OL) survival, tissue damage and functional impairment/recovery in white matter pathologies, including traumatic spinal cord injury (SCI). Accordingly, in OLs of OL-specific RiboTag mice, Atf4, Chop/Ddit3 and their downstream target gene transcripts were acutely upregulated at 2, but not 10, days post-contusive T9 SCI coinciding with maximal loss of spinal cord tissue. Unexpectedly, another, OL-specific upregulation of Atf4/Chop followed at 42 days post-injury. However, wild type versus OL-specific Atf4 −/− or Chop −/− mice showed similar white matter sparing and OL loss at the injury epicenter, as well as unaffected hindlimb function recovery as determined by the Basso mouse scale. In contrast, the horizontal ladder test revealed persistent worsening or improvement of fine locomotor control in OL-Atf4 −/− or OL-Chop −/− mice, respectively. Moreover, chronically, OL-Atf −/− mice showed decreased walking speed during plantar stepping despite greater compensatory forelimb usage. Therefore, ATF4 supports, while CHOP antagonizes, fine locomotor control during post-SCI recovery. No correlation between those effects and white matter sparing together with chronic activation of the OL ISR suggest that in OLs, ATF4 and CHOP regulate function of spinal cord circuitries that mediate fine locomotor control during post-SCI recovery.

Several preclinical studies have demonstrated that certain cytotoxic drugs enhance metastasis, but the importance of host responses triggered by chemotherapy in regulating cancer metastasis has not been fully explored. Here, we showed that multidose gemcitabine (GEM) treatment promoted breast cancer lung metastasis in a transgenic spontaneous breast cancer model. GEM treatment significantly increased accumulation of CCR2+ macrophages and monocytes in the lungs of tumor-bearing as well as tumor-free mice. These changes were largely caused by chemotherapy-induced reactive myelopoiesis biased toward monocyte development. Mechanistically, enhanced production of mitochondrial ROS was observed in GEM-treated BM Lin−Sca1+c-Kit+ cells and monocytes. Treatment with the mitochondria targeted antioxidant abrogated GEM-induced hyperdifferentiation of BM progenitors. In addition, GEM treatment induced upregulation of host cell–derived CCL2, and knockout of CCR2 signaling abrogated the pro-metastatic host response induced by chemotherapy. Furthermore, chemotherapy treatment resulted in the upregulation of coagulation factor X (FX) in lung interstitial macrophages. Targeting activated FX (FXa) using FXa inhibitor or F10 gene knockdown reduced the pro-metastatic effect of chemotherapy. Together, these studies suggest a potentially novel mechanism for chemotherapy-induced metastasis via the host response–induced accumulation of monocytes/macrophages and interplay between coagulation and inflammation in the lungs.

Summary: Natural IL-17-producing γδ T cells (γδT17 cells) are unconventional innate-like T cells that undergo functional programming in the fetal thymus. However, the intrinsic metabolic mechanisms of γδT17 cell development remain undefined. Here, we demonstrate that mTORC2, not mTORC1, selectively controls the functional fate commitment of γδT17 cells through regulating transcription factor c-Maf expression. scRNA-seq data suggest that fetal and adult γδT17 cells predominately utilize mitochondrial metabolism. mTORC2 deficiency results in impaired Drp1-mediated mitochondrial fission and mitochondrial dysfunction characterized by mitochondrial membrane potential (ΔΨm) loss, reduced oxidative phosphorylation (OXPHOS), and subsequent ATP depletion. Treatment with the Drp1 inhibitor Mdivi-1 alleviates imiquimod-induced skin inflammation. Reconstitution of intracellular ATP levels by ATP-encapsulated liposome completely rescues γδT17 defect caused by mTORC2 deficiency, revealing the fundamental role of metabolite ATP in γδT17 development. These results provide an in-depth insight into the intrinsic link between the mitochondrial OXPHOS pathway and γδT17 thymic programming and functional acquisition.

The advent of immunotherapy has revolutionised cancer therapeutics, but its application in the context of pancreatic ductal adenocarcinoma has been limited. Here authors explore the effect of innate trained responses to fungal β-glucan and assess its effect in a murine model of pancreatic ductal adenocarcinoma where they observe reduced tumour burden and enhanced survival.

Background: Cyclic nucleotides are second messengers, which play significant roles in numerous biological processes. Previous work has shown that cAMP and cGMP signaling regulates various pathways in liver cells, including Kupffer cells, hepatocytes, hepatic stellate cells, and cellular components of hepatic sinusoids. Importantly, it has been shown that cAMP levels and enzymes involved in cAMP homeostasis are affected by alcohol. Although the role of cyclic nucleotide signaling is strongly implicated in several pathological pathways in liver diseases, studies describing the changes in genes regulating cyclic nucleotide metabolism in ALD are lacking. Methods: Male C57B/6 mice were used in an intragastric model of alcohol-associated steatohepatitis (ASH). Liver injury, inflammation, and fibrogenesis were evaluated by measuring plasma levels of injury markers, liver tissue cytokines, and gene expression analyses. Liver transcriptome analysis was performed to examine the effects of alcohol on regulators of cyclic AMP and GMP levels and signaling. cAMP and cGMP levels were measured in mouse livers as well as in livers from healthy human donors and patients with alcohol-associated hepatitis (AH). Results: Our results show significant changes in several phosphodiesterases (PDEs) with specificity to degrade cAMP (Pde4a, Pde4d, and Pde8a) and cGMP (Pde5a, Pde6d, and Pde9a), as well as dual-specificity PDEs (Pde1a and Pde10a) in ASH mouse livers. Adenylyl cyclases (ACs) 7 and 9, which are responsible for cAMP generation, were also affected by alcohol. Importantly, adenosine receptor 1, which has been implicated in the pathogenesis of liver diseases, was significantly increased by alcohol. Adrenoceptors 1 and 3 (Adrb), which couple with stimulatory G protein to regulate cAMP and cGMP signaling, were significantly decreased. Additionally, beta arrestin 2, which interacts with cAMP-specific PDE4D to desensitize G-protein-coupled receptor to generate cAMP, was significantly increased by alcohol. Notably, we observed that cAMP levels are much higher than cGMP levels in the livers of humans and mice; however, alcohol affected them differently. Specifically, cGMP levels were higher in patients with AH and ASH mice livers compared with controls. As expected, these changes in liver cyclic nucleotide signaling were associated with increased inflammation, steatosis, apoptosis, and fibrogenesis. Conclusions: These data strongly implicate dysregulated cAMP and cGMP signaling in the pathogenesis of ASH. Future studies to identify changes in these regulators in a cell-specific manner could lead to the development of novel targeted therapies for ASH.

Abstract Key elements for viral pathogenesis include viral strains, viral load, co-infection, and host responses. Several studies analyzing these factors in the function of disease severity of have been published; however, no studies have shown how all of these factors interplay within a defined cohort. To address this important question, we sought to understand how these four key components interplay in a cohort of COVID-19 patients. We determined the viral loads and gene expression using high throughput sequencing and various virological methods. We found that viral loads in the upper respiratory tract in COVID-19 patients at an early phase of infection vary widely. While the majority of nasopharyngeal (NP) samples have a viral load lower than the limit of detection of infectious viruses, there are samples with an extraordinary amount of SARS-CoV-2 RNA and a high viral titer. No specific viral factors were identified that are associated with high viral loads. Host gene expression analysis showed that viral loads were strongly correlated with cellular antiviral responses. Interestingly, however, COVID-19 patients who experience mild symptoms have a higher viral load than those with severe complications, indicating that naso-pharyngeal viral load may not be a key factor of the clinical outcomes of COVID-19. The metagenomics analysis revealed that the microflora in the upper respiratory tract of COVID-19 patients with high viral loads were dominated by SARS-CoV-2, with a high degree of dysbiosis. Finally, we found a strong inverse correlation between upregulation of interferon responses and disease severity. Overall our study suggests that a high viral load in the upper respiratory tract may not be a critical factor for severe symptoms; rather, dampened antiviral responses may be a critical factor for a severe outcome from the infection.

Summary: Background: Psoriasis recurrence is a clinically challenging issue. However, the underlying mechanisms haven't been fully understood. Methods: RNAseq analysis from affected skin of psoriatic patients treated with topical glucocorticoid (GC) with different outcomes was performed. In addition, imiquimod (IMQ)-induced mouse psoriasis-like model was used to mimic GC treatment in human psoriasis patients. Skin tissues and draining and distant lymph nodes (LNs) were harvested for flow cytometry and histology analyses. Findings: RNAseq analysis revealed that chemokine and chemokine receptor gene expression was decreased in post-treated skin compared to pre-treated samples but was subsequently increased in the recurred skin. In IMQ-induced mouse psoriasis-like model, we found that γδT17 cells were decreased in the skin upon topical GC treatment but surprisingly increased in the draining and distant LNs. This redistribution pattern lasted even two weeks post GC withdrawal. Upon IMQ re-challenge on the same site, mice previously treated with GC developed more severe skin inflammation. There were γδT17 cells migrated from LNs to the skin. This dynamic trafficking was dependent on CCR6 as this phenomenon was completely abrogated in CCR6-deficient mice. In addition, inhibition of lymphocyte egress prevented this heightened skin inflammation induced by IMQ rechallenge. Interpretation: Redistribution of pathogenic γδT17 cells may be vital to prevent disease recurrence and this model of psoriasis-like dermatitis. Funding: This work was supported by National Natural Science Foundation of China 81830095/H1103, 81761128008/H10 (J.Z.) and the NIH R01AI128818 and the National Psoriasis Foundation (J.Y.).

Circular RNAs (circRNAs) are a recently discovered class of RNAs derived from protein-coding genes that have important biological and pathological roles. They are formed through backsplicing during co-transcriptional alternative splicing; however, the unified mechanism that accounts for backsplicing decisions remains unclear. Factors that regulate the transcriptional timing and spatial organization of pre-mRNA, including RNAPII kinetics, the availability of splicing factors, and features of gene architecture, have been shown to influence backsplicing decisions. Poly (ADP-ribose) polymerase I (PARP1) regulates alternative splicing through both its presence on chromatin as well as its PARylation activity. However, no studies have investigated PARP1’s possible role in regulating circRNA biogenesis. Here, we hypothesized that PARP1’s role in splicing extends to circRNA biogenesis. Our results identify many unique circRNAs in PARP1 depletion and PARylation-inhibited conditions compared to the wild type. We found that while all genes producing circRNAs share gene architecture features common to circRNA host genes, genes producing circRNAs in PARP1 knockdown conditions had longer upstream introns than downstream introns, whereas flanking introns in wild type host genes were symmetrical. Interestingly, we found that the behavior of PARP1 in regulating RNAPII pausing is distinct between these two classes of host genes. We conclude that the PARP1 pausing of RNAPII works within the context of gene architecture to regulate transcriptional kinetics, and therefore circRNA biogenesis. Furthermore, this regulation of PARP1 within host genes acts to fine tune their transcriptional output with implications in gene function.

Abstract Background Inadequate copper intake and increased fructose consumption represent two important nutritional problems in the USA. Dietary copper-fructose interactions alter gut microbial activity and contribute to the development of nonalcoholic fatty liver disease (NAFLD). The aim of this study is to determine whether dietary copper-fructose interactions alter gut microbial activity in a sex-differential manner and whether sex differences in gut microbial activity are associated with sex differences in hepatic steatosis. Methods Male and female weanling Sprague-Dawley (SD) rats were fed ad libitum with an AIN-93G purified rodent diet with defined copper content for 8 weeks. The copper content is 6 mg/kg and 1.5 mg/kg in adequate copper diet (CuA) and marginal copper diet (CuM), respectively. Animals had free access to either deionized water or deionized water containing 10% fructose (F) (w/v) as the only drink during the experiment. Body weight, calorie intake, plasma alanine aminotransferase, aspartate aminotransferase, and liver histology as well as liver triglyceride were evaluated. Fecal microbial contents were analyzed by 16S ribosomal RNA (16S rRNA) sequencing. Fecal and cecal short-chain fatty acids (SCFAs) were determined by gas chromatography-mass spectrometry (GC-MS). Results Male and female rats exhibit similar trends of changes in the body weight gain and calorie intake in response to dietary copper and fructose, with a generally higher level in male rats. Several female rats in the CuAF group developed mild steatosis, while no obvious steatosis was observed in male rats fed with CuAF or CuMF diets. Fecal 16S rRNA sequencing analysis revealed distinct alterations of the gut microbiome in male and female rats. Linear discriminant analysis (LDA) effect size (LEfSe) identified sex-specific abundant taxa in different groups. Further, total SCFAs, as well as, butyrate were decreased in a more pronounced manner in female CuMF rats than in male rats. Of note, the decreased SCFAs are concomitant with the reduced SCFA producers, but not correlated to hepatic steatosis. Conclusions Our data demonstrated sex differences in the alterations of gut microbial abundance, activities, and hepatic steatosis in response to dietary copper-fructose interaction in rats. The correlation between sex differences in metabolic phenotypes and alterations of gut microbial activities remains elusive.

Polychlorinated biphenyls (PCBs) are persistent organic pollutants associated with non-alcoholic fatty liver disease (NAFLD). Previously, we demonstrated that the PCB mixture, Aroclor1260, exacerbated NAFLD, reflective of toxicant-associated steatohepatitis, in diet-induced obese mice, in part through pregnane-xenobiotic receptor (PXR) and constitutive androstane receptor (CAR) activation. Recent studies have also reported PCB-induced changes in the gut microbiome that consequently impact NAFLD. Therefore, the objective of this study is to examine PCB effects on the gut-liver axis and characterize the role of CAR and PXR in microbiome alterations. C57Bl/6 (wildtype, WT), CAR and PXR knockout mice were fed a high fat diet and exposed to Aroclor1260 (20 mg/kg, oral gavage, 12 weeks). Metagenomics analysis of cecal samples revealed that CAR and/or PXR ablation increased bacterial alpha diversity regardless of exposure status. CAR and PXR ablation also increased bacterial composition (beta diversity) versus WT; Aroclor1260 altered beta diversity only in WT and CAR knockouts. Distinct changes in bacterial abundance at different taxonomic levels were observed between WT and knockout groups; however Aroclor1260 had modest effects on bacterial abundance within each genotype. Notably, both knockout groups displayed increased Actinobacteria and Verrucomicrobia abundance. In spite of improved bacterial diversity, the knockout groups however failed to show protection from PCB-induced hepato- and intestinal- toxicity including decreased mRNA levels of ileal permeability markers (occludin, claudin3). In summary, CAR and PXR ablation significantly altered gut microbiome in diet-induced obesity while Aroclor1260 compromised intestinal integrity in knockout mice, implicating interactions between PCBs and CAR, PXR on the gut-liver axis.

Vast amounts of plastic materials are produced in the modern world and despite recycling efforts, large amounts are disposed in water systems and landfills. Under these storage conditions, physical weathering and photochemical processes break down these materials into smaller particles of the micro- and nano-scale. In addition, ecosystems can be contaminated with plastic particles which are manufactured in these size ranges for commercial purposes. Independent of source, microplastics are abundant in the environment and have found their way into water supplies and the food cycle where human exposure is inevitable. Nevertheless, the health consequences of microplastic ingestion, inhalation, or absorption are largely unknown. In this study we sought to determine if ingestion of microplastics promoted pre-clinical cardiovascular disease (CVD). To do this, we supplied mice with normal drinking water or that supplemented with polystyrene beads of two different sizes (0.5 µm and 5 µm) and two different doses (0.1 μg/ml and 1 μg/ml) each for 12 weeks and measured several indices of metabolism and glucose homeostasis. As early as 3 weeks of consumption, we observed an accelerated weight gain with a corresponding increase in body fat for some exposure groups versus the control mice. Some exposure groups demonstrated increased levels of fasting plasma glucose. Those mice consuming the smaller sized beads (0.5 µm) at the higher dose (1 μg/ml), had increased levels of fasting plasma insulin and higher homeostatic model assessment of insulin resistance (HOMA-IR) scores as well. This was accompanied by changes in the gut microbiome consistent with an obese phenotype. Using samples of perivascular adipose tissue collected from the same group, we observed changes in gene expression consistent with increased adipogenesis. These results suggest that ingestion of polystyrene beads promotes a cardiometabolic disease phenotype and thus may be an unrecognized risk factor for CVD.

Abstract Background High-throughput RNA sequencing (RNA-seq) has evolved as an important analytical tool in molecular biology. Although the utility and importance of this technique have grown, uncertainties regarding the proper analysis of RNA-seq data remain. Of primary concern, there is no consensus regarding which normalization and statistical methods are the most appropriate for analyzing this data. The lack of standardized analytical methods leads to uncertainties in data interpretation and study reproducibility, especially with studies reporting high false discovery rates. In this study, we compared a recently developed normalization method, UQ-pgQ2, with three of the most frequently used alternatives including RLE (relative log estimate), TMM (Trimmed-mean M values) and UQ (upper quartile normalization) in the analysis of RNA-seq data. We evaluated the performance of these methods for gene-level differential expression analysis by considering the factors, including: 1) normalization combined with the choice of a Wald test from DESeq2 and an exact test/QL (Quasi-likelihood) F-Test from edgeR; 2) sample sizes in two balanced two-group comparisons; and 3) sequencing read depths. Results Using the MAQC RNA-seq datasets with small sample replicates, we found that UQ-pgQ2 normalization combined with an exact test can achieve better performance in term of power and specificity in differential gene expression analysis. However, using an intra-group analysis of false positives from real and simulated data, we found that a Wald test performs better than an exact test when the number of sample replicates is large and that a QL F-test performs the best given sample sizes of 5, 10 and 15 for any normalization. The RLE, TMM and UQ methods performed similarly given a desired sample size. Conclusion We found the UQ-pgQ2 method combined with an exact test/QL F-test is the best choice in order to control false positives when the sample size is small. When the sample size is large, UQ-pgQ2 with a QL F-test is a better choice for the type I error control in an intra-group analysis. We observed read depths have a minimal impact for differential gene expression analysis based on the simulated data.

Ethanol (EtOH)-induced alterations in intestinal homeostasis lead to multi-system pathologies, including liver injury. ω-6 PUFAs exert pro-inflammatory activity, while ω-3 PUFAs promote anti-inflammatory activity that is mediated, in part, through specialized pro-resolving mediators [e.g., resolvin D1 (RvD1)]. We tested the hypothesis that a decrease in the ω-6:ω-3 PUFA ratio would attenuate EtOH-mediated alterations in the gut-liver axis. ω-3 FA desaturase-1 (fat-1) mice, which endogenously increase ω-3 PUFA levels, were protected against EtOH-mediated downregulation of intestinal tight junction proteins in organoid cultures and in vivo. EtOH- and lipopolysaccharide-induced expression of INF-γ, Il-6, and Cxcl1 was attenuated in fat-1 and WT RvD1-treated mice. RNA-seq of ileum tissue revealed upregulation of several genes involved in cell proliferation, stem cell renewal, and antimicrobial defense (including Alpi and Leap2) in fat-1 versus WT mice fed EtOH. fat-1 mice were also resistant to EtOH-mediated downregulation of genes important for xenobiotic/bile acid detoxification. Further, gut microbiome and plasma metabolomics revealed several changes in fat-1versus WT mice that may contribute to a reduced inflammatory response. Finally, these data correlated with a significant reduction in liver injury. Our study suggests that ω-3 PUFA enrichment or treatment with resolvins can attenuate the disruption in intestinal homeostasis caused by EtOH consumption and systemic inflammation with a concomitant reduction in liver injury.

Despite entering an endemic phase, SARS-CoV-2 remains a significant burden to public health across the global community. Wastewater sampling has consistently proven utility to understanding SARS-CoV-2 prevalence trends and genetic variation as it represents a less biased assessment of the corresponding communities. Here, we report that ongoing monitoring of SARS-CoV-2 genetic variation in samples obtained from the wastewatersheds of the city of Louisville in Jefferson county Kentucky has revealed the periodic reemergence of the Delta strain in the presence of the presumed dominant Omicron strain. Unlike previous SARS-CoV-2 waves/emergence events, the Delta reemergence events were geographically restricted in the community and failed to spread into other areas as determined by wastewater analyses. Moreover, the reemergence of the Delta strain did not correlate with vaccination rates as communities with lower relative vaccination have been, to date, not affected. Importantly, Delta reemergence events correlate with increased public health burdens, as indicated by increased daily case rates and mortality relative to non-Delta wastewatershed communities. While the underlying reasons for the reemergence of the Delta variant remain unclear, these data reaffirm the ongoing importance of wastewater genomic analyses towards understanding SARS-CoV-2 as it enters the endemic phase.

SARS coronavirus 2 (SARS-CoV-2) is a novel viral pathogen that causes a clinical disease called coronavirus disease 2019 (COVID-19). Although most COVID-19 cases are asymptomatic or involve mild upper respiratory tract symptoms, a significant number of patients develop severe or critical disease. Patients with severe COVID-19 commonly present with viral pneumonia that may progress to life-threatening acute respiratory distress syndrome (ARDS). Patients with COVID-19 are also predisposed to venous and arterial thromboses that are associated with a poorer prognosis. The present study identified the emergence of a low-density inflammatory neutrophil (LDN) population expressing intermediate levels of CD16 (CD16Int) in patients with COVID-19. These cells demonstrated proinflammatory gene signatures, activated platelets, spontaneously formed neutrophil extracellular traps, and enhanced phagocytic capacity and cytokine production. Strikingly, CD16Int neutrophils were also the major immune cells within the bronchoalveolar lavage fluid, exhibiting increased CXCR3 but loss of CD44 and CD38 expression. The percentage of circulating CD16Int LDNs was associated with D-dimer, ferritin, and systemic IL-6 and TNF-α levels and changed over time with altered disease status. Our data suggest that the CD16Int LDN subset contributes to COVID-19–associated coagulopathy, systemic inflammation, and ARDS. The frequency of that LDN subset in the circulation could serve as an adjunct clinical marker to monitor disease status and progression.

Abstract Background Despite the convergence of rapid technological advances in genomics and the maturing field of ecoimmunology, our understanding of the genes that regulate immunity in wild populations is still nascent. Previous work to assess immune function has relied upon relatively crude measures of immunocompetence. However, with next-generation RNA-sequencing, it is now possible to create a profile of gene expression in response to an immune challenge. In this study, captive zebra finch (Taeniopygia guttata; adult males) were challenged with bacterial lipopolysaccharide (LPS) or vehicle to stimulate the innate immune system. 2 hours after injection, birds were euthanized and hypothalami, spleen, and red blood cells (RBCs) were collected. Taking advantage of the fully sequenced genome of zebra finch, total RNA was isolated, sequenced, and partially annotated in these tissue/cells. Results In hypothalamus, there were 707 significantly upregulated transcripts, as well as 564 and 144 in the spleen and RBCs, respectively, relative to controls. Also, 155 transcripts in the hypothalamus, 606 in the spleen, and 61 in the RBCs were significantly downregulated. More specifically, a number of immunity-related transcripts (e.g., IL-1β, RSAD2, SOCS3) were upregulated among tissues/cells. Additionally, transcripts involved in metabolic processes (APOD, LRAT, RBP4) were downregulated. Conclusions These results suggest a potential trade-off in expression of genes that regulate immunity and metabolism in birds challenged with LPS. This finding is consistent with a hypothermic response to LPS treatment in small birds. Unlike mammals, birds have nucleated RBCs, and these results support a novel transcriptomic response of avian RBCs to immune challenge.

Abstract Background Recently, we showed that PARP1 is involved in cotranscriptional splicing, possibly by bridging chromatin to RNA and recruiting splicing factors. It also can influence alternative splicing decisions through the regulation of RNAPII elongation. In this study, we investigated the effect of PARP1-mediated chromatin changes on RNAPII movement, during transcription and alternative splicing. Results We show that RNAPII pauses at PARP1–chromatin structures within the gene body. Knockdown of PARP1 abolishes this RNAPII pausing, suggesting that PARP1 may regulate RNAPII elongation. Additionally, PARP1 alters nucleosome deposition and histone post-translational modifications at specific exon–intron boundaries, thereby affecting RNAPII movement. Lastly, genome-wide analyses confirmed that PARP1 influences changes in RNAPII elongation by either reducing or increasing the rate of RNAPII elongation depending on the chromatin context. Conclusions These studies suggest a context-specific effect of PARP1–chromatin binding on RNA polymerase movement and provide a platform to delineate PARP1’s role in RNA biogenesis and processing.

Urolithins are microbial metabolites derived from food polyphenols. Here, Singh et al. show that urolithin A and a synthetic analogue enhance gut barrier function via Nrf2-dependent pathways and mitigate inflammation and colitis in mice, highlighting a potential application for inflammatory bowel diseases.

Periodontitis is an irreversible, bacteria-induced, chronic inflammatory disease that compromises the integrity of tooth-supporting tissues and adversely affects systemic health. As the immune system's first line of defense against bacteria, neutrophils use their microbicidal functions in the oral cavity to protect the host against periodontal disease. However, periodontal pathogens have adapted to resist neutrophil microbicidal mechanisms while still propagating inflammation, which provides essential nutrients for the bacteria to proliferate and cause disease. Advances in sequencing technologies have recognized several newly appreciated bacteria associated with periodontal lesions such as the Gram-positive anaerobic rod, Filifactor alocis. With the discovery of these oral bacterial species, there is also a growing need to assess their pathogenic potential and determine their contribution to disease progression. Currently, few studies have addressed the pathogenic mechanisms used by oral bacteria to manipulate the neutrophil functional responses at the level of the transcriptome. Thus, this study aims to characterize the global changes at the gene expression level in human neutrophils during infection with F. alocis. Our results indicate that the challenge of human neutrophils with F. alocis results in the differential expression of genes involved in multiple neutrophil effector functions such as chemotaxis, cytokine and chemokine signaling pathways, and apoptosis. Moreover, F. alocis challenges affected the expression of components from the TNF and MAPK kinase signaling pathways. This resulted in transient, dampened p38 MAPK activation by secondary stimuli TNFα but not by fMLF. Functionally, the F. alocis-mediated inhibition of p38 activation by TNFα resulted in decreased cytokine production but had no effect on the priming of the respiratory burst response or the delay of apoptosis by TNFα. Since the modulatory effect was characteristic of viable F. alocis only, we propose this as one of F. alocis' mechanisms to control neutrophils and their functional responses.

The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral genome is an RNA virus consisting of approximately 30,000 bases. As part of testing efforts, whole genome sequencing of human isolates has resulted in over 1,600 complete genomes publicly available from GenBank. We have performed a comparative analysis of the sequences, in order to detect common mutations within the population. Analysis of variants occurring within the assembled genomes yields 417 variants occurring in at least 1% of the completed genomes, including 229 within the 5' untranslated region (UTR), 152 within the 3'UTR, 2 within intergenic regions and 34 within coding sequences.

Exposure to ionizing radiation associated with highly energetic and charged heavy particles is an inherent risk astronauts face in long duration space missions. We have previously considered the transcriptional effects that three levels of radiation (0.3 Gy, 1.5 Gy, and 3.0 Gy) have at an immediate time point (1 hr) post-exposure [1]. Our analysis of these results suggest effects on transcript levels that could be modulated at lower radiation doses [2]. In addition, a time dependent effect is likely to be present. Therefore, in order to develop a lab-on-a-chip approach for detection of radiation exposure in terms of both radiation level and time since exposure, we developed a time- and dose-course study to determine appropriate sensitive and specific transcript biomarkers that are detectable in blood samples. The data described herein was developed from a study measuring exposure to 0.15 Gy, 0.30 Gy, and 1.5 Gy of radiation at 1 hr, 2 hr, and 6 hr post-exposure using Affymetrix® GeneChip® PrimeView™ microarrays. This report includes raw gene expression data files from the resulting microarray experiments representing typical radiation exposure levels an astronaut may experience as part of a long duration space mission. The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE63952. Keywords: Radiation exposure, Microarrays, Space flight, Gene expression, Lab-on-a-chip

The successful interaction between pathogen/parasite and host requires a delicate balance between fitness of the former and survival of the latter. To optimize fitness a parasite/pathogen must effectively create an environment conducive to reproductive success, while simultaneously avoiding or minimizing detrimental host defense response. The association between Microbotryum lychnidis-dioicae and its host Silene latifolia serves as an excellent model to examine such interactions. This fungus is part of a species complex that infects species of the Caryophyllaceae, replacing pollen with the fungal spores. In the current study, transcriptome analyses of the fungus and its host were conducted during discrete stages of bud development so as to identify changes in fungal gene expression that lead to spore development and to identify changes associated with infection in the host plant. In contrast to early biotrophic phase stages of infection for the fungus, the latter stages involve tissue necrosis and in the case of infected female flowers, further changes in the developmental program in which the ovary aborts and a pseudoanther is produced. Transcriptome analysis via Illumina RNA sequencing revealed enrichment of fungal genes encoding small secreted proteins, with hallmarks of effectors and genes found to be relatively unique to the Microbotryum species complex. Host gene expression analyses also identified interesting sets of genes up-regulated, including those involving stress response, host defense response, and several agamous-like MADS-box genes (AGL61 and AGL80), predicted to interact and be involved in male gametophyte development.

Abstract Background Since the introduction of microarrays in 1995, researchers world-wide have used both commercial and custom-designed microarrays for understanding differential expression of transcribed genes. Public databases such as ArrayExpress and the Gene Expression Omnibus (GEO) have made millions of samples readily available. One main drawback to microarray data analysis involves the selection of probes to represent a specific transcript of interest, particularly in light of the fact that transcript-specific knowledge (notably alternative splicing) is dynamic in nature. Results We therefore developed a framework for reannotating and reassigning probe groups for Affymetrix® GeneChip® technology based on functional regions of interest. This framework addresses three issues of Affymetrix® GeneChip® data analyses: removing nonspecific probes, updating probe target mapping based on the latest genome knowledge and grouping probes into gene, transcript and region-based (UTR, individual exon, CDS) probe sets. Updated gene and transcript probe sets provide more specific analysis results based on current genomic and transcriptomic knowledge. The framework selects unique probes, aligns them to gene annotations and generates a custom Chip Description File (CDF). The analysis reveals only 87% of the Affymetrix® GeneChip® HG-U133 Plus 2 probes uniquely align to the current hg38 human assembly without mismatches. We also tested new mappings on the publicly available data series using rat and human data from GSE48611 and GSE72551 obtained from GEO, and illustrate that functional grouping allows for the subtle detection of regions of interest likely to have phenotypical consequences. Conclusion Through reanalysis of the publicly available data series GSE48611 and GSE72551, we profiled the contribution of UTR and CDS regions to the gene expression levels globally. The comparison between region and gene based results indicated that the detected expressed genes by gene-based and region-based CDFs show high consistency and regions based results allows us to detection of changes in transcript formation.

Throughout the course of the ongoing SARS-CoV-2 pandemic there has been a need for approaches that enable rapid monitoring of public health using an unbiased and minimally invasive means. A major way this has been accomplished is through the regular assessment of wastewater samples by qRT-PCR to detect the prevalence of viral nucleic acid with respect to time and location. Further expansion of SARS-CoV-2 wastewater monitoring efforts to include the detection of variants of interest/concern through next-generation sequencing has enhanced the understanding of the SARS-CoV-2 outbreak. In this report, we detail the results of a collaborative effort between public health and metropolitan wastewater management authorities and the University of Louisville to monitor the SARS-CoV-2 pandemic through the monitoring of aggregate wastewater samples over a period of 28 weeks. Through the use of next-generation sequencing approaches the polymorphism signatures of Variants of Concern/Interest were evaluated to determine the likelihood of their prevalence within the community on the basis of their relative dominance within sequence datasets. Our data indicate that wastewater monitoring of water quality treatment centers and smaller neighborhood-scale catchment areas is a viable means by which the prevalence and genetic variation of SARS-CoV-2 within a metropolitan community of approximately one million individuals may be monitored, as our efforts detected the introduction and emergence of variants of concern in the city of Louisville. Importantly, these efforts confirm that regional emergence and spread of variants of interest/concern may be detected as readily in aggregate wastewater samples as compared to the individual wastewater sheds. Furthermore, the information gained from these efforts enabled targeted public health efforts including increased outreach to at-risk communities and the deployment of mobile or community-focused vaccination campaigns.

Brain-derived neurotrophic factor (BDNF) is a pleiotropic neuronal growth and survival factor that is indispensable in the brain, as well as in multiple other tissues and organs, including the cardiovascular system. In approximately 30% of the general population, BDNF harbors a nonsynonymous single nucleotide polymorphism that may be associated with cardiometabolic disorders, coronary artery disease, and Duchenne muscular dystrophy cardiomyopathy. We recently showed that transgenic mice with the human BDNF rs6265 polymorphism (Val66Met) exhibit altered cardiac function, and that cardiomyocytes isolated from these mice are also less contractile. To identify the underlying mechanisms involved, we compared cardiac function by echocardiography and performed deep sequencing of RNA extracted from whole hearts of all three genotypes (Val/Val, Val/Met, and Met/Met) of both male and female Val66Met mice. We found female-specific cardiac alterations in both heterozygous and homozygous carriers, including increased systolic (26.8%, p = 0.047) and diastolic diameters (14.9%, p = 0.022), increased systolic (57.9%, p = 0.039) and diastolic volumes (32.7%, p = 0.026), and increased stroke volume (25.9%, p = 0.033), with preserved ejection fraction and fractional shortening. Both males and females exhibited lower heart rates, but this change was more pronounced in female mice than in males. Consistent with phenotypic observations, the gene encoding SERCA2 (Atp2a2) was reduced in homozygous Met/Met mice but more profoundly in females compared to males. Enriched functions in females with the Met allele included cardiac hypertrophy in response to stress, with down-regulation of the gene encoding titin (Tcap) and upregulation of BNP (Nppb), in line with altered cardiac functional parameters. Homozygous male mice on the other hand exhibited an inflammatory profile characterized by interferon-γ (IFN-γ)-mediated Th1 immune responses. These results provide evidence for sex-based differences in how the BDNF polymorphism modifies cardiac physiology, including female-specific alterations of cardiac-specific transcripts and male-specific activation of inflammatory targets.

The length of untranslated regions at the 3′ end of transcripts (3′UTRs) is regulated by alternate polyadenylation (APA). 3′UTRs contain regions that harbor binding motifs for regulatory molecules. However, the mechanisms that coordinate the 3′UTR length of specific groups of transcripts are not well-understood. We therefore developed a method, CSI-UTR, that models 3′UTR structure as tandem segments between functional alternative-polyadenylation sites (termed cleavage site intervals—CSIs). This approach facilitated (1) profiling of 3′UTR isoform expression changes and (2) statistical enrichment of putative regulatory motifs. CSI-UTR analysis is UTR-annotation independent and can interrogate legacy data generated from standard RNA-Seq libraries. CSI-UTR identified a set of CSIs in human and rodent transcriptomes. Analysis of RNA-Seq datasets from neural tissue identified differential expression events within 3′UTRs not detected by standard gene-based differential expression analyses. Further, in many instances 3′UTR and CDS from the same gene were regulated differently. This modulation of motifs for RNA-interacting molecules with potential condition-dependent and tissue-specific RNA binding partners near the polyA signal and CSI junction may play a mechanistic role in the specificity of alternative polyadenylation.Source code, CSI BED files and example datasets are available at: https://github.com/UofLBioinformatics/CSI-UTR

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chronic alcohol consumption leads to disturbances in intestinal function which can be exacerbated by inflammation and modulated by different factors, e.g., polyunsaturated fatty acids (PUFAs). The mechanisms underlying these alterations are not well understood. In this study, RNA-seq analysis was performed on ileum tissue from WT and fat-1 transgenic mice (which have elevated endogenous n-3 PUFAs). Mice were chronically fed ethanol (EtOH) and challenged with a single lipopolysaccharide (LPS) dose to induce acute systemic inflammation. Both WT and fat-1 mice exhibited significant ileum transcriptome changes following EtOH + LPS treatment. Compared to WT, fat-1 mice had upregulated expression of genes associated with cell cycle and xenobiotic metabolism, while the expression of pro-inflammatory cytokines and pro-fibrotic genes was decreased. In response to EtOH + LPS, fat-1 mice had an increased expression of genes related to antibacterial B cells (APRIL and IgA), as well as an elevation in markers of pro-restorative macrophages and γδ T cells that was not observed in WT mice. Our study significantly expands the knowledge of regulatory mechanisms underlying intestinal alterations due to EtOH consumption and inflammation and identifies the beneficial transcriptional effects of n-3 PUFAs, which may serve as a viable nutritional intervention for intestinal damage resulting from excessive alcohol consumption.

The Suppressor of Clear, Caenorhabditis elegans Homolog (SHOC2) is a scaffold protein that positively modulates activity of the RAS/ERK1/2 MAP kinase signaling cascade. We set out to understand the ERK1/2 pathway transcriptional response transduced through the SHOC2 scaffolding module. This data article describes raw gene expression within triplicates of kidney fibroblast-like Cos1 cell line expressing non-targeting shRNA (Cos-NT) and triplicates of Cos1 cells depleted of SHOC2 using shRNA (Cos-LV1) upon activation of ERK1/2 pathway by the Epidermal Growth Factor Receptor (EGFR). The data referred here is available in NCBI׳s Gene Expression Omnibus (GEO), accession GEO: GSE67063 as well as NCBI׳s Sequence Read Archive (SRA), accession SRA: SRP056324. A complete analysis of the results can be found in “Shoc2-tranduced ERK1/2 motility signals – Novel insights from functional genomics”(Jeoung et al., 2016) [1].

Astronauts participating in long duration space missions are likely to be exposed to ionizing radiation associated with highly energetic and charged heavy particles. Previously proposed gene biomarkers for radiation exposure include phosphorylated H2A Histone Family, Member X (γH2AX), Tumor Protein 53 (TP53), and Cyclin-Dependent Kinase Inhibitor 1A (CDKN1A). However, transcripts of these genes may not be the most suitable biomarkers for radiation exposure due to a lack of sensitivity or specificity. As part of a larger effort to develop lab-on-a-chip methods for detecting radiation exposure events using blood samples, we designed a dose–course microarray study in order to determine coding and non-coding RNA transcripts undergoing differential expression immediately following radiation exposure. The main goal was to elicit a small set of sensitive and specific radiation exposure biomarkers at low, medium, and high levels of ionizing radiation exposure. Four separate levels of radiation were considered: 0 Gray (Gy) control; 0.3 Gy; 1.5 Gy; and 3.0 Gy with four replicates at each radiation level. This report includes raw gene expression data files from the resulting microarray experiments from all three radiation levels ranging from a lower, typical exposure than an astronaut might see (0.3 Gy) to high, potentially lethal, levels of radiation (3.0 Gy). The data described here is available in NCBI's Gene Expression Omnibus (GEO), accession GSE64375. Keywords: Ionizing radiation, Radiation exposure, Astronaut, Long duration space travel

Primary afferent collateral sprouting is a process whereby non-injured primary afferent neurons respond to some stimulus and extend new branches from existing axons. Neurons of both the central and peripheral nervous systems undergo this process, which contributes to both adaptive and maladaptive plasticity (e.g., [1–9]). In the model used here (the “spared dermatome” model), the intact sensory neurons respond to the denervation of adjacent areas of skin by sprouting new axon branches into that adjacent denervated territory. Investigations of gene expression changes associated with collateral sprouting can provide a better understanding of the molecular mechanisms controlling this process. Consequently, it can be used to develop treatments to promote functional recovery for spinal cord injury and other similar conditions. This report includes raw gene expression data files from microarray experiments in order to study the gene regulation in spared sensory ganglia in the initiation (7 days) and maintenance (14 days) phases of the spared dermatome model relative to intact (“naïve”) sensory ganglia. Data has been deposited into GEO (GSE72551).