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IntroductionStatus epilepticus (SE) is a seizure lasting more than 5 min that can have lethal consequences or lead to various neurological disorders, including epilepsy. Using a pilocarpine-induced SE model in mice we investigated temporal changes in the hippocampal transcriptome.MethodsWe performed mRNA-seq and microRNA-seq analyses at various times after drug treatment.ResultsAt 1 h after the start of seizures, hippocampal cells upregulated transcription of immediate early genes and genes involved in the IGF-1, ERK/MAPK and RNA-PolII/transcription pathways. At 8 h, we observed changes in the expression of genes associated with oxidative stress, overall transcription downregulation, particularly for genes related to mitochondrial structure and function, initiation of a stress response through regulation of ribosome and translation/EIF2 signaling, and upregulation of an inflammatory response. During the middle of the latent period, 36 h, we identified upregulation of membrane components, cholesterol synthesis enzymes, channels, and extracellular matrix (ECM), as well as an increased inflammatory response. At the end of the latent period, 120 h, most changes in expression were in genes involved in ion transport, membrane channels, and synapses. Notably, we also elucidated the involvement of novel pathways, such as cholesterol biosynthesis pathways, iron/BMP/ferroptosis pathways, and circadian rhythms signaling in SE and epileptogenesis.DiscussionThese temporal changes in metabolic reactions indicate an immediate response to injury followed by recovery and regeneration. CREB was identified as the main upstream regulator. Overall, our data provide new insights into molecular functions and cellular processes involved at different stages of seizures and offer potential avenues for effective therapeutic strategies.

Abstract Introduction The onset of puberty is associated with a shift in the circadian timing of sleep, leading to delayed sleep initiation [i.e., later sleep onset time (SOT)] due to later bedtimes and/or longer sleep onset latency (SOL). Several genome-wide association studies (GWAS) have identified genes that may be involved in the etiology of sleep phenotypes. However, circadian rhythms are also epigenetically regulated; therefore, epigenetic biomarkers may provide insight into the physiology of the pubertal sleep onset shift and the pathophysiology of prolonged or delayed sleep initiation. Results The gene-wide analysis indicated differential methylation within or around 1818 unique genes across the sleep initiation measurements using self-report, actigraphy (ACT), and polysomnography (PSG), while GWAS-informed analysis yielded 67 genes. Gene hits were identified for bedtime (PSG), SOL (subjective, ACT and PSG) and SOT (subjective and PSG). DNA methylation within 12 genes was associated with both subjective and PSG-measured SOL, 31 with both ACT- and PSG-measured SOL, 19 with both subjective and ACT-measured SOL, and one gene (SMG1P2) had methylation sites associated with subjective, ACT- and PSG-measured SOL. Conclusions Objective and subjective sleep initiation in adolescents is associated with altered DNA methylation in genes previously identified in adult GWAS of sleep and circadian phenotypes. Additionally, our data provide evidence for a potential epigenetic link between habitual (subjective and ACT) SOL and in-lab SOT and DNA methylation in and around genes involved in circadian regulation (i.e., RASD1, RAI1), cardiometabolic disorders (i.e., FADS1, WNK1, SLC5A6), and neuropsychiatric disorders (i.e., PRR7, SDK1, FAM172A). If validated, these sites may provide valuable targets for early detection and prevention of disorders involving prolonged or delayed SOT, such as insomnia, delayed sleep phase, and their comorbidity.

Abstract Fetal alcohol spectrum disorders (FASD) show behavioral problems due to prenatal alcohol exposure (PAE). A previous study reports changes in gene expressions linked to fatty acid (FA) metabolism in the cerebral cortex of the PAE mouse model. We find an increase of palmitic acid and arachidonic acid in phospholipid in the cerebral cortex of PAE at postnatal day 30. The increase of palmitic acid is consistent with increase of the producing enzyme, Fasn (fatty acid synthase). Decrease of 26:6 FA is also consistent with the increase of the enzyme which uses 26:6 as a substrate for making very long chain FAs, Elovl4 (elongation of very long chain fatty acids protein 4). However, there is no increase in the elongated products. Rather, lipid droplets (LDs) accumulated in the brain. Although FA-associated metabolic measurements are not affected by PAE, the abundance of FA-related gut microbiota is altered. This suggests that the gut microbiome could serve as a tool to facilitate uncovering the brain pathophysiology of FASD and a potential target to mitigate neurobehavioral problems.

Abstract Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate, where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone. The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate. Malnutrition is a leading cause of growth retardation in children; however, after recovery from nutrient deprivation, bone growth is accelerated beyond the normal rate, a phenomenon termed catch-up growth. Although nutritional status is a known regulator of long bone growth, it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability. Here, using fate-mapping analysis in Axin2Cre ERT2 mice, we showed that dietary restriction increased the number of Axin2+ chondroprogenitors in the resting zone and simultaneously inhibited their differentiation. Once nutrient deficiency was resolved, the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns, contributing to accelerated growth. Furthermore, we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors, decreasing their numbers. Our study of Axin2Cre ERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

Introduction: Wnt/β-catenin signaling controls cell division and lineage specification during embryonic development, and is crucial for stem cells maintenance and gut tissue regeneration in adults. Aberrant activation of Wnt/β-catenin signaling is also essential for the pathogenesis of a variety of malignancies. The RNA-binding protein IGF2BP1 is a transcriptional target of Wnt/β-catenin signaling, normally expressed during development and often reactivated in cancer cells, where it regulates the stability of oncogenic mRNA.Methods: In this study, we employed iCLIP and RNA sequencing techniques to investigate the role of IGF2BP1 in the post-transcriptional regulation of Wnt/β-catenin-induced genes at a global level within colorectal cancer (CRC) cells characterized by constitutively active Wnt/β-catenin signaling.Results and Discussion: In our study, we show that, in contrast to normal cells, CRC cells exhibit a much stronger dependency on IGF2BP1 expression for Wnt/β-catenin-regulated genes. We show that both untransformed and CRC cells have their unique subsets of Wnt/β-catenin-regulated genes that IGF2BP1 directly controls through binding to their mRNA. Our iCLIP analysis revealed a significant change in the IGF2BP1-binding sites throughout the target transcriptomes and a significant change in the enrichment of 6-mer motifs associated with IGF2BP1 binding in response to Wnt/β-catenin signaling. Our study also revealed a signature of IGF2BP1-regulated genes that are significantly associated with colon cancer-free survival in humans, as well as potential targets for CRC treatment. Overall, this study highlights the complex and context-dependent regulation of Wnt/β-catenin signaling target genes by IGF2BP1 in non-transformed and CRC cells and identifies potential targets for colon cancer treatment.

BackgroundCirculating small RNAs (smRNAs) originate from diverse tissues and organs. Previous studies investigating smRNAs as potential biomarkers for Parkinson’s disease (PD) have yielded inconsistent results. We investigated whether smRNA profiles from neuronally-enriched serum exosomes and microvesicles are altered in PD patients and discriminate PD subjects from controls.MethodsDemographic, clinical, and serum samples were obtained from 60 PD subjects and 40 age- and sex-matched controls. Exosomes and microvesicles were extracted and isolated using a validated neuronal membrane marker (CD171). Sequencing and bioinformatics analyses were used to identify differentially expressed smRNAs in PD and control samples. SmRNAs also were tested for association with clinical metrics. Logistic regression and random forest classification models evaluated the discriminative value of the smRNAs.ResultsIn serum CD171 enriched exosomes and microvesicles, a panel of 29 smRNAs was expressed differentially between PD and controls (false discovery rate (FDR) < 0.05). Among the smRNAs, 23 were upregulated and 6 were downregulated in PD patients. Pathway analysis revealed links to cellular proliferation regulation and signaling. Least absolute shrinkage and selection operator adjusted for the multicollinearity of these smRNAs and association tests to clinical parameters via linear regression did not yield significant results. Univariate logistic regression models showed that four smRNAs achieved an AUC ≥ 0.74 to discriminate PD subjects from controls. The random forest model had an AUC of 0.942 for the 29 smRNA panel.ConclusionCD171-enriched exosomes and microvesicles contain the differential expression of smRNAs between PD and controls. Future studies are warranted to follow up on the findings and understand the scientific and clinical relevance.

Abstract Pulmonary hypertension associated with bronchopulmonary dysplasia is a severe complication of preterm birth resulting in high mortality of up to 50% within the first 2 years of life. There is a direct relationship between bronchopulmonary dysplasia severity and incidence of associated pulmonary hypertension. However, it is challenging to clinically characterize severe bronchopulmonary dysplasia with and without pulmonary hypertension and there is need for better understanding of the two entities. Our main objective is to identify markers to help understand biological processes and characterize infants with pulmonary hypertension associated with bronchopulmonary dysplasia using tracheal aspirates. We conducted an unbiased multiomic analysis of tracheal aspirates via microRNA (miRNA) polymerase chain reaction arrays, RNA sequencing, and mass spectrometry proteomics in preterm infants with severe bronchopulmonary dysplasia with and without pulmonary hypertension (n = 46). Our pilot study analysis revealed 12 miRNAs (hsa‐miR‐29a, has‐miR‐542‐3p, has‐miR‐624, has‐miR‐183, hsa‐miR‐501‐3p, hsa‐miR‐101, hsa‐miR‐3131, hsa‐miR‐3683, hsa‐miR‐3193, hsa‐miR‐3672, hsa‐miR‐3128, and hsa‐miR‐1287), 6 transcripts (IL6, RPL35P5, HSD3B7, RNA5SP215, OR2A1‐AS1, and RNVU1‐19), and 5 proteins (CAPS, AAT, KRT5, SFTPB, and LGALS3BP) with significant differential expression in preterm infants with severe lung disease with pulmonary hypertension when compared with infants with severe lung disease but no pulmonary hypertension. Pathway analysis of the integrated multiomic expression signatures revealed NFkB, VEGF, SERPINA1, IL6, and ERK1/2 as target molecules and cellular development, cellular growth and proliferation, and cellular movement as key affected molecular functions. Our multiomic analysis of tracheal aspirates revealed a comprehensive thumbprint of miRNAs, mRNAs, and proteins that could help endotype infants with severe lung disease and pulmonary hypertension.

Consumption of a diet rich in saturated fatty acids and carbohydrates contributes to the accumulation of fat in the liver and development of non-alcoholic steatohepatitis (NASH). Herein we investigated the hypothesis that short-term consumption of a high fat/sucrose Western diet (WD) alters the genomic and translatomic profile of the liver in association with changes in signaling through the protein kinase mTORC1, and that such alterations contribute to development of NAFLD. The results identify a plethora of mRNAs that exhibit altered expression and/or translation in the liver of rats consuming a WD compared to a CD. In particular, consumption of a WD altered the abundance and ribosome association of mRNAs involved in lipid and fatty acid metabolism, as well as those involved in glucose metabolism and insulin signaling. Hepatic mTORC1 signaling was enhanced when rats were fasted overnight and then refed in the morning; however, this effect was blunted in rats fed a WD as compared to a CD. Despite similar plasma insulin concentrations, fatty acid content was elevated in the liver of rats fed a WD as compared to a CD. We found that feeding had a significant positive effect on ribosome occupancy of 49 mRNAs associated with hepatic steatosis (e.g., LIPE, LPL), but this effect was blunted in the liver of rats fed a WD. In many cases, changes in ribosome association were independent of alterations in mRNA abundance, suggesting a critical role for diet-induced changes in mRNA translation in the expression of proteins encoded by those mRNAs. Overall, the findings demonstrate that short-term consumption of a WD impacts hepatic gene expression by altering the abundance of many mRNAs, but also causes wide-spread variation in mRNA translation that potentially contribute to development of hepatic steatosis.

Hepatitis B virus (HBV) has a highly restricted host range and cell tropism. Other than the human sodium taurocholate cotransporting polypeptide (huNTCP), the HBV entry receptor, host determinants of HBV susceptibility are poorly understood. Woodchucks are naturally infected with woodchuck hepatitis virus (WHV), closely related to HBV, but not with HBV. Here, we investigated the capabilities of woodchuck hepatic and human non-hepatic cell lines to support HBV infection. DNA transfection assays indicated that all cells tested supported both HBV and WHV replication steps post entry, including the viral covalently closed circular DNA (cccDNA) formation, which is essential for establishing and sustaining infection. Ectopic expression of huNTCP rendered one, but not the other, woodchuck hepatic cell line and the non-hepatic human cell line competent to support productive HBV entry, defined here by cccDNA formation during de novo infection. All huNTCP-expressing cell lines tested became susceptible to infection with hepatitis D virus (HDV) that shares the same entry receptor and initial steps of entry with HBV, suggesting that a late entry/trafficking step(s) of HBV infection was defective in one of the two woodchuck cell lines. In addition, the non-susceptible woodchuck hepatic cell line became susceptible to HBV after fusion with human hepatic cells, suggesting the lack of a host cell-dependent factor(s) in these cells. Comparative transcriptomic analysis of the two woodchuck cell lines revealed widespread differences in gene expression in multiple biological processes that may contribute to HBV infection. In conclusion, other than huNTCP, neither human- nor hepatocyte-specific factors are essential for productive HBV entry. Furthermore, a late trafficking step(s) during HBV infection, following the shared entry steps with HDV and before cccDNA formation, is subject to host cell regulation and thus, a host determinant of HBV infection.

Abstract Background Harsh environments surrounding fetuses and children can induce cellular damage in the developing brain, increasing the risk of intellectual disability and other neurodevelopmental disorders such as schizophrenia. However, the mechanisms by which early damage leads to disease manifestation in later life remain largely unknown. Previously, we demonstrated that the activation of heat shock (HS) signaling can be utilized as a unique reporter to label the cells that undergo specific molecular/cellular changes upon exposure to environmental insults throughout the body. Since the activation of HS signaling is an acute and transient event, this approach was not intended for long-term tracing of affected cells after the activation has diminished. In the present study, we generated new reporter transgenic mouse lines as a novel tool to achieve systemic and long-term tracking of affected cells and their progeny. Methods The reporter transgenic mouse system was designed so that the activation of HS signaling through HS response element (HSE) drives flippase (FLPo)-flippase recognition target (FRT) recombination-mediated permanent expression of the red fluorescent protein (RFP), tdTomato. With a priority on consistent and efficient assessment of the reporter system, we focused on intraperitoneal (i.p.) injection models of high-dose, short prenatal exposure to alcohol (ethanol) and sodium arsenite (ethanol at 4.0 g/kg/day and sodium arsenite at 5.0 mg/kg/day, at embryonic day (E) 12 and 13). Long-term reporter expression was examined in the brain of reporter mice that were prenatally exposed to these insults. Electrophysiological properties were compared between RFP+ and RFP− cortical neurons in animals prenatally exposed to arsenite. Results We detected RFP+ neurons and glia in the brains of postnatal mice that had been prenatally exposed to alcohol or sodium arsenite. In animals prenatally exposed to sodium arsenite, we also detected reduced excitability in RFP+ cortical neurons. Conclusion The reporter transgenic mice allowed us to trace the cells that once responded to prenatal environmental stress and the progeny derived from these cells long after the exposure in postnatal animals. Tracing of these cells indicates that the impact of prenatal exposure on neural progenitor cells can lead to functional abnormalities in their progeny cells in the postnatal brain. Further studies using more clinically relevant exposure models are warranted to explore this mechanism.

Chronic olfactory inflammation (COI) in conditions such as chronic rhinosinusitis significantly impairs the functional and anatomical components of the olfactory system. COI induced by intranasal administration of lipopolysaccharide (LPS) results in atrophy, gliosis, and pro-inflammatory cytokine production in the olfactory bulb (OB). Although chronic rhinosinusitis patients have smaller OBs, the consequences of olfactory inflammation on OB neurons are largely unknown. In this study, we investigated the neurological consequences of COI on OB projection neurons, mitral cells (MCs) and tufted cells (TCs). To induce COI, we performed unilateral intranasal administration of LPS to mice for 4 and 10 weeks. Effects of COI on the OB were examined using RNA-sequencing approaches and immunohistochemical analyses. We found that repeated LPS administration upregulated immune-related biological pathways in the OB after 4 weeks. We also determined that the length of TC lateral dendrites in the OB significantly decreased after 10 weeks of COI. The axon initial segment of TCs decreased in number and in length after 10 weeks of COI. The lateral dendrites and axon initial segments of MCs, however, were largely unaffected. In addition, dendritic arborization and AIS reconstruction both took place following a 10-week recovery period. Our findings suggest that olfactory inflammation specifically affects TCs and their integrated circuitry, whereas MCs are potentially protected from this condition. This data demonstrates unique characteristics of the OBs ability to undergo neuroplastic changes in response to stress.

Mutations in the homologous recombination proteins BRCA1 and BRCA2 can sensitize cells to treatment with inhibitors of poly-ADP-ribose polymerase 1 (PARPi), but resistance to the treatment can occur. Here the authors by genome-wide CRISPR knockout and activation screens reveal novel pathways of PARPi resistance in BRCA2-deficient cells.

The molecular mechanisms driving proliferation in the pediatric brain cancer epdendymoma are poorly understood. Here the authors show that a YAP1- MAMLD1 fusion drives tumor formation in mice and show that the fusion protein can collaborate with the TEAD and NFI transcription factors.

Gain at chromosome 17q21 in neuroblastoma is associated with a poor prognosis, independent of MYCN amplification status. Several potential proto-oncogenes have been identified in this region, one of them—insulin-like growth-factor-2 mRNA binding protein (IGF2BP1)—is expressed at high levels in stage 4 tumors, and associated with overall lower patient survival. Here, we demonstrate that down-regulation of IGF2BP1 activity, either by transcript silencing or chemical inhibition, suppresses neuroblastoma cell growth. Furthermore, the combination of IGF2BP1 inhibition along with commonly used chemotherapeutics that broadly affect DNA synthesis, or cyclin-dependent kinase (CDK) inhibitors that disrupt signal transduction, have a synergistic effect on the suppression of neuroblastoma cell proliferation.

Synapse formation and regulation require signaling interactions between pre- and postsynaptic proteins, notably cell adhesion molecules (CAMs). It has been proposed that the functions of neuroligins (Nlgns), postsynaptic CAMs, rely on the formation of trans-synaptic complexes with neurexins (Nrxns), presynaptic CAMs. Nlgn3 is a unique Nlgn isoform that localizes at both excitatory and inhibitory synapses. However, Nlgn3 function mediated via Nrxn interactions is unknown. Here we demonstrate that Nlgn3 localizes at postsynaptic sites apposing vesicular glutamate transporter 3-expressing (VGT3+) inhibitory terminals and regulates VGT3+ inhibitory interneuron-mediated synaptic transmission in mouse organotypic slice cultures. Gene expression analysis of interneurons revealed that the αNrxn1+AS4 splice isoform is highly expressed in VGT3+ interneurons as compared with other interneurons. Most importantly, postsynaptic Nlgn3 requires presynaptic αNrxn1+AS4 expressed in VGT3+ interneurons to regulate inhibitory synaptic transmission. Our results indicate that specific Nlgn–Nrxn signaling generates distinct functional properties at synapses.

Background: MicroRNAs (miRNA) are small non-coding RNAs that regulate gene expression playing a key role in organogenesis. MiRNAs are studied in tracheal aspirates (TA) of preterm infants. However; this is difficult to obtain in infants who are not intubated. This study examines early salivary miRNA expression as non-invasive early biomarkers in extremely low gestational age newborns (ELGANs). Methods: Saliva was collected using DNA-genotek swabs, miRNAs were analyzed using RNA seq and RT PCR arrays. Salivary miRNA expression was compared to TA using RNA seq at 3 days of age, and longitudinal changes at 28 days of age were analyzed using RT PCR arrays in ELGANs. Results: Approximately 822 ng of RNA was extracted from saliva of 7 ELGANs; Of the 757 miRNAs isolated, 161 miRNAs had significant correlation in saliva and TA at 3 days of age (r = 0.97). Longitudinal miRNA analysis showed 29 miRNAs downregulated and 394 miRNAs upregulated at 28 days compared to 3 days of age (adjusted p < 0.1). Bioinformatic analysis (Ingenuity Pathway Analysis) of differentially expressed miRNAs identified organismal injury and abnormalities and cellular development as the top physiological system development and cellular function. Conclusion: Salivary miRNA expression are source for early biomarkers of underlying pathophysiology in ELGANs.

Surfactant protein A (SP-A) in addition to its surfactant-related functions interacts with alveolar macrophages (AM), the guardian cells of innate immunity in the lungs, and regulates many of its functions under basal condition and in response to various pressures, such as infection and oxidative stress. The human SP-A locus consists of two functional genes, SFTPA1 and SFTPA2, and one pseudogene. The functional genes encode human SP-A1 and SP-A2 proteins, respectively, and each has been identified with several genetic variants. SP-A variants differ in their ability to regulate lung function mechanics and survival in response to bacterial infection. Here, we investigated the effect of hSP-A variants on the AM gene expression profile in response to Klebsiella pneumoniae infection. We used four humanized transgenic (hTG) mice that each carried SP-A1 (6A2, 6A4) or SP-A2 (1A0, 1A3), and KO. AM gene expression profiling was performed after 6 h post-infection. We found: (a) significant sex differences in the expression of AM genes; (b) in response to infection, 858 (KO), 196 (6A2), 494 (6A4), 276 (1A0), and 397 (1A3) genes were identified (P < 0.05) and some of these were differentially expressed with ≥2 fold, specific to either males or females; (c) significant SP-A1 and SP-A2 variant-specific differences in AM gene expression; (d) via Ingenuity Pathway Analysis (IPA), key pathways and molecules were identified that had direct interaction with TP53, TNF, and cell cycle signaling nodes; (e) of the three pathways (TNF, TP-53, and cell cycle signaling nodes) studied here, all variants except SP-A2 (1A3) female, showed significance for at least 2 of these pathways, and KO male showed significance for all three pathways; (f) validation of key molecules exhibited variant-specific significant differences in the expression between sexes and a similarity in gene expression profile was observed between KO and SP-A1. These results reveal for the first time a large number of biologically relevant functional pathways influenced in a sex-specific manner by SP-A variants in response to infection. These data may assist in studying molecular mechanisms of SP-A-mediated AM gene regulation and potentially identify novel therapeutic targets for K. pneumoniae infection.

Extracellular vesicles (EVs) released by bone marrow stromal cells (BMSCs) have been shown to act as a transporter of bioactive molecules such as RNAs and proteins in the therapeutic actions of BMSCs in various diseases. Although EV therapy holds great promise to be a safer cell-free therapy overcoming issues related to cell therapy, manufacturing processes that offer scalable and reproducible EV production have not been established. Robust and scalable BMSC manufacturing methods have been shown to enhance EV production; however, the effects on EV quality remain less studied. Here, using human BMSCs isolated from nine healthy donors, we examined the effects of high-performance culture media that can rapidly expand BMSCs on EV production and quality in comparison with the conventional culture medium. We found significantly increased EV production from BMSCs cultured in the high-performance media without altering their multipotency and immunophenotypes. RNA sequencing revealed that RNA contents in EVs from high-performance media were significantly reduced with altered profiles of microRNA enriched in those related to cellular growth and proliferation in the pathway analysis. Given that pre-clinical studies at the laboratory scale often use the conventional medium, these findings could account for the discrepancy in outcomes between pre-clinical and clinical studies. Therefore, this study highlights the importance of selecting proper culture conditions for scalable and reproducible EV manufacturing.

Summary: Type 1 interferon (T1IFN) signaling promotes inflammation and lupus pathology, but its role in autoreactive B cell development in the antibody-forming cell (AFC) and germinal center (GC) pathways is unclear. Using a lupus model that allows for focused study of the AFC and GC responses, we show that T1IFN signaling is crucial for autoreactive B cell development in the AFC and GC pathways. Through bone marrow chimeras, DNA-reactive B cell transfer, and GC-specific Cre mice, we confirm that IFNαR signaling in B cells promotes autoreactive B cell development into both pathways. Transcriptomic analysis reveals gene expression alterations in multiple signaling pathways in non-GC and GC B cells in the absence of IFNαR. Finally, we find that T1IFN signaling promotes autoreactive B cell development in the AFC and GC pathways by regulating BCR signaling. These data suggest value for anti-IFNαR therapy in individuals with elevated T1IFN activity before clinical disease onset. : The B-cell-intrinsic mechanisms of type 1 interferon (T1IFN) signaling in regulating B cell tolerance is unclear. Domeier et al. show that T1IFN signaling in B cells causes loss of B cell tolerance, promoting autoreactive B cell development into the antibody-forming cell and germinal center pathways by regulating BCR signaling. Keywords: B cells, spontaneous germinal centers, antibody-forming cells, B cell tolerance, autoimmunity, systemic lupus erythematosus, type 1 interferon signaling

Summary: Homeostatic synaptic downscaling reduces neuronal excitability by modulating the number of postsynaptic receptors. Histone modifications and the subsequent chromatin remodeling play critical roles in activity-dependent gene expression. Histone modification codes are recognized by chromatin readers that affect gene expression by altering chromatin structure. We show that L3mbtl1 (lethal 3 malignant brain tumor-like 1), a polycomb chromatin reader, is downregulated by neuronal activity and is essential for synaptic response and downscaling. Genome-scale mapping of L3mbtl1 occupancies identified Ctnnb1 as a key gene downstream of L3mbtl1. Importantly, the occupancy of L3mbtl1 on the Ctnnb1 gene was regulated by neuronal activity. L3mbtl1 knockout neurons exhibited reduced Ctnnb1 expression. Partial knockdown of Ctnnb1 in wild-type neurons reduced excitatory synaptic transmission and abolished homeostatic downscaling, and transfecting Ctnnb1 in L3mbtl1 knockout neurons enhanced synaptic transmission and restored homeostatic downscaling. These results highlight a role for L3mbtl1 in regulating homeostasis of synaptic efficacy. : Synaptic homeostasis is crucial for maintaining proper neuronal excitability and excitatory/inhibitory balance in the brain. Mao et al. report that an activity-dependent chromatin reader protein is required for homeostatic control of synaptic strength through the regulation of downstream target gene Ctnnb1. Keywords: homeostatic plasticity, synaptic scaling, chromatin reader, glutamate receptor, synaptic transmission, hippocampus, neuronal activity

Abstract Background Influenza A viruses cause life-threatening pneumonia and lung injury in the lower respiratory tract. Application of high GM-CSF levels prior to infection has been shown to reduce morbidity and mortality from pathogenic influenza infection in mice, but the mechanisms of protection and treatment efficacy have not been established. Methods Mice were infected intranasally with influenza A virus (PR8 strain). Supra-physiologic levels of GM-CSF were induced in the airways using the double transgenic GM-CSF (DTGM) or littermate control mice starting on 3 days post-infection (dpi). Assessment of respiratory mechanical parameters was performed using the flexiVent rodent ventilator. RNA sequence analysis was performed on FACS-sorted airway macrophage subsets at 8 dpi. Results Supra-physiologic levels of GM-CSF conferred a survival benefit, arrested the deterioration of lung mechanics, and reduced the abundance of protein exudates in bronchoalveolar (BAL) fluid to near baseline levels. Transcriptome analysis, and subsequent validation ELISA assays, revealed that excess GM-CSF re-directs macrophages from an “M1-like” to a more “M2-like” activation state as revealed by alterations in the ratios of CXCL9 and CCL17 in BAL fluid, respectively. Ingenuity pathway analysis predicted that GM-CSF surplus during IAV infection elicits expression of anti-inflammatory mediators and moderates M1 macrophage pro-inflammatory signaling by Type II interferon (IFN-γ). Conclusions Our data indicate that application of high levels of GM-CSF in the lung after influenza A virus infection alters pathogenic “M1-like” macrophage inflammation. These results indicate a possible therapeutic strategy for respiratory virus-associated pneumonia and acute lung injury.

Prenatal exposure to environmental stressors is known to impair cortical development. Here the authors show that upon exposure to stressors, the activation of Hsf1-Hsp signalling is highly variable among cells in the embryonic cortex of mice, and either too much or too little activation can result in defects in cortical development.

In humans there are two surfactant protein A (SP-A) functional genes SFTPA1 and SFTPA2 encoding innate immune molecules, SP-A1 and SP-A2, respectively, with numerous genetic variants each. SP-A interacts and regulates many of the functions of alveolar macrophages (AM). It is shown that SP-A variants differ in their ability to regulate the AM miRNome in response to oxidative stress (OxS). Because humans have both SP-A gene products, we were interested to determine the combined effect of co-expressed SP-A1/SP-A2 (co-ex) in response to ozone (O3) induced OxS on AM miRNome. Human transgenic (hTG) mice, carrying both SP-A1/SP-A2 (6A2/1A0, co-ex) and SP-A- KO were utilized. The hTG and KO mice were exposed to filtered air (FA) or O3 and miRNA levels were measured after AM isolation with or without normalization to KO. We found: (i) The AM miRNome of co-ex males and females in response to OxS to be largely downregulated after normalization to KO, but after Bonferroni multiple comparison analysis only in females the AM miRNome remained significantly different compared to control (FA); (ii) The targets of the significantly changed miRNAs were downregulated in females and upregulated in males; (iii) Several of the validated mRNA targets were involved in pro-inflammatory response, anti-apoptosis, cell cycle, cellular growth and proliferation; (iv) The AM of SP-A2 male, shown, previously to have major effect on the male AM miRNome in response to OxS, shared similarities with the co-ex, namely in pathways involved in the pro-inflammatory response and anti-apoptosis but also exhibited differences with the cell-cycle, growth, and proliferation pathway being involved in co-ex and ROS homeostasis in SP-A2 male. We speculate that the presence of both gene products vs. single gene products differentially impact the AM responses in males and females in response to OxS.

Background: Hypoalgesic inflammatory bowel disease (IBD), a condition in which patients with active disease do not perceive and/or report abdominal pain, is associated with serious complications and there is a lack of cost-effective, reliable diagnostic methods to identify “at-risk” patients. The voltage-gated sodium channels (VGSC's), Nav1.7, Nav1.8, and Nav1.9, are preferentially expressed on nociceptive neurons, and have been implicated in visceral inflammatory pain. At least 29 VGSC single nucleotide polymorphisms (SNPs) have been implicated in chronic somatic pain syndromes, but little is known about their role in human visceral sensation. We hypothesized that disruptive VGSC polymorphisms result in anti-nociceptive behavior in IBD.Methods and Findings: We performed targeted exome sequencing and/or TaqMan genotyping to evaluate the Nav1.7, Nav1.8, and Nav1.9 genes (SCN9A, SCN10A and SCN11A) in 121 IBD patients (including 41 “hypoalgesic” IBD patients) and 86 healthy controls. Allelic and genotypic frequencies of polymorphisms were compared among study groups who had undergone characterization of intestinal inflammatory status and abdominal pain experience. Forty-nine total exonic SNPs were identified. The allelic frequency of only one non-synonymous SNP (rs6795970 [SCN10A]) approached significance in hypoalgesic IBD patients when compared to other IBD patients (p = 0.096, Fisher's exact test). Hypoalgesic IBD patients were more likely to be homozygous for this polymorphism (46 vs. 22%, p = 0.01, Fisher's exact test).Conclusions: This is the first human study to demonstrate a link between a genetic variant of SCN10A and abdominal pain perception in IBD. These findings provide key insights into visceral nociceptive physiology and new diagnostic and therapeutic targets to consider in IBD and other gastrointestinal conditions associated with chronic abdominal pain. Further studies are required to elucidate the precise pathophysiological impact of the rs6795970 polymorphism on human gastrointestinal nociception.

Tissue-resident memory CD8 T (TRM) cells defend against microbial reinfections at mucosal barriers; determinants driving durable TRM cell responses in non-mucosal tissues, which often harbor opportunistic persistent pathogens, are unknown. JC polyomavirus (JCPyV) is a ubiquitous constituent of the human virome. With altered immunological status, JCPyV can cause the oft-fatal brain demyelinating disease progressive multifocal leukoencephalopathy (PML). JCPyV is a human-only pathogen. Using the mouse polyomavirus (MuPyV) encephalitis model, we demonstrate that CD4 T cells regulate development of functional antiviral brain-resident CD8 T cells (bTRM) and renders their maintenance refractory to systemic CD8 T cell depletion. Acquired CD4 T cell deficiency, modeled by delaying systemic CD4 T cell depletion until MuPyV-specific CD8 T cells have infiltrated the brain, impacted the stability of CD8 bTRM, impaired their effector response to reinfection, and rendered their maintenance dependent on circulating CD8 T cells. This dependence of CD8 bTRM differentiation on CD4 T cells was found to extend to encephalitis caused by vesicular stomatitis virus. Together, these findings reveal an intimate association between CD4 T cells and homeostasis of functional bTRM to CNS viral infection.

Despite progress, our understanding of psychiatric and neurological illnesses remains poor, at least in part due to the inability to access neurons directly from patients. Currently, there are in vitro models available but significant work remains, including the search for a less invasive, inexpensive and rapid method to obtain neuronal-like cells with the capacity to deliver reproducible results. Here, we present a new protocol to transdifferentiate human circulating monocytes into neuronal-like cells in 20 days and without the need for viral insertion or reprograming. We have thoroughly characterized these monocyte-derived-neuronal-like cells (MDNCs) through various approaches including immunofluorescence (IF), flow cytometry, qRT-PCR, single cell mRNA sequencing, electrophysiology and pharmacological techniques. These MDNCs resembled human neurons early in development, expressed a variety of neuroprogenitor and neuronal genes as well as several neuroprogenitor and neuronal proteins and also presented electrical activity. In addition, when these neuronal-like cells were exposed to either dopamine or colchicine, they responded similarly to neurons by retracting their neuronal arborizations. More importantly, MDNCs exhibited reproducible differentiation rates, arborizations and expression of dopamine 1 receptors (DR1) on separate sequential samples from the same individual. Differentiation efficiency measured by cell morphology was on average 11.9 ± 1.4% (mean, SEM, n = 38,819 cells from 15 donors). To provide context and help researchers decide which in vitro model of neuronal development is best suited to address their scientific question,we compared our results with those of other in vitro models currently available and exposed advantages and disadvantages of each paradigm.

Here we describe isolation and characterization of macrophage-tumor cell fusions (MTFs) from the blood of pancreatic ductal adenocarcinoma (PDAC) patients. The MTFs were generally aneuploidy, and immunophenotypic characterizations showed that the MTFs express markers characteristic of PDAC and stem cells, as well as M2-polarized macrophages. Single cell RNASeq analyses showed that the MTFs express many transcripts implicated in cancer progression, LINE1 retrotransposons, and very high levels of several long non-coding transcripts involved in metastasis (such as MALAT1). When cultured MTFs were transplanted orthotopically into mouse pancreas, they grew as obvious well-differentiated islands of cells, but they also disseminated widely throughout multiple tissues in "stealth" fashion. They were found distributed throughout multiple organs at 4, 8, or 12 weeks after transplantation (including liver, spleen, lung), occurring as single cells or small groups of cells, without formation of obvious tumors or any apparent progression over the 4 to 12 week period. We suggest that MTFs form continually during PDAC development, and that they disseminate early in cancer progression, forming "niches" at distant sites for subsequent colonization by metastasis-initiating cells.

Genetic and epigenetic alterations observed at end stage OSCC formation could be considered as a consequence of cancer development and thus changes in normal or premalignant tissues which had been exposed to oral carcinogens such as Dibenzo[def,p]chrysene (DBP) may better serve as predictive biomarkers of disease development. Many types of DNA damage can induce epigenetic changes which can occur early and in the absence of evident morphological abnormalities. Therefore we used ERRBS to generate genome-scale, single-base resolution DNA methylomes from histologically normal oral tissues of mice treated with DBP under experimental conditions known to induce maximum DNA damage which is essential for the development of OSCC induced by DBP in mice. After genome-wide correction, 30 and 48 differentially methylated sites (DMS) were identified between vehicle control and DBP treated mice using 25% and 10% differences in methylation, respectively. RT-PCR was further performed to examine the expressions of nine selected genes. Among them, Fgf3, a gene frequently amplified in head and neck cancer, showed most prominent and significant gene expression change (2.4× increases), despite the hypomethylation of Fgf3 was identified at >10kb upstream of transcription start site. No difference was observed in protein expression between normal oral tissues treated with DBP or vehicle as examined by immunohistochemistry. Collectively, our results indicate that Fgf3 hypomethylation and gene overexpression, but not protein expression, occurred in the early stage of oral carcinogenesis induced by DBP. Thus, Fgf3 hypomethylation may serve as a potential biomarker for early detection of OSCC.

The hallmark of the cerebral neocortex is its organization into six layers, each containing a characteristic set of cell types and synaptic connections. The transcriptional events involved in laminar development and function still remain elusive. Here, we employed deep sequencing of mRNA and small RNA species to gain insights into transcriptional differences among layers and their temporal dynamics during postnatal development of the mouse primary somatosensory neocortex. We identify a number of coding and noncoding transcripts with specific spatiotemporal expression and splicing patterns. We also identify signature trajectories and gene coexpression networks associated with distinct biological processes and transcriptional overlap between these processes. Finally, we provide data that allow the study of potential miRNA and mRNA interactions. Overall, this study provides an integrated view of the laminar and temporal expression dynamics of coding and noncoding transcripts in the mouse neocortex and a resource for studies of neurodevelopment and transcriptome.

Second generation antipsychotics (SGAs), like olanzapine, exhibit acute metabolic side effects leading to metabolic inflexibility, hyperglycemia, adiposity and diabetes. Understanding how SGAs affect the skeletal muscle transcriptome could elucidate approaches for mitigating these side effects. Male Sprague-Dawley rats were infused intravenously with vehicle or olanzapine for 24h using a dose leading to a mild hyperglycemia. RNA-Seq was performed on gastrocnemius muscle, followed by alignment of the data with the Rat Genome Assembly 5.0. Olanzapine altered expression of 1347 out of 26407 genes. Genes encoding skeletal muscle fiber-type specific sarcomeric, ion channel, glycolytic, O2- and Ca2+-handling, TCA cycle, vascularization and lipid oxidation proteins and pathways, along with NADH shuttles and LDH isoforms were affected. Bioinformatics analyses indicate that olanzapine decreased the expression of slower and more oxidative fiber type genes (e.g., type 1), while up regulating those for the most glycolytic and least metabolically flexible, fast twitch fiber type, IIb. Protein turnover genes, necessary to bring about transition, were also up regulated. Potential upstream regulators were also identified. Olanzapine appears to be rapidly affecting the muscle transcriptome to bring about a change to a fast-glycolytic fiber type. Such fiber types are more susceptible than slow muscle to atrophy, and such transitions are observed in chronic metabolic diseases. Thus these effects could contribute to the altered body composition and metabolic disease olanzapine causes. A potential interventional strategy is implicated because aerobic exercise, in contrast to resistance exercise, can oppose such slow to fast fiber transitions.

Severity of neurobehavioral deficits in children born from adverse pregnancies, such as maternal alcohol consumption and diabetes, does not always correlate with the adversity's duration and intensity. Therefore, biological signatures for accurate prediction of the severity of neurobehavioral deficits, and robust tools for reliable identification of such biomarkers, have an urgent clinical need. Here, we demonstrate that significant changes in the alternative splicing (AS) pattern of offspring lymphocyte RNA can function as accurate peripheral biomarkers for motor learning deficits in mouse models of prenatal alcohol exposure (PAE) and offspring of mother with diabetes (OMD). An aptly trained deep-learning model identified 29 AS events common to PAE and OMD as superior predictors of motor learning deficits than AS events specific to PAE or OMD. Shapley-value analysis, a game-theory algorithm, deciphered the trained deep-learning model's learnt associations between its input, AS events, and output, motor learning performance. Shapley values of the deep-learning model's input identified the relative contribution of the 29 common AS events to the motor learning deficit. Gene ontology and predictive structure-function analyses, using Alphafold2 algorithm, supported existing evidence on the critical roles of these molecules in early brain development and function. The direction of most AS events was opposite in PAE and OMD, potentially from differential expression of RNA binding proteins in PAE and OMD. Altogether, this study posits that AS of lymphocyte RNA is a rich resource, and deep-learning is an effective tool, for discovery of peripheral biomarkers of neurobehavioral deficits in children of diverse adverse pregnancies. [ABSTRACT FROM AUTHOR]

Background: Circulating small RNAs (smRNAs) originate from diverse tissues and organs. Previous studies investigating smRNAs as potential biomarkers for Parkinson's disease (PD) have yielded inconsistent results. We investigated whether smRNA profiles from neuronally-enriched serum exosomes and microvesicles are altered in PD patients and discriminate PD subjects from controls. Methods: Demographic, clinical, and serum samples were obtained from 60 PD subjects and 40 age- and sex-matched controls. Exosomes and microvesicles were extracted and isolated using a validated neuronal membrane marker (CD171). Sequencing and bioinformatics analyses were used to identify differentially expressed smRNAs in PD and control samples. SmRNAs also were tested for association with clinical metrics. Logistic regression and random forest classification models evaluated the discriminative value of the smRNAs. Results: In serum CD171 enriched exosomes and microvesicles, a panel of 29 smRNAs was expressed differentially between PD and controls (false discovery rate (FDR) < 0.05). Among the smRNAs, 23 were upregulated and 6 were downregulated in PD patients. Pathway analysis revealed links to cellular proliferation regulation and signaling. Least absolute shrinkage and selection operator adjusted for the multicollinearity of these smRNAs and association tests to clinical parameters via linear regression did not yield significant results. Univariate logistic regression models showed that four smRNAs achieved an AUC ≥ 0.74 to discriminate PD subjects from controls. The random forest model had an AUC of 0.942 for the 29 smRNA panel. Conclusion: CD171-enriched exosomes and microvesicles contain the differential expression of smRNAs between PD and controls. Future studies are warranted to follow up on the findings and understand the scientific and clinical relevance. [ABSTRACT FROM AUTHOR]