A roadmap to human hippocampal neurogenesis in adulthood, aging and AD.

In the rodent, hippocampal neurogenesis plays critical roles in learning and memory1,2, is tightly regulated by inhibitory neurons3-7 and contributes to memory dysfunction in Alzheimer's disease (AD) mouse models8-10. In contrast, the mechanisms regulating neurogenesis in the adult human hippocampus, the dynamic shifts in the transcriptomic and epigenomic profiles in aging and AD and putative niche interactions within the cellular environment, remain largely unknown. Using single nuclei multi-omics of postmortem human hippocampi we map the molecular mechanisms of hippocampal neurogenesis across aging, cognitive decline, and AD neuropathology. Transcriptomic and epigenetic profiling of neural stem cells (NSCs), neuroblasts and immature neurons suggests that the earliest shift in the characteristics of neurogenesis takes place in NSCs in aging. Cognitive impairment was associated with changes in neuroblast profile. In AD, there was a widespread cessation of the transcription machinery in immature neurons, with robust downregulation of genes regulating ribosomal and mitochondrial function. Further, there was substantial loss of parvalbumin+ inhibitory neurons in the hippocampus in aging. The number of the rest of inhibitory neurons were reduced as a function of age and diagnosis. Notably, a similar system-level effect was observed between immature and inhibitory neurons in the transition from aging to AD, manifested by common molecular pathways that were ultimately lost in AD. The numbers of neuroblasts, immature and GABAergic neurons inversely correlated with extent of neuropathology. Using CellChat and NeuronChat, we inferred the ligands and receptors by which neurogenic cells communicate with their cellular environment. Loss of synaptic adhesion molecules and neurotransmitters, either sent or received by neurogenic cells, was observed in AD. Together, this study delineates the molecular mechanisms and dynamics of human neurogenesis, functional association with inhibitory neurons and a mechanism of hippocampal hyperexcitability in AD.

Oleuropein has hypophagic effects in broiler chicks.

Oleuropein, a phenolic compound derived from olives, has known glucoregulatory effects in mammalian models but effects in birds are unknown. We investigated effects of dietary supplementation and exogenous administration of oleuropein on broiler chick feed intake and glucose homeostasis during the first 7 days post-hatch. One hundred and forty-eight day-of-hatch broiler chicks were randomly allocated to one of four dietary treatments with varying oleuropein concentrations (0, 250, 500, or 1,000 mg/kg). Body weight and breast muscle and liver weights were recorded on day 7. In the next experiment, chicks received intraperitoneal (IP) injections of oleuropein at doses of 0 (vehicle), 50, 100, or 200 mg/kg on day 4 post-hatch, with feed intake and blood glucose levels measured thereafter. Lastly, chicks fed a control diet were fasted and administered intracerebroventricular (ICV) injections of oleuropein at doses of 0, 50, 100, or 200 µg, after which feed intake was recorded. Results indicated that IP and ICV injections led to decreased feed intake, primarily at 60 min post-injection, with effects diminishing by 90 min in the IP study. Blood glucose levels decreased 1-h post-IP injection at higher oleuropein doses. These findings suggest that oleuropein acts as a mild appetite suppressant and influences energy metabolism in broiler chickens.

MLK3 promotes prooncogenic signaling in hepatocellular carcinoma via TGFß pathway.

Advanced hepatocellular carcinoma (HCC) is a lethal disease, with limited therapeutic options. Mixed Lineage Kinase 3 (MLK3) is a key regulator of liver diseases, although its role in HCC remains unclear. Analysis of TCGA databases suggested elevated MAP3K11 (MLK3 gene) expression, and TMA studies showed higher MLK3 activation in human HCCs. To understand MLK3's role in HCC, we utlized carcinogen-induced HCC model and compared between wild-type and MLK3 knockout (MLK3-/-) mice. Our studies showed that MLK3 kinase activity is upregulated in HCC, and MLK3 deficiency alleviates HCC progression. MLK3 deficiency reduced proliferation in vivo and MLK3 inhibition reduced proliferation and colony formation in vitro. To obtain further insight into the mechanism and identify newer targets mediating MLK3-induced HCCs, RNA-sequencing analysis was performed. These showed that MLK3 deficiency modulates various gene signatures, including EMT, and reduces TGFB1&2 expressions. HCC cells overexpressing MLK3 promoted EMT via autocrine TGFß signaling. Moreover, MLK3 deficiency attenuated activated hepatic stellate cell (HSC) signature, which is increased in wild-type. Interestingly, MLK3 promotes HSC activation via paracrine TGFß signaling. These findings reveal TGFß playing a key role at different steps of HCC, downstream of MLK3, implying MLK3-TGFß axis to be an ideal drug target for advanced HCC management.

BRWD1 establishes epigenetic states for germinal center initiation, maintenance, and function.

Germinal center (GC) B cells segregate into three subsets that compartmentalize the antagonistic molecular programs of selection, proliferation, and somatic hypermutation. In bone marrow, the epigenetic reader BRWD1 orchestrates and insulates the sequential stages of cell proliferation and Igk recombination. We hypothesized BRWD1 might play similar insulative roles in the periphery. In Brwd1 -/- follicular B cells, GC initiation and class switch recombination following immunization were inhibited. In contrast, in Brwd1 -/- GC B cells there was admixing of chromatin accessibility across GC subsets and transcriptional dysregulation including induction of inflammatory pathways. This global molecular GC dysregulation was associated with specific defects in proliferation, affinity maturation, and tolerance. These data suggest that GC subset identity is required for some but not all GC-attributed functions. Furthermore, these data demonstrate a central role for BRWD1 in orchestrating epigenetic transitions at multiple steps along B cell developmental and activation pathways.

Zymosan and lipopolysaccharide decrease gene expression of neuronal nitric oxide synthase in peripheral organs in chicks.

Nitric oxide (NO) is gaseous bioactive molecule that is synthesized by NO synthase (NOS). Inducible NOS (iNOS) expression occurs in response to pathogenic challenges, resulting in the production of large amounts of NO. However, there is a lack of knowledge regarding neuronal NOS (nNOS) and endothelial NOS (eNOS) in birds during pathogenic challenge. Therefore, the present study was conducted to determine the influence of intraperitoneal (IP) injection of zymosan (cell wall component of yeast) and lipopolysaccharide (LPS, a cell wall component of gram-negative bacteria) on NOS expression in chicks (Gallus gallus). Furthermore, the effect of NOS inhibitors on the corresponding behavioral and physiological parameters was investigated. Zymosan and LPS injections induced iNOS mRNA expression in several organs. Zymosan had no effect on eNOS mRNA expression in the organs investigated, whereas LPS increased its expression in the pancreas. Zymosan and LPS decreased nNOS mRNA expression in the lung, heart, kidney, and pancreas. The decreased nNOS mRNA expression in pancreas was probably associated with the NO from iNOS provided that such effect was reproduced by IP injection of sodium nitroprusside, which is a NO donor. Furthermore, pancreatic nNOS mRNA expression decreased following subcutaneous injection of corticosterone. Furthermore, IP injections of a nonspecific NOS inhibitor, NG-nitro-L-arginine methyl ester, and an nNOS-specific inhibitor, 7-nitroindazole, resulted in the significant decreases in food intake, cloacal temperature, and feed passage via the digestive tract in chicks. Collectively, the current findings imply the decreased nNOS expression because of fungal and bacterial infections, which affects food intake, body temperature, and the digestive function in birds.

Effect of platelet-activating factor on food intake, cloacal temperature, voluntary activity and crop emptying rate in chicks.

Platelet-activating factor (PAF) plays a significant role in several leucocyte functions, including platelet aggregation and inflammation. Additionally, PAF has a role in the behavioral and physiological changes in mammals. However, the effect of PAF has not been well studied in birds. Therefore, the study aimed to determine if PAF affects feeding behavior, voluntary activity, cloacal temperature, and feed passage through the digestive tract in chicks (Gallus gallus). We also studied the involvement of PAF in the innate immune system induced by lipopolysaccharide (LPS), a cell wall component of gram-negative bacteria. Both intraperitoneal (IP) and intracerebroventricular (ICV) injections of PAF significantly decreased food intake. IP injection of PAF significantly decreased voluntary activity and slowed the feed passage from the crop, whereas ICV injection had no effect. Conversely, ICV injection of PAF significantly increased the cloacal temperature, but IP injection had no effect. The IP injection of LPS significantly reduced the mRNA expression of lysophosphatidylcholine acyltransferase 2, an enzyme responsible for PAF production in the heart and pancreas. On the other hand, LPS significantly increased the mRNA expression of the PAF receptor in the peripheral organs. The present study shows that PAF influences behavioral and physiological responses and is related to the response against bacterial infections in chicks.

ERBB3 Overexpression is Enriched in Diverse Patient Populations with Castration-sensitive Prostate Cancer and is Associated with a Unique AR Activity Signature.

PURPOSE: Despite successful clinical management of castration-sensitive prostate cancer (CSPC), the 5-year survival rate for men with castration-resistant prostate cancer is only 32%. Combination treatment strategies to prevent disease recurrence are increasing, albeit in biomarker-unselected patients. Identifying a biomarker in CSPC to stratify patients who will progress on standard-of-care therapy could guide therapeutic strategies. EXPERIMENTAL DESIGN: Targeted deep sequencing was performed for the University of Illinois (UI) cohort (n = 30), and immunostaining was performed on a patient tissue microarray (n = 149). Bioinformatic analyses identified pathways associated with biomarker overexpression (OE) in the UI cohort, consolidated RNA sequencing samples accessed from Database of Genotypes and Phenotypes (n = 664), and GSE209954 (n = 68). Neutralizing antibody patritumab and ectopic HER3 OE were utilized for functional mechanistic experiments. RESULTS: We identified ERBB3 OE in diverse patient populations with CSPC, where it was associated with advanced disease at diagnosis. Bioinformatic analyses showed a positive correlation between ERBB3 expression and the androgen response pathway despite low dihydrotestosterone and stable expression of androgen receptor (AR) transcript in Black/African American men. At the protein level, HER3 expression was negatively correlated with intraprostatic androgen in Black/African American men. Mechanistically, HER3 promoted enzalutamide resistance in prostate cancer cell line models and HER3-targeted therapy resensitized therapy-resistant prostate cancer cell lines to enzalutamide. CONCLUSIONS: In diverse patient populations with CSPC, ERBB3 OE was associated with high AR signaling despite low intraprostatic androgen. Mechanistic studies demonstrated a direct link between HER3 and enzalutamide resistance. ERBB3 OE as a biomarker could thus stratify patients for intensification of therapy in castration-sensitive disease, including targeting HER3 directly to improve sensitivity to AR-targeted therapies.

Ten-eleven translocation (Tet) methylcytosine dioxygenase-dependent viral DNA demethylation mediates in vivo hepatitis B virus (HBV) biosynthesis.

Liver-specific ten-eleven translocation (Tet) methylcytosine dioxygenases 2 and 3 (Tet2 plus Tet3)-deficient hepatitis B virus (HBV) transgenic mice fail to support viral biosynthesis. The levels of viral transcription and replication intermediates are dramatically reduced. Hepatitis B core antigen is only observed in a very limited number of pericentral hepatocytes in a pattern that is similar to glutamate-ammonia ligase (Glul), a ß-catenin target gene. HBV transcript abundance in adult Tet-deficient mice resembles that observed in wild-type neonatal mice. Furthermore, the RNA levels of several ß-catenin target genes including Glul, Lhpp, Notun, Oat, Slc1a2, and Tbx3 in Tet-deficient mice were also similar to that observed in wild-type neonatal mice. As HBV transcription is regulated by ß-catenin, these findings support the suggestion that neonatal Tet deficiency might limit ß-catenin target gene expression, limiting viral biosynthesis. Additionally, HBV transgene DNA displays increased 5-methylcytosine (5mC) frequency at CpG sequences consistent with neonatal Tet deficiency being responsible for decreased developmental viral DNA demethylation mediated by 5mC oxidation to 5-hydroxymethylcytosine, a process that might be responsible for the reduction in cellular ß-catenin target gene expression and viral transcription and replication.IMPORTANCEChronic hepatitis B virus (HBV) infection causes significant worldwide morbidity and mortality. There are no curative therapies available to resolve chronic HBV infections, and the small viral genome limits molecular targets for drug development. An alternative approach to drug development is to target cellular genes essential for HBV biosynthesis. In the liver, ten-eleven translocation (Tet) genes encode cellular enzymes that are not essential for postnatal mouse development but represent essential activities for viral DNA demethylation and transcription. Consequently, Tet inhibitors may potentially be developed into therapeutic agents capable of inducing and/or maintaining HBV covalently closed circular DNA methylation, resulting in transcriptional silencing and the resolution of chronic viral infection.

Associations Between Gut Microbial Features and Sickness Symptoms in Kidney Transplant Recipients.

PURPOSE: The study investigated the relationship of gut microbiome features and sickness symptoms in kidney transplant recipients. METHODS: Employing a prospective, longitudinal design, we collected data from 19 participants who had undergone living-donor kidney transplant at three timepoints (pre-transplant and 1 week and 3 months post-transplant). Sickness symptom data and fecal specimens were collected at each timepoint. Participants were grouped either as high or low sickness symptom severity at baseline. Shotgun metagenomics sequencing characterized gut microbial structure and functional gene content. Fecal microbial features, including alpha (evenness and richness within samples) and beta (dissimilarities between samples) diversity and relative abundances, were analyzed using R statistical packages. Cross-sectional and longitudinal analyses examined relationships between gut microbial features and sickness symptoms. RESULTS: Although our exploratory findings revealed no significant differences in alpha and beta diversity between groups, the high-severity group showed lower microbial richness and evenness than the low-severity group. The high-severity group had enriched relative abundance of bacteria from the genera Citrobacter and Enterobacter and reduced relative abundance of bacteria from the genus Akkermansia across timepoints. No functional genes differed significantly between groups or timepoints. CONCLUSIONS: Kidney transplant recipients with high symptom burden displayed increased putative proinflammatory bacteria and decreased beneficial bacteria. This study provides an effect size that future large cohort studies can employ to confirm associations between gut microbial features and sickness symptom experiences in the kidney transplant population. The study findings also have implications for future interventional studies aiming to alleviate the sickness symptom burden in this population.

BRWD1 orchestrates small pre-B cell chromatin topology by converting static to dynamic cohesin.

Lymphocyte development consists of sequential and mutually exclusive cell states of proliferative selection and antigen receptor gene recombination. Transitions between each state require large, coordinated changes in epigenetic landscapes and transcriptional programs. How this occurs remains unclear. Here we demonstrate that in small pre-B cells, the lineage and stage-specific epigenetic reader bromodomain and WD repeat-containing protein 1 (BRWD1) reorders three-dimensional chromatin topology to affect the transition between proliferative and gene recombination molecular programs. BRWD1 regulated the switch between poised and active enhancers interacting with promoters, and coordinated this switch with Igk locus contraction. BRWD1 did so by converting chromatin-bound static to dynamic cohesin competent to mediate long-range looping. ATP-depletion revealed cohesin conversion to be the main energetic mechanism dictating dynamic chromatin looping. Our findings provide a new mechanism of cohesin regulation and reveal how cohesin function can be dictated by lineage contextual mechanisms to facilitate specific cell fate transitions.

trans-Endothelial neutrophil migration activates bactericidal function via Piezo1 mechanosensing.

The regulation of polymorphonuclear leukocyte (PMN) function by mechanical forces encountered during their migration across restrictive endothelial cell junctions is not well understood. Using genetic, imaging, microfluidic, and in vivo approaches, we demonstrated that the mechanosensor Piezo1 in PMN plasmalemma induced spike-like Ca2+ signals during trans-endothelial migration. Mechanosensing increased the bactericidal function of PMN entering tissue. Mice in which Piezo1 in PMNs was genetically deleted were defective in clearing bacteria, and their lungs were predisposed to severe infection. Adoptive transfer of Piezo1-activated PMNs into the lungs of Pseudomonas aeruginosa-infected mice or exposing PMNs to defined mechanical forces in microfluidic systems improved bacterial clearance phenotype of PMNs. Piezo1 transduced the mechanical signals activated during transmigration to upregulate nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 4, crucial for the increased PMN bactericidal activity. Thus, Piezo1 mechanosensing of increased PMN tension, while traversing the narrow endothelial adherens junctions, is a central mechanism activating the host-defense function of transmigrating PMNs.

Specific Polo-Like Kinase 1 Expression in Nodular Lymphocyte-Predominant Hodgkin Lymphoma Suggests an Intact Immune Surveillance Program.

Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL) is a rare and relatively indolent B-cell lymphoma. Characteristically, the [lymphocyte-predominant (LP)] tumor cells are embedded in a microenvironment enriched in lymphocytes. More aggressive variants of mature B-cell and peripheral T-cell lymphomas exhibit nuclear expression of the polo-like kinase 1 (PLK1) protein, stabilizing MYC (alias c-myc) and associated with worse clinical outcomes. This study demonstrated expression of PLK1 in the LP cells in 100% of NLPHL cases (n = 76). In contrast, <5% of classic Hodgkin lymphoma cases (n = 70) showed PLK1 expression within the tumor cells. Loss-of-function approaches demonstrated that the expression of PLK1 promoted cell proliferation and increased MYC stability in NLPHL cell lines. Correlation with clinical parameters revealed that the increased expression of PLK1 was associated with advanced-stage disease in patients with NLPHL. A multiplex immunofluorescence panel coupled with artificial intelligence algorithms was used to correlate the composition of the tumor microenvironment with the proliferative stage of LP cells. The results showed that LP cells with PLK1 (high) expression were associated with increased numbers of cytotoxic and T-regulatory T cells. Overall, the findings demonstrate that PLK1 signaling increases NLPHL proliferation and constitutes a potential vulnerability that can be targeted with PLK1 inhibitors. An active immune surveillance program in NLPHL may be a critical mechanism limiting PLK1-dependent tumor growth.

Single-cell RNA sequencing of human prostate basal epithelial cells reveals zone-specific cellular populations and gene expression signatures.

Despite evidence of genetic signatures in normal tissue correlating with disease risk, prospectively identifying genetic drivers and cell types that underlie subsequent pathologies has historically been challenging. The human prostate is an ideal model to investigate this phenomenon because it is anatomically segregated into three glandular zones (central, peripheral, and transition) that develop differential pathologies: prostate cancer in the peripheral zone (PZ) and benign prostatic hyperplasia (BPH) in the transition zone (TZ), with the central zone (CZ) rarely developing disease. More specifically, prostatic basal cells have been implicated in differentiation and proliferation during prostate development and regeneration; however, the contribution of zonal variation and the critical role of basal cells in prostatic disease etiology are not well understood. Using single-cell RNA sequencing of primary prostate epithelial cultures, we elucidated organ-specific, zone-specific, and cluster-specific gene expression differences in basal cells isolated from human prostate and seminal vesicle (SV). Aggregated analysis identified ten distinct basal clusters by Uniform Manifold Approximation and Projection. Organ specificity compared gene expression in SV with the prostate. As expected, SV cells were distinct from prostate cells by clustering, gene expression, and pathway analysis. For prostate zone specificity, we identified two CZ-specific clusters, while the TZ and PZ populations clustered together. Despite these similarities, differential gene expression was identified between PZ and TZ samples that correlated with gene expression profiles in prostate cancer and BPH, respectively. Zone-specific profiles and cell type-specific markers were validated using immunostaining and bioinformatic analyses of publicly available RNA-seq datasets. Understanding the baseline differences at the organ, zonal, and cellular level provides important insight into the potential drivers of prostatic disease and guides the investigation of novel preventive or curative treatments. Importantly, this study identifies multiple prostate basal cell populations and cell type-specific gene signatures within prostate basal epithelial cells that have potential critical roles in driving prostatic diseases. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Possible role of neuropeptide Y on zymosan- and lipopolysaccharide-induced change in gastrointestinal feed passage via the medulla oblongata in chicks.

Zymosan is a fungi-derived pathogen-associated molecular pattern. It activates the immune system and induces the reduction of feed passage rate in the gastrointestinal tract of vertebrates including birds. However, the mechanism mediating the zymosan-induced inhibition of feed passage in the gastrointestinal tract remains unknown. Since the medulla oblongata regulates the digestive function, it is plausible that the medulla oblongata is involved in the zymosan-induced inhibition of feed passage. The present study was performed to identify the genes that were affected by zymosan within the medulla oblongata of chicks (Gallus gallus) using an RNA sequencing approach. We found that mRNAs of several bioactive molecules including neuropeptide Y (NPY) were increased with an intraperitoneal (IP) injection of zymosan. The increase of mRNA expression of NPY in the medulla oblongata was also observed after the IP injection of lipopolysaccharide, derived from gram-negative bacteria. These results suggest that medullary NPY is associated with physiological changes during fungal and bacterial infection. Furthermore, we found that intracerebroventricular injection of NPY and its receptor agonists reduced the feed passage from the crop. Additionally, the injection of NPY reduced the feed passage from the proventriculus to lower digestive tract. NPY also suppressed the activity of duodenal activities of amylase and trypsin. The present study suggests that fungi- and bacteria-induced activation of the immune system may activate the NPY neurons in the medulla oblongata and thereby reduce the digestive function in chicks.

D-Galactosamine Causes Liver Injury Synergistically with Lipopolysaccharide but not Zymosan in Chicks.

The pathogen-associated molecular patterns (PAMPs) lipopolysaccharide (LPS) and zymosan, derived from gram-negative bacteria and fungi, respectively, activate the innate immune system and cause injury to multiple organs, including the liver and intestine, in mammals. In rodents, PAMP-induced injury has been demonstrated to be potentiated by co-administration of D-galactosamine (D-GalN) in rodents. However, whether PAMPs and D-GalN collectively cause organ injury in birds remains unclear. The present study aimed to measure the effects of intraperitoneal injection of D-GalN with LPS or zymosan on parameters related to hepatic injury in chicks (Gallus gallus). Plasma aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) activities were not affected by intraperitoneal injection of D-GalN alone. Although these activities were not affected by LPS injection alone, they were increased by combining LPS with D-GalN. In contrast, plasma AST, ALT, and LDH activities were not affected by zymosan, both alone and with D-GalN. The expression of mRNAs for interleukin-6 (IL-6) and inducible nitric oxide synthase (iNOS) in the liver was significantly increased by the combination of LPS and D-GalN. In contrast, combining zymosan with D-GalN significantly increased iNOS mRNA expression, irrespective of hepatic injury. These results suggest that IL-6 may be the cause and/or result of hepatic injury in chicks. Additionally, chicks are tolerant to the hepatic effects of D-GalN, LPS, or zymosan alone.

Effect of the LSD1 inhibitor RN-1 on γ-globin and global gene expression during erythroid differentiation in baboons (Papio anubis).

Elevated levels of Fetal Hemoglobin interfere with polymerization of sickle hemoglobin thereby reducing anemia, lessening the severity of symptoms, and increasing life span of patients with sickle cell disease. An affordable, small molecule drug that stimulates HbF expression in vivo would be ideally suited to treat the large numbers of SCD patients that exist worldwide. Our previous work showed that administration of the LSD1 (KDM1A) inhibitor RN-1 to normal baboons increased Fetal Hemoglobin (HbF) and was tolerated over a prolonged treatment period. HbF elevations were associated with changes in epigenetic modifications that included increased levels of H3K4 di-and tri-methyl lysine at the γ-globin promoter. While dramatic effects of the loss of LSD1 on hematopoietic differentiation have been observed in murine LSD1 gene deletion and silencing models, the effect of pharmacological inhibition of LSD1 in vivo on hematopoietic differentiation is unknown. The goal of these experiments was to investigate the in vivo mechanism of action of the LSD1 inhibitor RN-1 by determining its effect on γ-globin expression in highly purified subpopulations of bone marrow erythroid cells enriched for varying stages of erythroid differentiation isolated directly from baboons treated with RN-1 and also by investigating the effect of RN1 on the global transcriptome in a highly purified population of proerythroblasts. Our results show that RN-1 administered to baboons targets an early event during erythroid differentiation responsible for γ-globin repression and increases the expression of a limited number of genes including genes involved in erythroid differentiation such as GATA2, GFi-1B, and LYN.

End binding-3 inhibitor activates regenerative program in age-related macular degeneration.

Wet age-related macular degeneration (AMD), characterized by leaky neovessels emanating from the choroid, is a main cause of blindness. As current treatments for wet AMD require regular intravitreal injections of anti-vascular endothelial growth factor (VEGF) biologics, there is a need for the development of less invasive treatments. Here, we designed an allosteric inhibitor of end binding-3 (EB3) protein, termed EBIN, which reduces the effects of environmental stresses on endothelial cells by limiting pathological calcium signaling. Delivery of EBIN via eye drops in mouse and non-human primate (NHP) models of wet AMD prevents both neovascular leakage and choroidal neovascularization. EBIN reverses the epigenetic changes induced by environmental stresses, allowing an activation of a regenerative program within metabolic-active endothelial cells comprising choroidal neovascularization (CNV) lesions. These results suggest the therapeutic potential of EBIN in preventing the degenerative processes underlying wet AMD.

Possible role of corticosterone on behavioral, physiological, and immune responses in chicks.

Glucocorticoids are one of steroid hormone and have a variety of functions including stress response, carbohydrate metabolism, and modulation of immune system in vertebrates. Corticosterone is the main glucocorticoid in birds, although the precise role of the glucocorticoid during immune challenge is not fully understood. Therefore, the purpose of the present study was to determine if a single subcutaneous injection of corticosterone could affect inflammation-related gene expressions in the spleen and liver of chicks (Gallus gallus). In addition, the effects of corticosterone injection on the food intake, cloacal temperature, formation of conditioned visual aversion, and plasma constituents were also measured. Corticosterone did not affect the food intake or cloacal temperature and did not cause conditioned visual aversion in chicks. The corticosterone injection was associated with a significant decrease in gene expression of several pro-inflammatory cytokines including inducible nitric oxide synthase and cyclooxygenase-2 in the spleen and liver at 1 and 3 h post-injection. Corticosterone increased the plasma glucose and uric acid concentrations and the antioxidant capacity. In summary, the present study suggests that corticosterone is likely not associated with food intake, cloacal temperature or the development of aversive sensation, but suppresses the synthesis of inflammation-associated bioactive molecules and increases the antioxidant capacity in chicks.

Trained immunity of alveolar macrophages enhances injury resolution via KLF4-MERTK-mediated efferocytosis.

Recent studies suggest that training of innate immune cells such as tissue-resident macrophages by repeated noxious stimuli can heighten host defense responses. However, it remains unclear whether trained immunity of tissue-resident macrophages also enhances injury resolution to counterbalance the heightened inflammatory responses. Here, we studied lung-resident alveolar macrophages (AMs) prechallenged with either the bacterial endotoxin or with Pseudomonas aeruginosa and observed that these trained AMs showed greater resilience to pathogen-induced cell death. Transcriptomic analysis and functional assays showed greater capacity of trained AMs for efferocytosis of cellular debris and injury resolution. Single-cell high-dimensional mass cytometry analysis and lineage tracing demonstrated that training induces an expansion of a MERTKhiMarcohiCD163+F4/80low lung-resident AM subset with a proresolving phenotype. Reprogrammed AMs upregulated expression of the efferocytosis receptor MERTK mediated by the transcription factor KLF4. Adoptive transfer of these trained AMs restricted inflammatory lung injury in recipient mice exposed to lethal P. aeruginosa. Thus, our study has identified a subset of tissue-resident trained macrophages that prevent hyperinflammation and restore tissue homeostasis following repeated pathogen challenges.

Conserved role for PCBP1 in altered RNA splicing in the hippocampus after chronic alcohol exposure.

We previously discovered using transcriptomics that rats undergoing withdrawal after chronic ethanol exposure had increased expression of several genes encoding RNA splicing factors in the hippocampus. Here, we examined RNA splicing in the rat hippocampus during withdrawal from chronic ethanol exposure and in postmortem hippocampus of human subjects diagnosed with alcohol use disorder (AUD). We found that expression of the gene encoding the splicing factor, poly r(C) binding protein 1 (PCBP1), was elevated in the hippocampus of rats during withdrawal after chronic ethanol exposure and AUD subjects. We next analyzed the rat RNA-Seq data for differentially expressed (DE) exon junctions. One gene, Hapln2, had increased usage of a novel 3' splice site in exon 4 during withdrawal. This splice site was conserved in human HAPLN2 and was used more frequently in the hippocampus of AUD compared to control subjects. To establish a functional role for PCBP1 in HAPLN2 splicing, we performed RNA immunoprecipitation (RIP) with a PCBP1 antibody in rat and human hippocampus, which showed enriched PCBP1 association near the HAPLN2 exon 4 3' splice site in the hippocampus of rats during ethanol withdrawal and AUD subjects. Our results indicate a conserved role for the splicing factor PCBP1 in aberrant splicing of HAPLN2 after chronic ethanol exposure. As the HAPLN2 gene encodes an extracellular matrix protein involved in nerve conduction velocity, use of this alternative splice site is predicted to result in loss of protein function that could negatively impact hippocampal function in AUD.