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Rationale: Brain-derived neurotrophic factor precursor (proBDNF) is expressed in the central nervous system (CNS) and the immune system. However, the role of proBDNF in the pathogenesis of multiple sclerosis (MS) is unknown. Methods: Peripheral blood and post-mortem brain and spinal cord specimens were obtained from multiple sclerosis patients to analyze proBDNF expression in peripheral lymphocytes and infiltrating immune cells in the lesion site. The proBDNF expression profile was also examined in the experimental autoimmune encephalomyelitis (EAE) mouse model, and polyclonal and monoclonal anti-proBDNF antibodies were used to explore their therapeutic effect in EAE. Finally, the role of proBDNF in the inflammatory immune activity of peripheral blood mononuclear cells (PBMCs) was verified in vitro experiments. Results: High proBDNF expression was detected in the circulating lymphocytes and infiltrated inflammatory cells at the lesion sites of the brain and spinal cord in MS patients. In the EAE mouse model, proBDNF was upregulated in CNS and in circulating and splenic lymphocytes. Systemic but not intracranial administration of anti-proBDNF blocking antibodies attenuated clinical scores, limited demyelination, and inhibited proinflammatory cytokines in EAE mice. Immuno-stimulants treatment increased the proBDNF release and upregulated the expression of p75 neurotrophic receptors (p75 NTR ) in lymphocytes. The monoclonal antibody against proBDNF inhibited the inflammatory response of PBMCs upon stimulations. Conclusion: The findings suggest that proBDNF from immune cells promotes the immunopathogenesis of MS. Monoclonal Ab-proB may be a promising therapeutic agent for treating MS., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Early-life multiple anesthetics exposure causes neurotoxicity and hence cognitive dysfunction on developing brain. However, the effects of early-life multiple sevoflurane exposures on emotional changes, especially upon stress, are far beyond understood. In young male C57BL6/J mice, the present study showed that 3% sevoflurane inhalation for 2 h in three consecutive days did not influence anxiety-like behaviors as measured by open field test, light dark transition, and elevated plus maze test. In addition, foot shocks stress induced both the short- and long-term anxiety-like behaviors. However, triple sevoflurane exposures ameliorated the long-term anxiety-like behaviors induced by the foot shocks. In parallel, foot shocks stress upregulated the expression of phosphorylated extracellular signal-regulated kinase (p-ERK) and brain-derived neurotrophic factor precursor (proBDNF) in the anterior cingulate cortex (ACC), which were significantly inhibited by triple sevoflurane exposures. Immunofluorescence further indicated that the increased p-ERK was mainly expressed in the proBDNF-positive staining cells. Intra-ACC injection of recombinant proBDNF protein upregulated the p-ERK expression and blocked the anxiolytic effect of sevoflurane exposure on long-term anxiety-like behaviors. Therefore, our study demonstrated that multiple sevoflurane exposures alleviate long-term anxiety-like behaviors upon acute stress in young mice by inhibiting proBDNF-ERK signaling in the ACC.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is erupting and spreading globally. Cardiovascular complications secondary to the infection have caught notice. This study aims to delineate the relationship of cardiac biomarkers and outcomes in severe cases of corona virus disease 2019 (COVID-19). One hundred forty-eight critically ill adult patients with COVID-19 were enrolled. From these patients, the demographic data, symptoms, cardiac biomarkers, treatments, and clinical outcomes were collected. Data were compared between survivors and non-survivors. Four patients in the non-survivor group were selected, and their cardiac biomarkers were collected and analyzed. Among the 148 patients, the incidence of cardiovascular complications was 19 (12.8%). Five of them were survivors (5.2%), and 14 of them were non-survivors (26.9%). Compared with the survivors, the non-survivors had higher levels of high-sensitivity cardiac troponin I, creatine kinase isoenzyme-MB, myoglobin, and N-terminal pro-brain natriuretic peptide (P < 0.05). The occurrence of cardiovascular events began at 11-15 days after the onset of the disease and reached a peak at 14-20 days. COVID-19 not only is a respiratory disease but also causes damage to the cardiovascular system. Cardiac biomarkers have the potential for early warning and prognostic evaluation in patients with COVID-19. It is recommended that cardiac biomarker monitoring in patients with COVID-19 should be initiated at least from the 11th day of the disease course.

Oral antigen administration to induce regulatory T cells (Treg) takes advantage of regulatory mechanisms that the gastrointestinal tract utilizes to promote unresponsiveness against food antigens or commensal microorganisms. Recently, antigen-based oral immunotherapies (OITs) have shown efficacy as treatment for food allergy and autoimmune diseases. Similarly, OITs appear to prevent anti-drug antibody responses in replacement therapy for genetic diseases. Intestinal epithelial cells and microbiota possibly condition dendritic cells (DC) toward a tolerogenic phenotype that induces Treg via expression of several mediators, e.g. IL-10, transforming growth factor-β, retinoic acid. Several factors, such as metabolites derived from microbiota or diet, impact the stability and expansion of these induced Treg, which include, but are not limited to, FoxP3 + Treg, LAP + Treg, and/or Tr1 cells. Here, we review various orally induced Treg, their plasticity and cooperation between the Treg subsets, as well as underlying mechanisms controlling their induction and role in oral tolerance., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Chloroplasts have multiple suborganellar membranes. Correct and efficient translocation of chloroplast proteins from their site of synthesis into or across membranes to their functional compartments are fundamental processes. In recent years, several new components and regulatory mechanisms involved in chloroplast protein import and sorting have been explored. Moreover, the formation of liquid-liquid phase transition (LLPT) has been recently reported as a novel mechanism for regulating chloroplast protein sorting. Here, we overview the recent advances of both nuclear- and chloroplast-encoded protein trafficking to their final destination within chloroplasts, and discuss the novel components and regulatory mechanisms of intrachloroplast sorting. Furthermore, we propose that LLPT may be a universal and conserved mechanism for driving organelle protein trafficking and organelle biogenesis., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Radical prostatectomy induces some degree of cavernous nerve injury (CNI) and causes denervation-induced pathologic changes in cavernous vasculature, regardless of the advances in surgical techniques and robotic procedures. The precursor for nerve growth factor (proNGF) is known to be involved in neuronal cell apoptosis and microvascular dysfunction through its receptor p75 NTR ., Objectives: To determine the expression of proNGF/p75 NTR and the efficacy of proNGF neutralizing antibody (anti-proNGF-Ab) in a mouse model of ED induced by CNI., Materials and Methods: Age-matched 12-week-old C57BL/6 mice were distributed into three groups: sham group and bilateral CNI group treated with intracavernous injections of PBS (20 μL) or of anti-proNGF-Ab (20 µg in 20 μL of PBS) on days -3 and 0. Two weeks after treatment, erectile function was measured by electrical stimulation of cavernous nerve. Penis tissues from a separate group of animals were harvested for further analysis. We also determined the efficacy of anti-proNGF-Ab on neural preservation in major pelvic ganglion (MPG) ex vivo., Results: We observed increased penile expression of proNGF and p75 NTR after CNI. Intracavernous administration of anti-proNGF-Ab increased nNOS and neurofilament expression probably by enhancing the production of neurotrophic factors, such as neurotrophin-3, NGF, and brain-derived neurotrophic factor. Anti-proNGF-Ab preserved the integrity of cavernous sinusoids, such as pericytes, endothelial cells, and endothelial cell-to-cell junctions, possibly by controlling angiogenic factors (angiopoietin-1, angiopoietin-2, and vascular endothelial growth factor) and induced endogenous eNOS phosphorylation in CNI mice. And finally, treatment with anti-proNGF-Ab rescued erectile function in CNI mice. Anti-proNGF-Ab also enhanced neurite sprouting from MPG exposed to lipopolysaccharide., Discussion and Conclusion: The preservation of damaged cavernous neurovasculature through inhibition of the proNGF/p75 NTR pathway may be a novel strategy to treat radical prostatectomy-induced erectile dysfunction., (© 2020 American Society of Andrology and European Academy of Andrology.)

Chloroplast stromal factors involved in regulating thylakoid protein targeting are poorly understood. We previously reported that in Arabidopsis thaliana, the stromal-localized chaperone HSP90C (plastid heat shock protein 90) interacted with the nuclear-encoded thylakoid lumen protein PsbO1 (PSII subunit O isoform 1) and suggested a role for HSP90C in aiding PsbO1 thylakoid targeting. Using in organello transport assays, particularly with model substrates naturally expressed in stroma, we showed that light, exogenous ATP, and HSP90C activity were required for Sec-dependent transport of green fluorescent protein (GFP) led by the PsbO1 thylakoid targeting sequence. Using a previously identified PsbO1T200A mutant, we provided evidence that a stronger interaction between HSP90C and PsbO1 better facilitated its stroma-thylakoid trafficking. We also demonstrated that SecY1, the channel protein of the thylakoid SEC translocase, specifically interacted with HSP90C in vivo. Inhibition of the chaperone ATPase activity suppressed the association of the PsbO1GFP-HSP90C complex with SecY1. Together with analyzing the expression and accumulation of a few other thylakoid proteins that utilize the SRP, TAT, or SEC translocation pathways, we propose a model in which HSP90C forms a guiding complex that interacts with thylakoid protein precursors and assists in their specific targeting to the thylakoid SEC translocon., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Overweight and obese individuals may have leaky intestinal barrier and microbiome dysbiosis. The aim of this study was to determine whether body mass reduction with diet and synbiotics in an adult person with excess body mass has an influence on the gut microbiota and zonulin concentration. The study was a single blinded trial. 60 persons with excess body mass were examined. Based on randomization, patients were qualified either to the intervention group (Synbiotic group) or to the control group (Placebo group). Anthropometric measurements, microbiological assessment of faecal samples and zonulin concentration in the stool were performed before and after observation. After 3-months, an increase in the variety of intestinal bacteria (increase in the Shannon-Weaver index and the Simpson index) and a decrease in concentration of zonulin in faecal samples were observed in the Synbiotic group. Also, statistically significant correlation between zonulin and Bifidobacterium spp. (Spearman test, R=-0.51; p=0.0040) was noticed. There were no significant relationships between the body mass, BMI and changes in the intestinal microbiota or zonulin concentrations. The use of diet and synbiotics improved the condition of the microbiota and intestinal barrier in patients in the Synbiotic group.

Objective: Hepatocellular carcinoma (HCC) is a primary liver tumor derived from metabolic or viral chronic hepatitis, with few treatment options in advanced cases. New biomarkers that allow improving diagnosis and staging are widely desired. Here, we aim to evaluate the performance of Protein Induced by Vitamin K Absence or Antagonist-II (PIVKA-II) in combination with α-fetoprotein (AFP), in the diagnosis of HCC in patients with metabolic or viral hepatitis., Patients and Methods: We enrolled 60 HCC patients (20 metabolic and 40 viral) and 20 healthy subjects (HS) as negative controls. PIVKA-II, AFP, Matrix metalloproteinase-9 (MMP-9) and Fibroblast growth factor (FGF) serum levels were assessed by immunoassays., Results: AFP and PIVKA-II levels were obviously higher in patients than in HS. AFP displayed a better diagnostic performance than PIVKA-II for viral HCC while PIVKA-II was better for metabolic HCC. The combination of the two biomarkers did not improve the discriminating ability., Conclusions: PIVKA-II may be considered an independent predictor of macrovascular invasion from HCC cells and it can be used to better stratify HCC patients and should be evaluated in prospective studies for early detection of advanced HCC in metabolic subjects.

Introduction: A large volume of data indicates that the known thymic hormones, thymulin, thymopoietin, thymosin-α, thymosin-β, and thymic humoral factor-y2, exhibit different spectra of activities. Although large in volume, available data are rather fragmented, resulting in a lack of understanding of the role played by thymic hormones in immune homeostasis., Area Covered: Existing data compartmentalizes the effect of thymic peptides into 2 categories: influence on immune cells and interconnection with neuroendocrine systems. The current study draws attention to a third aspect of the thymic peptide effect that has not been clarified yet, wherein ubiquitous and highly abundant intranuclear precursors of so called 'thymic peptides' play a fundamental role in all somatic cells., Expert Opinion: Our analysis indicated that, under certain stress-related conditions, these precursors are cleaved to form immunologically active peptides that rapidly leave the nucleus and intracellular spaces, to send 'distress signals' to the immune system, thereby acting as stress sensors. We propose that these peptides may form a link between somatic cells and immune as well as neuroendocrine systems. This model may provide a better understanding of the mechanisms underlying immune homeostasis, leading thereby to the development of new therapeutic regimes utilizing the characteristics of thymic peptides.

Androgens can affect the reproductive axis of both sexes. In healthy women, as in men, elevated exogenous androgens decrease gonad function and lower gonadotropin levels; such circumstances occur with anabolic steroid abuse or in transgender men (genetic XX individuals) taking androgen supplements. The neuroendocrine mechanisms by which endogenous or exogenous androgens regulate gonadotropin release, including aspects of pulsatile luteinizing hormone (LH) secretion, remain unknown. Because animal models are valuable for interrogating neural and pituitary mechanisms, we studied effects of androgens in the normal male physiological range on in vivo LH secretion parameters in female mice and in vitro LH secretion patterns from isolated female pituitaries. We also assessed androgen effects on hypothalamic and gonadotrope gene expression in female mice, which may contribute to altered LH secretion profiles. We used a nonaromatizable androgen, dihydrotestosterone (DHT), to isolate effects occurring specifically via androgen receptor (AR) signaling. Compared with control females, DHT-treated females exhibited markedly reduced in vivo LH pulsatility, with decreases in pulse frequency, amplitude, peak, and basal LH levels. Correlating with reduced LH pulsatility, DHT-treated females also exhibited suppressed arcuate nucleus Kiss1 and Tac2 expression. Separate from these neural effects, we determined in vitro that the female pituitary is directly inhibited by AR signaling, resulting in lower basal LH levels and reduced LH secretory responses to gonadotropin-releasing hormone pulses, along with lower gonadotropin gene expression. Thus, in normal adult females, male levels of androgen acting via AR can strongly inhibit the reproductive axis at both the neural and pituitary levels., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Endocrine Society. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Research Question: Does the relative distribution of anti-Müllerian hormone (AMH) isoforms differ between patients depending on their body mass index (BMI) and polycystic ovary syndrome (PCOS) status in serum and follicular fluid?, Design: Obese and normal weight patients (PCOS [n = 70]; non-PCOS [n = 37]) were selected for this case-control study in the serum. Between 2018 and 2019, obese (n = 19) and normal weight (n = 20) women with or without PCOS who were receiving IVF treatment were included in the follicular fluid study. The bio-banked serums and follicular fluid were tested for total AMH (proAMH and AMH N,C combined) and proAMH using an automatic analyzer. The AMH prohormone index (API = [proAMH]/[total AMH]x 100) was calculated as an inverse marker of conversion of proAMH to AMH N,C , with only the latter isoform that could bind to the AMH receptor complex., Results: The API was not significantly different between controls and women with PCOS, whereas obese women had a lower API compared with their normal weight counterparts. Grouping PCOS and controls, a lower API was found in obese versus normal weight women, suggesting a greater conversion of proAMH to AMH N,C . The API in the serum was significantly correlated with metabolic parameters. In the follicular fluid, API is not different between obese and normal weight women independently of PCOS and is higher than in the concomitant serum., Conclusions: The proportion of inactive form of AMH in the serum is higher in normal weight versus obese women but not in the follicular fluid, independently of PCOS. The conversion of proAMH into the cleaved isoform is likely to occur in extra-ovarian tissues and to exacerbate in obese individuals., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Hepatocellular carcinoma (HCC) is different from other solid tumors because it is commonly associated with the occurrence of intrahepatic metastasis. Additionally, the liver, unlike other organs, is the main site of coagulation and fibrinolytic factor production. Therefore, it was speculated that coagulation and fibrinolytic factors could be associated with intrahepatic metastasis of HCC. Do the coagulation and fibrinolytic systems protect HCC cells against anoikis during infiltration and metastasis? Conversely, do the coagulation and fibrinolytic systems lead to immune escape of HCC cells by affecting the immune microenvironment of patients? The current review aimed to present a number of novel hypotheses for the treatment of HCC by exploring the mechanisms of coagulation and fibrinolytic factors in the regulation of cancer growth.

Hepatitis B virus (HBV), a major global health problem, can cause chronic hepatitis, liver cirrhosis, and hepatocellular carcinomas in chronically infected patients. However, before HBV infection can be adequately controlled, many mysteries about the HBV life cycle must be solved. In this study, TIMM29, an inner mitochondrial membrane protein, was identified as an interaction partner of the preS1 region of the HBV large S protein. The interaction was verified by both an immunoprecipitation with preS1 peptides and a GST-pulldown assay. Immunofluorescence studies also showed colocalization of preS1 and TIMM29. Moreover, it was determined that the preS1 bound with amino acids 92-189 of the TIMM29 protein. Infection of HBV in TIMM29-overexpressing NTCP/G2 cells resulted in a significant decrease of HBeAg and both extracellular particle-associated and core particle-associated HBV DNA without affecting cccDNA formation. Comparable results were obtained with TIMM29-overexpressing HB611 cells, which constitutively produce HBV. In contrast, knockout of TIMM29 in NTCP/G2 cells led to a higher production of HBV including HBeAg expression, as did knockout of TIMM29 in HB611. Collectively, these results suggested that TIMM29 interacts with the preS1 region of the HBV large S protein and modulates HBV amplification., (© 2020 The Societies and John Wiley & Sons Australia, Ltd.)

The modification of galactose with α1,2-fucose is involved in symbiosis with intestinal bacteria and elimination of pathogenic bacteria. It is postulated that α1,2-fucosylated mucin secreted from goblet cells is involved in defending an organism against infections, but the detailed molecular mechanisms are yet to be elucidated. It was previously reported that Paneth cells of the small intestine were positive for UEA-1 lectin staining. However, glycoproteins in Paneth cells carrying α1,2-fucose have not yet been identified. Glycoproteomic analysis of ileal lysates identified 3212 O-linked and 2962 N-linked glycopeptides. In particular, cryptdin-related sequence 1 (CRS1) expressed in Paneth cells was found to be α1,2-fucosylated. Unlike other antimicrobial α-defensin proteins, CRS1 contains unique Thr residues, which are modified with O-glycans, with 3HexNAc2Hex1Fuc1NeuAc being the main glycoform. Identification of α1,2-fucose on the O-glycans of CRS1 expressed in Paneth cells will pave the way for a mechanistic understanding of α1,2-fucose-dependent symbiosis with intestinal bacteria and elimination of pathogenic bacteria in the intestine., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Brain-derived neurotrophic factor (BDNF) plays a role in synaptic plasticity and neuroprotection. BDNF has well-established pro-survival effects, whereas its precursor protein, proBDNF, induces apoptosis. Thus, it has been suggested that the proBDNF/BDNF ratio could be an indicator of neuronal health. Access to neurons is, understandably, limited. Because of their similarities, platelets have been put forward as a non-invasive biomarker of neuronal health; indeed, they store large quantities of BDNF and can release it into circulation upon activation, similarly to neurons. However, whether platelets also express the precursor proBDNF protein remains unknown. We therefore sought to characterize proBDNF levels in human platelets and plasma., Methods: The presence of proBDNF was assessed by immunoblotting, cell fractionation, flow cytometry, and confocal microscopy in washed platelets from 10 healthy volunteers. Platelets from 20 independent healthy volunteers were activated with several classical agonists and the release of BDNF and proBDNF into plasma was quantified by ELISA., Results: Platelets expressed detectable levels of proBDNF (21 ± 13 fmol/250 x 10 6 platelets). ProBDNF expression was mainly localized in the intracellular compartment. The proBDNF to BDNF molar ratio was ~1:5 in platelets and 10:1 in plasma. In stark contrast to the release of BDNF during platelet activation, intraplatelet and plasma concentrations of proBDNF remained stable following stimulation with classical platelet agonists, consistent with non-granular expression., Conclusions: Platelets express both the mature and the precursor form of BDNF. Whether the intraplatelet proBDNF to BDNF ratio could be used as a non-invasive biomarker of cognitive health warrants further investigation., Competing Interests: ML has received speaker fees from Bayer; has participated in industry-funded trials from Idorsia; has served on advisory boards for Servier; and has received in-kind and financial support for investigator-initiated grants from Leo Pharma, Roche Diagnostics, Aggredyne, and Fujimori Kogyo. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2020 Le Blanc, Fleury, Boukhatem, Bélanger, Welman and Lordkipanidzé.)

Alcohol use disorders (AUD) are chronic relapsing brain disorder characterized by compulsive and heavy alcohol consumption. During acute withdrawal, patients with AUD display excessive daytime sleepiness, a condition linked to serious life-threatening complications, however, the mechanism is not known. Orexin and melanin-concentrating hormone (MCH) are the two hypothalamic neuropeptides that regulate many behaviors including sleep-wakefulness, and alcohol consumption, reinforcement, and reinstatement. Importantly, loss of orexin neurons causes narcolepsy, a severe sleep disorder with excessive daytime sleepiness. Does acute alcohol withdrawal reduce orexin gene expression? To investigate this, male Sprague-Dawley rats were divided in two groups: Rats were either administered with alcohol, mixed with infant formula (alcohol group) or control mixture containing water and infant formula (Controls) by gastric intubation every 8 h for 4 days using Majchrowicz's chronic binge drinking protocol. The doses of alcohol were adjusted depending on degree of intoxication, exhibited by animals, prior to each dose. The animals were euthanized after 12 h of last alcohol/water administration. During withdrawal, the hypothalamus was rapidly dissected out, and the expressions of orexin and MCH genes were examined by Real-time PCR. There was a significant reduction in orexin gene expression in rats subjected to alcohol withdrawal as compared to controls. No such change was observed in the MCH gene expression. These results suggest that downregulation of orexin gene expression may be a possible mechanism responsible for excessive daytime sleepiness associated with alcohol withdrawal in patients with AUD., (Published by Elsevier B.V.)

Feeding-regulatory peptides such as thyrotropin-releasing hormone (TRH), α-melanocyte-stimulating hormone (α-MSH) and their receptors are expressed in brain regions involved in the homeostatic and hedonic control of food intake, such as the hypothalamus and the mesolimbic system, respectively. The nucleus accumbens (NAc) is part of the latter, a brain circuit involved in processing reward stimuli and the appetitive motivation of feeding. When TRH or α-MSH are administered in the NAc, both decrease food intake, through activating their respective receptors, TRH-R1 and MC4R. The actions of α-MSH as a homeostatic feeding-regulator involves the increase of hypothalamic TRH expression, thus, we aimed to identify whether TRH signaling in the NAc was also participating in α-MSH-induced reduction of food intake. α-MSH administration in the NAc of 48 h fasted rats reduced their food intake during the 2-h period of refeeding, increased accumbal TRH mRNA expression and decreased that of MC4R. Such downregulated MC4R mRNA levels implied a compensatory decrease of α-MSH actions in the NAc after the previous pathway stimulation. The co-administration of α-MSH along with an antisense oligonucleotide directed against pro-TRH mRNA in the NAc impaired the α-MSH-induced feeding reduction, supporting that the accumbal TRHergic pathway is downstream of α-MSH actions to inhibit feeding. Our results suggested that TRH in the NAc mediates some effects of α-MSH on inhibition of food intake; this supports the role of TRH not only as a homeostatic regulator but also as modulating the motivational aspects of feeding., (Copyright © 2020. Published by Elsevier B.V.)

Most mitochondrial proteins are synthesized as precursors that carry N-terminal presequences. After they are imported into mitochondria, these targeting signals are cleaved off by the mitochondrial processing peptidase (MPP). Using the mitochondrial tandem protein Arg5,6 as a model substrate, we demonstrate that MPP has an additional role in preprotein maturation, beyond the removal of presequences. Arg5,6 is synthesized as a polyprotein precursor that is imported into mitochondria and subsequently separated into two distinct enzymes. This internal processing is performed by MPP, which cleaves the Arg5,6 precursor at its N-terminus and at an internal site. The peculiar organization of Arg5,6 is conserved across fungi and reflects the polycistronic arginine operon in prokaryotes. MPP cleavage sites are also present in other mitochondrial fusion proteins from fungi, plants, and animals. Hence, besides its role as a "ticket canceller" for removal of presequences, MPP exhibits a second conserved activity as an internal processing peptidase for complex mitochondrial precursor proteins.

Motivational states consist of cognitive, emotional, and physiological components controlled by multiple brain regions. An integral component of this neural circuitry is the bed nucleus of the stria terminalis (BNST). Here, we identify that neurons within BNST that express the gene prepronociceptin (Pnoc BNST ) modulate rapid changes in physiological arousal that occur upon exposure to motivationally salient stimuli. Using in vivo two-photon calcium imaging, we find that Pnoc BNST neuronal responses directly correspond with rapid increases in pupillary size when mice are exposed to aversive and rewarding odors. Furthermore, optogenetic activation of these neurons increases pupillary size and anxiety-like behaviors but does not induce approach, avoidance, or locomotion. These findings suggest that excitatory responses in Pnoc BNST neurons encode rapid arousal responses that modulate anxiety states. Further histological, electrophysiological, and single-cell RNA sequencing data reveal that Pnoc BNST neurons are composed of genetically and anatomically identifiable subpopulations that may differentially tune rapid arousal responses to motivational stimuli., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Gut microbiota are suspected to affect brain functions and behavior as well as lowering inflammation status. Therefore, an effect on depression has already been suggested by recent research. The aim of this randomized double-blind controlled trial was to evaluate the effect of probiotic treatment in depressed individuals. Within inpatient care, 82 currently depressed individuals were randomly assigned to either receive a multistrain probiotic plus biotin treatment or biotin plus placebo for 28 days. Clinical symptoms as well as gut microbiome were analyzed at the begin of the study, after one and after four weeks. After 16S rRNA analysis, microbiome samples were bioinformatically explored using QIIME, SPSS, R and Piphillin. Both groups improved significantly regarding psychiatric symptoms. Ruminococcus gauvreauii and Coprococcus 3 were more abundant and β-diversity was higher in the probiotics group after 28 days. KEGG-analysis showed elevated inflammation-regulatory and metabolic pathways in the intervention group. The elevated abundance of potentially beneficial bacteria after probiotic treatment allows speculations on the functionality of probiotic treatment in depressed individuals. Furthermore, the finding of upregulated vitamin B6 and B7 synthesis underlines the connection between the quality of diet, gut microbiota and mental health through the regulation of metabolic functions, anti-inflammatory and anti-apoptotic properties. Concluding, four-week probiotic plus biotin supplementation, in inpatient individuals with a major depressive disorder diagnosis, showed an overall beneficial effect of clinical treatment. However, probiotic intervention compared to placebo only differed in microbial diversity profile, not in clinical outcome measures.

Major depressive disorder (MDD) presents with two primary symptoms: depressed mood and anhedonia, which suggests that distinct neuronal circuits may regulate MDD. However, the underlying circuits of these individual symptoms linked to depression remain elusive. Herein, we identify a discrete circuit of tachykinin precursor 1 (Tac1)-expressing neurons in the nucleus accumbens (NAc) lateral shell, which project to ventral pallidum and contribute to stress-induced anhedonia-like behavior. Selective inhibition and activation of Tac1 NAc neurons bidirectionally modulate stress susceptibility, revealing that Tac1 neurons in the NAc are critical for regulating anhedonia-like behaviors. We find that a subpopulation of VP neurons receives inhibitory inputs from Tac1 NAc neurons and exhibits decreased excitability in susceptible mice. Furthermore, the inhibition of the neurokinin 1 receptor promotes susceptibility to social stress. Overall, our study reveals a discrete circuit regulating anhedonia-like behavior in mice., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Hepatocellular carcinoma (HCC) is increasing in prevalence and is the third leading cause of cancer-related death worldwide. Unlike other malignancies, HCC can be diagnosed with dynamic imaging with very high accuracy, and tissue diagnosis is not needed for cancer therapy. There is a unique role of established as well as developing biomarkers in diagnosis, prognosis, and management of HCC. Sequencing HCC tumors has yielded substantial insights into HCC tumor biology and has raised the possibility of precision oncology in which therapy decisions are guided by cancer genetics. However, it is not ready for prime time yet., Competing Interests: Disclosure V.L. Chen and P. Sharma: No conflicts of interest., (Published by Elsevier Inc.)

Accumulation of unfolded proteins in the endoplasmic reticulum (ER) causes ER stress and activates a signaling network known as the unfolded protein response (UPR). Here we characterize how ER stress and the UPR inhibit insulin signaling. We find that ER stress inhibits insulin signaling by depleting the cell surface population of the insulin receptor. ER stress inhibits proteolytic maturation of insulin proreceptors by interfering with transport of newly synthesized insulin proreceptors from the ER to the plasma membrane. Activation of AKT, a major target of the insulin signaling pathway, by a cytosolic, membrane-bound chimera between the AP20187-inducible F V 2E dimerization domain and the cytosolic protein tyrosine kinase domain of the insulin receptor was not affected by ER stress. Hence, signaling events in the UPR, such as activation of the JNK mitogen-activated protein (MAP) kinases or the pseudokinase TRB3 by the ER stress sensors IRE1α and PERK, do not contribute to inhibition of signal transduction in the insulin signaling pathway. Indeed, pharmacologic inhibition and genetic ablation of JNKs, as well as silencing of expression of TRB3, did not restore insulin sensitivity or rescue processing of newly synthesized insulin receptors in ER-stressed cells. [Media: see text].

Atrial natriuretic peptide (ANP) is of major importance in the maintenance of electrolyte balance and normal blood pressure. Reduced plasma ANP levels are associated with the increased risk of cardiovascular disease. Corin is a type II transmembrane serine protease that converts the ANP precursor to mature ANP. Corin deficiency prevents ANP generation and alters electrolyte and body fluid homeostasis. Corin is synthesized as a zymogen that is proteolytically activated on the cell surface. Factors that disrupt corin folding, intracellular trafficking, cell surface expression, and zymogen activation are expected to impair corin function. To date, CORIN variants that reduce corin activity have been identified in hypertensive patients. In addition to the heart, corin expression has been detected in non-cardiac tissues, where corin and ANP participate in diverse physiological processes. In this review, we summarize the current knowledge in corin biosynthesis and post-translational modifications. We also discuss tissue-specific corin expression and function in physiology and disease., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Paneth cells contribute to intestinal innate immunity by sensing bacteria and secreting α-defensin. In Institute of Cancer Research (ICR) mice, α-defensin termed cryptdin (Crp) in Paneth cells consists of six major isoforms, Crp1 to 6. Despite accumulating evidences that α-defensin functions in controlling the intestinal microbiota, topographical localization of Paneth cells in the small intestine in relation to functions of α-defensin remains to be determined. In this study, we examined the expression level of messenger RNA (mRNA) encoding six Crp-isoforms and Crp immunoreactivities using singly isolated crypts together with bactericidal activities of Paneth cell secretions from isolated crypts of duodenum, jejunum, and ileum. Here we showed that levels of Crp mRNAs in the single crypt ranged from 5 x 10 3 to 1 x 10 6 copies per 5 ng RNA. For each Crp isoform, the expression level in ileum was 4 to 50 times higher than that in duodenum and jejunum. Furthermore, immunohistochemical analysis of isolated crypts revealed that the average number of Paneth cell per crypt in the small intestine increased from proximal to distal, three to seven-fold, respectively. Both Crp1 and 4 expressed greater in ileal Paneth cells than those in duodenum or jejunum. Bactericidal activities in secretions of ileal Paneth cell exposed to bacteria were significantly higher than those of duodenum or jejunum. In germ-free mice, Crp expression in each site of the small intestine was attenuated and bactericidal activities released by ileal Paneth cells were decreased compared to those in conventional mice. Taken together, Paneth cells and their α-defensin in adult mouse appeared to be regulated topographically in innate immunity to control intestinal integrity., (Copyright © 2020 Nakamura, Yokoi, Fukaya, Ohira, Shinozaki, Nishida, Kikuchi and Ayabe.)

Touch and itch sensations are crucial for evoking defensive and emotional responses, and light tactile touch may induce unpleasant itch sensations (mechanical itch or alloknesis). The neural substrate for touch-to-itch conversion in the spinal cord remains elusive. We report that spinal interneurons expressing Tachykinin 2-Cre (Tac2 Cre ) receive direct Aβ low threshold mechanoreceptor (LTMR) input and form monosynaptic connections with GRPR neurons. Ablation or inhibition markedly reduces mechanical but not acute chemical itch nor noxious touch information. Chemogenetic inhibition of Tac2 Cre neurons also displays pronounced deficit in chronic dry skin itch, a type of chemical itch in mice. Consistently, ablation of gastrin-releasing peptide receptor (GRPR) neurons, which are essential for transmitting chemical itch, also abolishes mechanical itch. Together, these results suggest that innocuous touch and chemical itch information converge on GRPR neurons and thus map an exquisite spinal circuitry hard-wired for converting innocuous touch to irritating itch.

Background: The optimal duration of antibiotic treatment for community-acquired pneumonia (CAP) is not well established. The aim of this study was to assess the impact of reducing the duration of antibiotic treatment on long-term prognosis in patients hospitalized with CAP., Methods: This was a multicenter study assessing complications developed during 1 year of patients previously hospitalized with CAP who had been included in a randomized clinical trial concerning the duration of antibiotic treatment. Mortality at 90 days, at 180 days and at 1 year was analyzed, as well as new admissions and cardiovascular complications. A subanalysis was carried out in one of the hospitals by measuring C-reactive protein (CRP), procalcitonin (PCT) and proadrenomedullin (proADM) at admission, at day 5 and at day 30., Results: A total of 312 patients were included, 150 in the control group and 162 in the intervention group. Ninety day, 180 day and 1-year mortality in the per-protocol analysis were 8 (2.57%), 10 (3.22%) and 14 (4.50%), respectively. There were no significant differences between both groups in terms of 1-year mortality (p = 0.94), new admissions (p = 0.84) or cardiovascular events (p = 0.33). No differences were observed between biomarker level differences from day 5 to day 30 (CRP p = 0.29; PCT p = 0.44; proADM p = 0.52)., Conclusions: Reducing antibiotic treatment in hospitalized patients with CAP based on clinical stability criteria is safe, without leading to a greater number of long-term complications.

The beneficial effects of brain-derived neurotrophic factor (BDNF)-a member of the neurotrophin family-on cognitive function or dementia are well established in both rodents and human beings. In contrast, little is known about the association of proBDNF-a precursor protein with opposing neuronal effects of BDNF-with cognitive function in non-demented older adults. We analyzed brain magnetic resonance imaging findings of 256 community-dwelling older adults (mean age of 68.4 years). Serum BDNF and proBDNF levels were measured by quantitative enzyme-linked immunosorbent assay. Logistic regression analysis revealed that older age, less physical activity, hippocampal atrophy, and lower BDNF levels were independently associated with memory impairment determined by the Rivermead Behavioral Memory Test. Path analysis based on structural equation modeling indicated that age, sport activity, hippocampal atrophy and BDNF but not proBDNF were individually associated with Rivermead Behavioral Memory Test scores. These findings suggest that impaired BDNF function, in addition to physical inactivity and hippocampal atrophy, is associated with age-related memory impairment. Therefore, BDNF may be a potential target for dementia prevention.

The hair bundle of cochlear hair cells is the site of auditory mechanoelectrical transduction. It is formed by three rows of stiff microvilli-like protrusions of graduated heights, the short, middle-sized, and tall stereocilia. In developing and mature sensory hair cells, stereocilia are connected to each other by various types of fibrous links. Two unconventional cadherins, protocadherin-15 (PCDH15) and cadherin-23 (CDH23), form the tip-links, whose tension gates the hair cell mechanoelectrical transduction channels. These proteins also form transient lateral links connecting neighboring stereocilia during hair bundle morphogenesis. The proteins involved in anchoring these diverse links to the stereocilia dense actin cytoskeleton remain largely unknown. We show that the long isoform of whirlin (L-whirlin), a PDZ domain-containing submembrane scaffold protein, is present at the tips of the tall stereocilia in mature hair cells, together with PCDH15 isoforms CD1 and CD2; L-whirlin localization to the ankle-link region in developing hair bundles moreover depends on the presence of PCDH15-CD1 also localizing there. We further demonstrate that L-whirlin binds to PCDH15 and CDH23 with moderate-to-high affinities in vitro. From these results, we suggest that L-whirlin is part of the molecular complexes bridging PCDH15-, and possibly CDH23-containing lateral links to the cytoskeleton in immature and mature stereocilia.

Major depression is a leading cause of morbidity and disease burden in modern society. Current drug treatment is only effective in a fraction of patients as underlying mechanisms of depression are not fully understood. ProBDNF, a precursor of brain-derived neurotrophic factor (BDNF), and its receptor p75 NTR are highly upregulated in patients with major depression and in animal models of depression induced by chronic stress. Here, we hypothesise that proBDNF may be a pathogenic factor triggering depression. C57BL/6 mice were injected in the bilateral gluteus maximus muscle with AAV-proBDNF or AAV-EGFP. Four weeks after the injection, AAV-proBDNF injected animals developed depression-like behaviours, which were evident for 4-8 weeks and then returned to the control level after 12 weeks. In the second experiment, mice were divided into three groups; one group was treated with sheep anti-proBDNF antibody after AAV-proBDNF injection whereas the other two groups received PBS injection after the AAV-proBDNF or AAV-EGFP delivery. The group that was injected with AAV-proBDNF showed a time-dependent increase in immobility time in the tail suspension test and forced swim test, reduced sucrose consumption and decreased grooming time after sucrose spraying. Treatment with sheep anti-proBDNF antibody alleviated the depressive-like symptoms. Peripheral AAV-proBDNF delivery also resulted in a reduction of density and length of dendritic spines in the dentate gyrus and amygdala. Thus, we conclude that peripheral proBDNF is a primary pathogenic factor triggering depression-like behavioural changes in mice likely by reducing dendritic spine plasticity.

As the population ages, obesity and metabolic complications as well as neurological disorders are becoming more prevalent, with huge economic burdens on both societies and families. New therapeutics are urgently needed. Nerve growth factor (NGF), first discovered in 1950s, is a neurotrophic factor involved in regulating cell proliferation, growth, survival, and apoptosis in both central and peripheral nervous systems. NGF and its precursor, proNGF, bind to TrkA and p75 receptors and initiate protein phosphorylation cascades, resulting in changes of cellular functions, and are associated with obesity, diabetes and its complications, and Alzheimer's disease. In this article, we summarize changes in NGF levels in metabolic and neuronal disorders, the signal transduction initiated by NGF and proNGF, the physiological and pathophysiological relevance, and therapeutic potential in treating chronic metabolic diseases and cognitive decline., Competing Interests: Declaration of competing interest The authors declare that there is no conflict of interest with this manuscript., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Five prosomatostatin genes (PSST1, PSST2, PSST3, PSST5, and PSST6) have been recently identified in elasmobranchs (Tostivint et al., General and Comparative Endocrinology, 2019, 279, 139-147). In order to gain insight into the contribution of each somatostatin to specific nervous systems circuits and behaviors in this important jawed vertebrate group, we studied the distribution of neurons expressing PSST mRNAs in the central nervous system (CNS) of Scyliorhinus canicula using in situ hybridization. Additionally, we combined in situ hybridization with tyrosine hydroxylase (TH) immunochemistry for better characterization of PSST1 and PSST6 expressing populations. We observed differential expression of PSST1 and PSST6, which are the most widely expressed PSST transcripts, in cell populations of many CNS regions, including the pallium, subpallium, hypothalamus, diencephalon, optic tectum, midbrain tegmentum, and rhombencephalon. Interestingly, numerous small pallial neurons express PSST1 and PSST6, although in different populations judging from the colocalization of TH immunoreactivity and PSST6 expression but not with PSST1. We observed expression of PSST1 in cerebrospinal fluid-contacting (CSF-c) neurons of the hypothalamic paraventricular organ and the central canal of the spinal cord. Unlike PSST1 and PSST6, PSST2, and PSST3 are only expressed in cells of the hypothalamus and in some hindbrain lateral reticular neurons, and PSST5 in cells of the region of the entopeduncular nucleus. Comparative data of brain expression of PSST genes indicate that the somatostatinergic system of sharks is the most complex reported in any fish., (© 2020 Wiley Periodicals, Inc.)

Context: Type 1 diabetes (T1D) is classified into 3 subtypes: acute-onset (AT1D), slowly progressive (SP1D), and fulminant (FT1D). The differences in the type of cellular autoimmunity within each subtype remain largely undetermined., Objective: To determine the type and frequency of islet antigen-specific CD4+ T cells in each subtype of T1D., Participants: Twenty patients with AT1D, 17 with SP1D, 18 with FT1D, and 17 persons without diabetes (ND)., Methods: We performed an integrated assay to determine cellular immune responses and T-cell repertoires specific for islet antigens. This assay included an ex vivo assay involving a 48-hour stimulation of peripheral blood mononuclear cells with antigen peptides and an expansion assay involving intracytoplasmic cytokine analysis., Results: The results of the ex vivo assay indicated that glutamic acid decarboxylase 65 (GAD65)-specific interleukin-6 and interferon-inducible protein-10 (IP-10) responses and preproinsulin (PPI)-specific IP-10 responses were significantly upregulated in AT1D compared with those of ND. Furthermore, GAD65- and PPI-specific granulocyte colony-stimulating factor responses were significantly upregulated in FT1D. Expansion assay revealed that GAD65- and PPI-specific CD4+ T cells were skewed toward a type 1 helper T (Th1)- cell phenotype in AT1D, whereas GAD65-specific Th2 cells were prevalent in SP1D. GAD65-specific Th1 cells were more abundant in SP1D with human leukocyte antigen-DR9 than in SP1D without DR9. FT1D displayed significantly less type 1 regulatory T (Tr1) cells specific for all 4 antigens than ND., Conclusions: The phenotypes of islet antigen-specific CD4+ T cells differed among the three T1D subtypes. These distinct T-cell phenotypes may be associated with the manner of progressive β-cell destruction., (© Endocrine Society 2020. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Glucose homeostasis plays a key role in maintaining stable physiological conditions, and its dysfunction causes severe chronic health issues including diabetes. In this study, we have characterized goldfish adipocytes as cells with properties similar to that of pancreatic β-cells: they express considerable high levels of preproinsulin mRNAs, possess the necessary machinery for processing preproinsulin (prohormone convertases 1 and 2, carboxypeptidase E and trypsin) and responding to extracellular glucose (glucokinase and the glucose transporters 1, 2, and 4), produce insulin in a glucose-responsive manner and express key transcription factors typically involved in pancreas development (Pdx1, Neurogenin3, Nkx2.2, Pax6, and FOXO1A). These findings reinforce the feature of fish adipocytes as alternate sources of active insulin, holding the promise that they could eventually be developed as transplantable sources of this vital hormone., (© 2020 Wiley Periodicals, Inc.)

The effect of estrogens on Oryzias curvinotus juveniles were investigated by sequencing the transcriptome of O. curvinotus juveniles exposed to 17 β - estradiol for 24 h. A total of 69,071,524 and 71,210,528 raw reads were obtained for the control group (NC) and 17 β - estradiol exposure group (E2), respectively. After de novo assembly, total 133,210 unigenes were identified, and 85,837 unigenes (64.44% of 133,210) were annotated. Analysis of the transcriptome showed that exposure to 2 μg/L 17 β - estradiol led to the up-regulation of 19 genes and down-regulation of 18 genes. The eef1b and rps4x was most suitable as controls for quantitative real-time PCR (qPCR) using Reffinder. Different expression genes enrichment analysis found that exposed to 2 μg/L 17 β - estradiol affected various physiological processes, including spliceosome, phototransduction, amino sugar and nuclear sugar metabolism, hypotaurine metabolism, and renin-angiotensin system, etc. Exposing O. curvinotus juveniles to increasing concentrations of 17 β - estradiol (2 ng/L, 20 ng/L, 200 ng/L and 2 μg/L) led to significant up-regulation of vitellogenins (vtgs) and choriogenins (chgs) mRNA expression. The present study is the first high-throughput transcriptome sequencing of O. curvinotus juveniles, which will be useful for future functional analysis of genes related to environmental estrogen exposed, and development of biomarkers., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020. Published by Elsevier Inc.)

Extracellular calcium (Ca 2+ ) and store-operated Ca 2+ entry (SOCE) govern homoeostasis in the mammalian epidermis. Multiple microRNAs (miRNA) also regulate epidermal differentiation, and raised external Ca 2+ modulates the expression of several such miRNAs in keratinocytes. However, little is known about the regulation of miR-184 in keratinocytes or the roles of miR-184 in keratinocyte differentiation. Here we report that exogenous Ca 2+ stimulates miR-184 expression in primary epidermal keratinocytes and that this occurs in a SOCE-dependent manner. Levels of miR-184 were raised by about 30-fold after exposure to 1.5 mM Ca 2+ for 5 days. In contrast, neither phorbol ester nor 1,25-dihydroxyvitamin D 3 had any effect on miR-184 levels. Pharmacologic and genetic inhibitors of SOCE abrogated Ca 2+ -dependent miR-184 induction by 70% or more. Ectopic miR-184 inhibited keratinocyte proliferation and led to a fourfold increase in the expression of involucrin, a marker of early keratinocyte differentiation. Exogenous miR-184 also triggered a threefold rise in levels of cyclin E and doubled the levels of γH2AX, a marker of DNA double-strand breaks. The p21 cyclin-dependent kinase inhibitor, which supports keratinocyte growth arrest, was also induced by miR-184. Together our findings point to an SOCE:miR-184 pathway that targets a cyclin E/DNA damage regulatory node to facilitate keratinocyte differentiation., (© 2020 Wiley Periodicals, Inc.)

The discovery of insulin in 1921 has been one of greatest scientific achievements of the 20th century. Since then, the availability of insulin has shifted the focus of diabetes treatment from trying to keep patients alive to saving and improving the life of millions. Throughout this time, basic and clinical research has advanced our understanding of insulin synthesis and action, both in healthy and pathological conditions. Yet, multiple aspects of insulin production remain unknown. In this review, we focus on the most recent findings on insulin synthesis, highlighting their relevance in diabetes. Graphical abstract.

Psoriasis is one of the most common chronic inflammatory skin diseases, it is characterized by hyperproliferation of keratinocytes and infiltration of inflammatory cells. Several in vitro studies have reported that interleukin (IL)‑22 is involved in excessive proliferation and abnormal differentiation of human keratinocytes. However, the association between IL‑22 and CCAAT enhancer binding protein α (C/EBPα) in the pathogenesis of psoriasis remains unclear. Therefore, the present study aimed to investigate the association between IL‑22 and C/EBPα, and the effects of IL‑22 on the proliferation and differentiation of keratinocytes. Keratinocytes were treated with different concentrations of IL‑22 (30, 60 and 90 ng/ml) and subsequently cells were collected at different time intervals. The expression levels of the key molecules of the mitogen‑activated protein kinase (MAPK) signaling pathway were detected using western blot analysis. In addition, the effect of IL‑22 on the proliferation rate of keratinocytes and the mRNA expression levels of C/EBPα were determined using a Cell Counting Kit‑8 assay and reverse transcription‑quantitative PCR, respectively. Furthermore, keratinocytes were transfected with C/EBPα small interfering (si)RNA or control using Lipofectamine® 2000. The results revealed that IL‑22 significantly induced the proliferation of keratinocytes and the expression of phosphorylated (p)‑JNK, p‑ERK and p‑p38 (P<0.05). Additionally, IL‑22 significantly inhibited the differentiation of keratinocytes, and the mRNA and protein expression of C/EBPα (P<0.05). Furthermore, downregulation of C/EBPα increased the proliferation rate of keratinocytes and reduced the expression levels of cytokeratin 10 and involucrin. Therefore, these results suggested that the effect of IL‑22 on the proliferation and differentiation of keratinocytes may be mediated via the regulation of the MAPK signaling pathway and the expression of C/EBPα.

Background: Profilaggrin belongs to the S100 fused-type protein family expressed in keratinocytes and is important for skin barrier integrity. Its N-terminus contains an S100 ("A") domain and a unique "B" domain with a nuclear localization sequence., Objective: To determine whether profilaggrin B domain cooperates with the S100 domain to bind macromolecules. To characterize the biochemical and structural properties of the profilaggrin N-terminal "AB" domain and compare it to other S100 fused-type proteins., Methods: We used biochemical (protease protection, light scattering, fluorescence spectroscopy, pull-down assays) and computational techniques (sequence analysis, molecular modeling with crystallographic structures) to examine human profilaggrin and S100 fused-type proteins., Results: Comparing profilaggrin S100 crystal structure with models of the other S100 fused-type proteins demonstrated each has a unique chemical composition of solvent accessible surface around the hydrophobic binding pocket. S100 fused-type proteins exhibit higher pocket hydrophobicity than soluble S100 proteins. The inter-EF-hand linker in S100 fused-type proteins contains conserved hydrophobic residues involved in binding substrates. Profilaggrin B domain cooperates with the S100 domain to bind annexin II and keratin intermediate filaments in a calcium-dependent manner using exposed cationic surface. Using molecular modeling we demonstrate profilaggrin B domain likely interacts with annexin II domains I and II. Steric clash analysis shows annexin II N-terminal peptide is favored to bind profilaggrin among S100 fused-type proteins., Conclusion: The N-terminal S100 and B domains of profilaggrin cooperate to bind substrate molecules in granular layer keratinocytes to provide epidermal barrier functions., Competing Interests: Declaration of Competing Interest The authors have no conflicts of interest to declare., (Copyright © 2020 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

High-risk pregnancies, such as pregnancies with gestational diabetes mellitus (GDM), are becoming more common and as such, have become important public health issues worldwide. GDM increases the risks of macrosomia, premature infants, and preeclampsia. Although placental dysfunction, including fibrosis is associated with the development of GDM, factors that link these observations remain unknown. Prothymosin α (ProTα) is expressed in the placenta and is involved in cell proliferation and immunomodulation. It also plays an important role in insulin resistance and fibrosis. However, the role of ProTα in GDM is still unclear. In the present study, we found that fibrosis-related protein expressions, such as type I collagen (Col-1) were significantly increased in the placentae of ProTα transgenic mice. With elevated fibrosis-related protein expressions, placental weights significantly increased in GDM group. In addition, placental and circulating ProTα levels were significantly higher in patients with GDM (n=39), compared with the healthy group (n=102), and were positively correlated with Col-1 expression. Mice with streptozotocin (STZ)-induced GDM had increased ProTα, fasting blood glucose, Col-1, and placental weight, whereas plasma insulin levels were decreased. ProTα overexpression enhanced nuclear factor κB (NFκB) activation to increase fibrosis-related protein expressions in 3A-Sub-E trophoblasts, while treatment with an NFκB inhibitor reversed the effect of ProTα on fibrosis-related protein expressions. We further investigated whether ProTα is regulated by hyperglycemia-induced reactive oxygen species (ROS). In conclusion, ProTα increases the amount of placental connective tissue and thus contributes to the pathogenesis of placental fibrosis in GDM. Therefore, ProTα may be a novel therapeutic target for GDM., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Germline telomere maintenance defects are associated with an increased incidence of inflammatory diseases in humans, yet whether and how telomere dysfunction causes inflammation are not known. Here, we show that telomere dysfunction drives pATM/c-ABL-mediated activation of the YAP1 transcription factor, up-regulating the major pro-inflammatory factor, pro-IL-18. The colonic microbiome stimulates cytosolic receptors activating caspase-1 which cleaves pro-IL-18 into mature IL-18, leading to recruitment of interferon (IFN)-γ-secreting T cells and intestinal inflammation. Correspondingly, patients with germline telomere maintenance defects exhibit DNA damage (γH2AX) signaling together with elevated YAP1 and IL-18 expression. In mice with telomere dysfunction, telomerase reactivation in the intestinal epithelium or pharmacological inhibition of ATM, YAP1, or caspase-1 as well as antibiotic treatment, dramatically reduces IL-18 and intestinal inflammation. Thus, telomere dysfunction-induced activation of the ATM-YAP1-pro-IL-18 pathway in epithelium is a key instigator of tissue inflammation.

The onset establishment and maintenance of gonadotropin-releasing hormone (GnRH) secretion is an important phenomenon regulating pubertal development and reproduction. GnRH neurons as well as other neurons in the hypothalamus have high-energy demands and require a constant energy supply from their mitochondria machinery to maintain active functioning. However, the involvement of mitochondrial function in GnRH neurons is still unclear. In this study, we examined the role of NADH Dehydrogenase (Ubiquinone) Fe-S protein 4 (Ndufs4), a member of the mitochondrial complex 1, on GnRH neurons using Ndufs4-KO mice and Ndufs4-KO GT1-7 cells. Ndufs4 was highly expressed in GnRH neurons in the medial preoptic area (MPOA) and NPY/AgRP and POMC neurons in the arcuate (ARC) nucleus in WT mice. Conversely, there was a significant decrease in GnRH expression in MPOA and median eminence of Ndufs4-KO mice, followed by impaired peripheral endocrine system. In Ndufs4-KO GT1-7 cells, Gnrh1 expression was significantly decreased with or without stimulation with either kisspeptin or NGF, whereas, stimulation significantly increased Gnrh1 expression in control cells. In contrast, there was no difference in cell signaling activity including ERK and CREB as well as the expression of GPR54, TrkA and p75 NTR , suggesting that Ndufs4 is involved in the transcriptional regulation system for GnRH production. These findings may be useful in understanding the mitochondrial function in GnRH neuron., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Actin filaments and microtubules create diverse cellular protrusions, but intermediate filaments, the strongest and most stable cytoskeletal elements, are not known to directly participate in the formation of protrusions. Here we show that keratin intermediate filaments directly regulate the morphogenesis of microridges, elongated protrusions arranged in elaborate maze-like patterns on the surface of mucosal epithelial cells. We found that microridges on zebrafish skin cells contained both actin and keratin filaments. Keratin filaments stabilized microridges, and overexpressing keratins lengthened them. Envoplakin and periplakin, plakin family cytolinkers that bind F-actin and keratins, localized to microridges, and were required for their morphogenesis. Strikingly, plakin protein levels directly dictate microridge length. An actin-binding domain of periplakin was required to initiate microridge morphogenesis, whereas periplakin-keratin binding was required to elongate microridges. These findings separate microridge morphogenesis into distinct steps, expand our understanding of intermediate filament functions, and identify microridges as protrusions that integrate actin and intermediate filaments., Competing Interests: YI, VC, Av, LC, AS No competing interests declared, (© 2020, Inaba et al.)

Gonadotropin-releasing hormone (GnRH) is a reproductive neuropeptide, which controls vertebrate reproduction. In most vertebrates, there are more than two GnRH orthologs in the brain. In cichlid fish, the Nile tilapia ( Oreochromis niloticus ), GnRH1 is the primary hypophysiotropic hormone, while GnRH2 and GnRH3 are non-hypophysiotropic but neuromodulatory in function. Hypophysiotropic GnRH neurons are thought to inter-communicate, while it remains unknown if hypophysiotropic and non-hypophysiotropic GnRH systems communicate with each other. In the present study, we examined interrelationship between three GnRH types using specific antibodies raised against their respective GnRH associated peptide (GAP) sequence. Double-immunofluorescence labeling coupled with confocal microscopy revealed that in sexually mature males, GnRH-GAP1-immunoreactive (-ir) processes are in proximities of GnRH-GAP3-ir cell somata in the terminal nerve, while GnRH-GAP1-ir cell somata were also accompanied by GnRH-GAP3-ir processes in the preoptic area. However, such interaction was not seen in immature males. Further, there was no interaction between GnRH-GAP2 and GnRH-GAP1 or GnRH-GAP3 neurons. Single cell gene expression analysis revealed co-expression of multiple GnRH receptor genes ( gnrhr1 and gnrhr2 ) in three GnRH-GAP cell types. In mature males, high levels of gnrhr2 mRNA were expressed in GnRH-GAP1-ir cells. In immature males, gnrhr1 and gnrhr2 mRNAs are highly expressed in GnRH-GAP3-ir cells. These results suggest heterologous interactions between the three GnRH-GAP cell types and their potential functional interaction during different reproductive stages., (Copyright © 2020 Ogawa and Parhar.)

The rules governing Medicinal Products in the European Union necessitates the production of cell-based therapy in good manufacturing practice facilities. The produced cells may need several hours in transportation to reach the application sites. In this study, we investigated four candidate solutions for transporting human keratinocytes. The solutions are (1) normal saline, (2) saline with 2.5% human serum albumin (Saline + HSA), (3) chemically defined, xeno-free keratinocyte media and (4) keratinocyte media with pituitary bovine extract (PBE-media). One million keratinocytes from three donors were suspended in each solution and kept at 4 °C for up to 24 h. Cells kept in Saline + HSA showed higher viability after 1, 3 and 24 h. Then, equal number of viable cells were seeded on collagenous matrix and cultured for 48 h. The adhesion and colonization were higher in the cells kept in PBE-media, while the keratinocyte surface marker, cytokeratin 14, was present in all studied groups. These results confirmed the suitability of Saline + HSA as a cell transportation solution for clinical use, which will be the choice for the planned clinical trial. Keratinocyte PBE-media can be an alternative for cells transported for research purpose, if the same media type is going to be used in the following experiments.

Regulatory T (Treg) cells, expressing CD25 (interleukin-2 receptor α chain) and Foxp3 transcription factor, maintain immunological self-tolerance and suppress various immune responses. Here we report a feature of skin Treg cells expanded by ultraviolet B (UVB) exposure. We found that skin Treg cells possessing a healing function are expanded by UVB exposure with the expression of an endogenous opioid precursor, proenkephalin (PENK). Upon UVB exposure, skin Treg cells were expanded with a unique TCR repertoire. Also, they highly expressed a distinctive set of genes enriched in "wound healing involved in inflammatory responses" and the "neuropeptide signaling pathway," as indicated by the high expression of Penk. We found that not only was PENK expression at the protein level detected in the UVB-expanded skin Treg (UVB-skin Treg) cells, but that a PENK-derived neuropeptide, methionine enkephalin (Met-ENK), from Treg cells promoted the outgrowth of epidermal keratinocytes in an ex vivo skin explant assay. Notably, UVB-skin Treg cells also promoted wound healing in an in vivo wound closure assay. In addition, UVB-skin Treg cells produced amphiregulin (AREG), which plays a key role in Treg-mediated tissue repair. Identification of a unique function of PENK + UVB-skin Treg cells provides a mechanism for maintaining skin homeostasis., Competing Interests: The authors declare no competing interest.

Skin aging is influenced by several genetic, physiological, and environmental factors. In particular, ultraviolet (UV) exposure is an important factor involved in inducing skin photoaging. Autophagy controlling homeostatic balance between the synthesis, degradation, and recycling of cellular organelles and proteins plays important regulatory roles in several biological processes, including aging. The opioid neuropeptide α-neoendorphin (named NEP) is an endogenous decapeptide (N-YGGFLRKYPK-C) that activates the kappa opioid receptor and exhibits certain anti-aging and anti-wrinkling effects on skin cells; however, its action mechanism has not yet been elucidated. Therefore, the aim of this study was to determine the effects of NEP on anti-skin aging and autophagy activation in human dermal fibroblast cells. Western blot results showed that NEP down-regulates the production of phospho-mammalian target of rapamycin (p-mTOR), whereas increases the expression of key autophagy-related molecules such as Beclin-1, Atg5-Atg12, and LC3-II. The immunocytochemical analysis performed with anti-LC3-II antibody also showed that the autophagic indicators, autophagosomes are formed by NEP. These results suggest that NEP can activate cellular autophagy through mTOR-Beclin-1-mediated signaling pathway. It was also revealed by CM-H 2 DCF-DA assay and Western blottings that NEP can reduce the production of ultraviolet B (UVB)-induced reactive oxygen species (ROS) like with N-acetylcysteine (NAC), resulting in decreasing the expression levels of skin aging-related proteins, such as phospho-ERK (p-ERK), phospho-p38 (p-p38), and phospho-JNK (p-JNK). Furthermore, NEP could increase the type I procollagen production, while decreasing MMP-1, MMP-2, and MMP-9 activities. Taken together, the results demonstrate that NEP can reduce UVB-induced photoaging by activating autophagy., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

HOXA13 overexpression has been detected in human ESCC tissue and high HOXA13 protein expression is correlated with a shorter median survival time in ESCC patients. Although aberrant expression of HOXA13 in ESCC has thus been established, little is known regarding the functional consequences thereof. The present study aimed to examine to what extent aberrant HOXA13 might drive carcinogenesis in esophageal keratinocytes. To this end, we overexpressed HOXA13 in a non-transformed human esophageal cell line EPC2-hTERT, performed gene expression profiling to identify key processes and functions, and performed functional experiments. We found that HOXA13 expression confers oncogenic hallmarks to esophageal keratinocytes. It provides proliferation advantage to keratinocytes, reduces sensitivity to chemical agents, regulates MHC class I expression and differentiation status and promotes cellular migration. Our data indicate a crucial role of HOXA13 at early stages of esophageal carcinogenesis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Authors. Published by Elsevier B.V. All rights reserved.)