National Institutes Of Health Guide Research Articles

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Erratum: Eyestalk Ablation to Increase Ovarian Maturation in Mud Crabs.

Novel biomarkers in healthy aging: Tongue and jaw kinematics as a potential way to further explore Alzheimer’s‐induced biological changes

AbstractBackgroundMany age‐related oral health problems, such as masticatory dysfunction and dysphagia, have been associated with Alzheimer’s Disease (AD). However, how oromotor dysfunctions in healthy aging differ from those found in AD is still largely unknown. Here, we aim to assess changes in tongue and jaw kinematics in healthy aging to understand how these biomarkers may provide early signatures of AD.MethodData were collected from two male monkeys (Macaca mulatta), one young (8 years) and one older (20 years). Using high‐resolution bilateral videoradiography (200 frames/s) and the X‐Ray Reconstruction of Moving Morphology (XROMM) workflow, we quantified the 3D position of tongue and jaw radio‐opaque markers while monkeys engaged in a natural feeding behavior. To evaluate age‐related changes, we analyzed tongue and jaw kinematics relative to specific events in the feeding cycle during 10‐s feeding trials (20‐60 trials). All protocols were approved by the University of Chicago Animal Care and Use Committee and complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. ResultDuring chews and swallows, both monkeys exhibited cyclical tongue motions, however, the tongue motions in the older monkey were substantially less stereotyped. The distributions of x‐y‐z positions of the tongue during start/end of chews and swallows were tightly clustered in the young monkey but were more spread out in the older (Fig. 1). In addition, tongue movements in the rostro‐caudal direction lagged behind jaw movements to a greater degree in the older monkey than in the younger (Fig. 2). Indeed, in the young monkey, tongue movements in the rostro‐caudal direction were highly correlated with supero‐inferior jaw movements, with peak correlations occurring at zero lag (Fig. 3).ConclusionThese results show that tongue and jaw kinematics may change with age, particularly with regards to stereotypy of tongue movement and tongue‐jaw coordination. Understanding oromotor changes in healthy aging may serve as diagnostic measures that may be helpful in treating or delaying AD progression and in reducing the severity of debilitating oromotor dysfunctions.

Protective Role of Adipose-Derived Circulating Exosomal miR-122-5p on the Pulmonary Endothelial Barrier Through Inhibiting EndMT via Downregulation of the TGFβ1/TGFβr1/Smad2 Pathway: A Potential Explanation for the Obesity Paradox in ARDS

Background: The “obesity paradox in acute respiratory distress syndrome (ARDS)” refers to the phenomenon that obesity is associated with a lower mortality in patients with ARDS. Endothelial to mesenchymal transition (EndMT) represents a key link in the interaction between endothelial disruption and mesenchymal fibrosis under inflammatory conditions, which are intersectional pathophysiologies of ARDS. Adipose tissue is reported to constitute the major source of circulating exosomal miRNAs, thereby these miRNAs act as genetic forms of adipokines for cell-cell crosstalk. However, the regulation of adipose-derived exosomal miRNAs in the obesity paradox in ARDS are largely unknown.Methods High-fat induced-obese mice and lean control mice were subjected to ARDS insult to investigate the effects of obesity on ARDS and microarray analysis was performed to screen the different circulating microRNAs. Mice and pulmonary endothelial cells were administered to adipose-derived exosomal miR-122-5p to further investigate the molecular mechanism. Findings: We found that high-fat diet-induced obesity protected against ARDS by reinforcing the endothelial barrier and attenuating fibroproliferation in mice. Circulating exosomes produced in the obese state mediated this protective effect by inhibiting EndMT. Mechanistically, adipose-derived exosomal miR-122-5p promoted the integrity and function of pulmonary endothelial barrier and alleviated fibrogenesis by suppressing EndMT through downregulation of the TGFβ1/TGFβR1/Smad2 pathway in vivo and in vitro. Interpretation Our findings propose a potential explanation for the obesity paradox in ARDS and indicate promising prospects for adipose-derived exosomes in cell-free therapies for ARDS. Funding: This work was supported by the NSFC of China (Grant NO. 81800083) and China Postdoctoral Science Foundation (2019M653832XB). Declaration of Interest: The authors declare no conflict of interest. Ethical Approval: Animal procedures were approved by the ethics committee of Chongqing Medical University and implemented according to the instructions of the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

P2X7 Receptor Deficiency Ameliorates STZ-Induced Cardiac Damage and Remodeling Through PKCβ and ERK

Background: Diabetic cardiomyopathy (DCM) is a complication of diabetes mellitus which result in cardiac remodeling and subsequent heart failure. However, the role of the P2X7 receptor (P2X7R) in DCM has yet to be elucidated. The principal objective of this study was to investigate whether P2X7R participates in the pathogenesis of DCM. Methods: The C57BL/6 mice were induced by STZ and treated with a P2X7R inhibitor (A438079). Cardiac dysfunction and remodeling were detected by two-dimensional B-ultrasound, Western blot (WB), quantitative PCR (qPCR), immunofluorescence, immunohistochemistry and so on. P2X7R knockout (P2X7R-/-) mouse models were used to assess the role of the P2X7R in diabetic cardiomyopathy. Cell hypertrophy, apoptosis, and fibrosis were assessed in H9c2 cells and primary rat cardiomyocytes treated with A438079. Protein kinase C-β (PKC-β) and extracellular regulated protein kinases (ERK) activation was examined by immunoblotting. Findings: Cardiac dysfunction and remodeling were attenuated by the intraperitoneal injection of A438079 or P2X7R deficiency. In vitro, A438079 reduced high-glucose induced cell damage in H9c2 cells and primary rat cardiomyocytes. Furthermore, HG/STZ-induced P2X7R activation mediated downstream protein kinase C-β (PKC-β) and extracellular regulated protein kinases (ERK)activation. Funding: This study was supported by the National Natural Science Foundation of China (grant numbers 81670227 and 82070446), the Wenzhou Science and Technology Bureau (grant no. Y2020242 and Y20180099), and the Key Research and Development Program of Zhejiang (grant no. 2019C03012). Declaration of Interest: The authors have declared no conflicts of interest. Ethical Approval: Animals: All operations were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Animal care and experimental protocols were approved by the Committee on Animal Care of Wenzhou Medical University.

Assessing the Association of Mitochondrial Function and Inflammasome Activation in Murine Macrophages Exposed to Select Mitotoxic Tri-Organotin Compounds.

Background:Mitochondrial function is implicated as a target of environmental toxicants and found in disease or injury models, contributing to acute and chronic inflammation. One mechanism by which mitochondrial damage can propagate inflammation is via activation of the nucleotide-binding oligomerization domain (NOD)-like receptor (NLR) family, pyrin domain-containing receptor (NLRP)3 inflammasome, a protein complex that processes mature interleukin . plays an important role in the innate immune response and dysregulation is associated with autoinflammatory disorders.Objective:The objective was to evaluate whether mitochondrial toxicants recruit inflammasome activation and processing.Method:Murine macrophages (RAW 264.7) exposed to tri-organotins (triethyltin bromide (TETBr), trimethyltin hydroxide (TMTOH), triphenyltin hydroxide (TPTOH), bis(tributyltin)oxide) [Bis(TBT)Ox] were examined for pro-inflammatory cytokine induction. TMTOH and TETBr were examined in RAW 264.7 and bone marrow-derived macrophages for mitochondrial bioenergetics, reactive oxygen species (ROS) production, and inflammasome activation via visualization of aggregate formation, caspase-1 flow cytometry, enzyme-linked immunosorbent assay and Western blots, and microRNA (miRNA) and mRNA arrays.Results:TETBr and TMTOH induced inflammasome aggregate formation and release in lipopolysaccharide (LPS)-primed macrophages. Mitochondrial bioenergetics and mitochondrial ROS were suppressed. Il1a and Il1b induction with LPS or challenge was diminished. Differential miRNA and mRNA profiles were observed. Lower miR-151-3p targeted cyclic adenosine monophosphate (cAMP)-mediated and AMP-activated protein kinase signaling pathways; higher miR-6909-5p, miR-7044-5p, and miR-7686-5p targeted Wnt beta-catenin signaling, retinoic acid receptor activation, apoptosis, signal transducer and activator of transcription 3, IL-22, IL-12, and IL-10 signaling. Functional enrichment analysis identified apoptosis and cell survival canonical pathways.Conclusion:Select mitotoxic tri-organotins disrupted murine macrophage transcriptional response to LPS, yet triggered inflammasome activation. The differential response pattern suggested unique functional changes in the inflammatory response that may translate to suppressed host defense or prolong inflammation. We posit a framework to examine immune cell effects of environmental mitotoxic compounds for adverse health outcomes. https://doi.org/10.1289/EHP8314

Heme Oxygenase-1-Modified Bone Marrow Mesenchymal Stem Cells Perfusion Using a Normothermic Machine Perfusion System Reduces the Acute Rejection of Liver Transplantation by Regulating Natural Killer T Cell Co-Inhibitory Receptors

Background: Acute rejection (ACR) remains an important risk factor affecting the survival of recipients following liver transplantation (LT). Immunosuppressive agents are associated with many complications. Bone marrow mesenchymal stem cells (BMMSCs) are used in the treatment of organ transplantation; however, there is limited colonization in the target organs and a short survival time following systemic BMMSCs application. Methods: BMMSCs were isolated from rat bone marrow and modified with the adenovirus for heme oxygenase 1 (HO-1) gene. HO-1/BMMSCs were perfused into the donor liver in vitro using a normothermic machine perfusion (NMP) system, followed by LT. The severity of ACR was evaluated and Gene chip technology was used to detect differential gene expression, then the flow cytometry was used to analyze changes in natural killer (NK) T cells. Findings: NMP can induce BMMSCs to colonize the donor liver during in vitro preservation, and the survival of HO-1/BMMSCs in the liver after transplantation was significantly longer than that of BMMSCs. When the donor liver contained HO-1/BMMSCs, the ACR of LT is obviously controlled, and the survival time was significantly prolonged. Interpretation: The application of HO-1/BMMSCs reduces the number of NKT cells in the liver following transplantation, increases the expression of the NKT cell co-inhibitory receptors, BTLA and CD160, and reduces the level of NKT cell expression of IFN-γ. Thus, NK cell and CD8+ T cell activation was inhibited, which reduced the ACR. Funding Information: The work was supported by the National Natural Science Foundation of China (No. 82070639, 81670574, 81441022 and 81270528). Declaration of Interests: All of the authors declared no conflict of interest. Ethics Approval Statement: All animal protocols were based on the National Institutes of Health Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 85 - 23, Bethesda, MD). All procedures were approved by the Ethics Committee of Tianjin First Central Hospital (license number: 2016-03-A1).

Combined Metabolic Activators Improves Cognitive Functions in Alzheimer's Disease

Alzheimer’s disease (AD) is associated with metabolic abnormalities linked to critical elements of neurodegeneration. Here, we analysed the brain transcriptomics data of more than 600 AD patients using genome-scale metabolic models and provided supporting evidence of mitochondrial dysfunction related to the pathophysiologic mechanisms of AD progression. Subsequently, we investigated, in a rat model of AD, the oral administration of Combined Metabolic Activators (CMA), consisting of NAD+ and glutathione precursors, to explore the effect for improvement of biological functions in AD. CMA includes L-serine, nicotinamide riboside, N-acetyl-L-cysteine, and L-carnitine tartrate, salt form of L-carnitine. The study revealed that supplementation of the CMA improved the AD-associated histological parameters in the animals. Finally, we designed a randomized, double-blinded, placebo-controlled human phase 2 clinical trial and showed that the administration of CMA improves cognitive functions in AD patients. As decreased AD Assessment Scale-cognitive subscale (ADAS-Cog) score is the indicator of the improved cognitive function in AD patients, we observed a significant decrease of ADAS-Cog scores on Day 84 vs Day 0 (Log2FC= -0.37, (29% improvement), p-value=0.00001) in the CMA group. We also observed a significant decrease in the placebo group on Day 84 vs Day 0 (Log2FC= -0.19, (14% improvement), p-value=0.001) possibly due to the recommendations of exercise and Mediterranean diet to all AD patients and/or a placebo effect apparent in the early stages of AD clinical trials. A comprehensive analysis of the human plasma metabolome and proteome revealed that plasma levels of proteins and metabolites associated with redox metabolism are significantly improved after treatment. In conclusion, our results show that treating AD patients with CMA leads to enhanced cognitive functions, suggesting a role for such a therapeutic regime in treating AD and other neurodegenerative diseases. Funding: This work was financially supported by ScandiBio Therapeutics and Knut and Alice Wallenberg Foundation. The authors would like to thank the Metabolon Inc. (Durham, USA) for generation of metabolomics data, and ChromaDex Inc. (Irvine, CA, USA) for providing NR. AM and HY acknowledge support from the PoLiMeR Innovative Training Network (Marie Sklodowska-Curie Grant Agreement No. 812616) which has received funding from the European Union’s Horizon 2020 research and innovation programme. Declaration of Interest: AM, JB and MU are the founder and shareholders of ScandiBio Therapeutics. The other authors declare no competing interests. Ethical Approval: All experiments for the treatment of the animals were approved by the Ethics Committee of Ataturk University, and were conducted following the National Institutes of Health Guide for Care and Use of Laboratory Animals.

Exposure to High Altitude Environment Is Associated With Drug Metabolism Change: miRNA-873-5p-Mediated Alteration of Function and Expression Levels of Drug Transporters Under Hypoxia

Background: Hypoxia is the main characteristic of a high-altitude environment, affecting drug metabolism. However, little is known about the role of microRNA (miRNA) in drug metabolism under high-altitude hypoxia. This study aims to investigate the functions and the expression levels of multidrug resistance mutation 1 (MDR1), multidrug resistance-associated protein 2 (MRP2), breast cancer resistance protein (BCRP), peptide transport 1 (PEPT1), and organic anion-transporting polypeptides 2B1 (OATP2B1) in vivo and in vitro after exposure to high-altitude hypoxia. Methods: Western blot and qPCR analyses were used to analyze the protein and mRNA expression levels of MDR1, MRP2, BCRP, PEPT1, and OATP2B1. The functions of MDR1, MRP2, BCRP, PEPT1, and OATP2B1 were evaluated by determining the effective intestinal permeability and absorption rate constants of their specific substrates in rats after exposure to high-altitude hypoxia, and uptake and transport studies were performed in Caco-2 cells. To clarify whether miRNA-873-5p regulates MDR1 and pregnane X receptor (PXR) under hypoxia, Caco-2 cells were transfected with mimics or inhibitors of miRNA-873-5p and negative control (NC). Findings: Hypoxia increased the expression and activity of most of the drug transporters in vivo and in vitro. We found that miRNA-873-5p regulated MDR1 and PXR expression, thereby affecting drug metabolism. Interpretation: Herein, it was shown that miRNA might affect the expression of drug transporter and nuclear receptor under hypoxia. miRNA-873-5p play important roles in the process of drug metabolism under hypoxia. Funding Information: This work was supported by the National Natural Science Foundation of China (No. 81760673 and 81460568), the Qinghai Innovation Platform Construction Project (No. 2021-ZJ-T03), and the Key Research Foundation of Development and Transformation of Qinghai Province (2021-SF-158). Declaration of Interests: None of the authors has any potential conflict of interest or financial interests to disclose. Ethics Approval Statement: All experimental procedures were performed strictly according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals. The protocol was approved by the Animal Ethics Committee of The Qinghai University.

Prior SARS-CoV-2 Infection Prevents Acute Disease and Lung Pathology in Reinfected Syrian Hamsters but not Virus Replication in the Upper Respiratory Tract

Human cases of SARS-CoV-2 reinfection have been documented throughout the pandemic but are likely underreported. In the current study, we used the Syrian hamster SARS-CoV-2 model to assess reinfection with homologous (Washington WA1) and heterologous (United Kingdom B.1.1.7 (alpha) and South Africa B.1.351 (beta)) SARS-CoV-2 variants over time. Upon primary infection with SARS-CoV-2 WA1, hamsters rapidly developed a strong and long-lasting humoral immune response. Following reinfection with homologous and heterologous SARS-CoV-2 variants, this immune response protected hamsters from clinical disease, virus replication in the lower respiratory tract and acute lung pathology. However, reinfection led to SARS-CoV-2 replication in the upper respiratory tract with the potential for virus shedding. Our findings indicate that reinfection results in restricted SARS-CoV-2 replication despite substantial levels of humoral immunity, denoting the potential for transmission through reinfected asymptomatic individuals.Funding: This study was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases (NIAID) at the National Institutes of Health (NIH). Declaration of Interests: The authors declare no competing interests.Ethics Approval Statement: Work with infectious SARS-CoV-2 was approved by the Institutional Biosafety Committee (IBC) and performed in the high biocontainment facilities at Rocky Mountain Laboratories (RML), NIAID, NIH. Sample removal from high biocontainment followed IBC-approved Standard Operating Protocols (44). Animal work was approved by the Institutional Animal Care and Use Committee and performed by certified staff in an Association for Assessment and Accreditation of Laboratory Animal Care International accredited facility. Work followed the institution’s guidelines for animal use, the guidelines and basic principles in the NIH Guide for the Care and Use of Laboratory Animals, the Animal Welfare Act, United States Department of Agriculture and the United States Public Health Service Policy on Humane Care and Use of Laboratory Animals.

SOX2-Upregulated microRNA-30e Promotes Cell Invasion in Esophageal Cancer via USP4/SMAD4/CK2 Axis

Background: Both SOX2 and microRNA-30e (miR-30e) were reported to be involved in esophageal cancer (EC) progression. However, whether there is correlation between SOX2 and miR-30e in EC cells remains undiscovered. Herein, this study was intended to explore the correlation between SOX2 and miR-30e in EC and the effect of this correlation on EC cell invasion. Methods: Following collection of EC tissues and cells, miR-30e, SOX2, miR-30e, USP4, SMAD4, and CK2 expression was determined by RT-qPCR, western blot analysis and immunohistochemistry. Gain- and loss-of-function approaches were conducted in screened cell lines, followed by determination of cell proliferation, migration, invasion, and expression of epithelial-mesenchymal transition (EMT)-related genes. The relationship among miR-30e, USP4, SMAD4 and CK2 was assessed by dual-luciferase reporter assay, in vitro protein purification, immunoprecipitation, and ubiquitination experiments, and ChIP assay. The role of SOX2 and CK2 in EC in vivo was evaluated by tumor xenograft in nude mice. Results: EC tissues and cells contained high expression of SOX2, miR-30e, and CK2 as well as low expression of USP4 and SMAD4. miR-30e was upregulated by SOX2, and specifically targeted USP4. Additionally, USP4 induced the deubiquitination of SMAD4 to upregulate SMAD4. SMAD4 was enriched in CK2 promoter region and inhibited CK2 expression. Silencing of SMAD4 or USP4 or upregulation of SOX2, miR-30e or CK2 triggered enhancement in EC cell proliferation, migration, invasion, and EMT in vitro, and promotion of tumor growth and lung metastasis in vivo. Conclusion: Taken together, our work reveals a novel SOX2-mediated regulatory axis that plays critical roles in the invasiveness of EC cells. Funding Statement: This work was supported by Youth Foundation of the National Natural Science Foundation of China (No. 81702971), Key Scientific Research Projects of Colleges and Universities of Henan Province (No. 18A320054), Science and Technology Research Projects of Henan Province (No. 182102310116) and Youth Talent Lifting Projects of Henan Province (No. 2020HYTP050) Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The ethics committee of the First Affiliated Hospital of Zhengzhou University provided Ethical Approval for the experiments involved human beings in this study which were guided by the intentions of the Declaration of Helsinki. Ethical agreements were obtained from the donors or their relatives by written informed consent. The experiments involved animals were implemented in accordance with the principles embodied in the National Institutes of Health Guide for the Care and Use of Laboratory. Efforts were made to avoid all unnecessary distress to the animals.

Fecal Microbiota Transplantation from Patients with Autoimmune Encephalitis Modulates Immune Response and Relevant Behaviors in Mice

Background: The significance of the microbiota-gut-brain axis has been increasingly recognized as a major modulator of autoimmunity. However, little is known about the effects of the gut microbiome on immune function and behaviors in autoimmune encephalitis. In this study, we aim to take advantage of a large cohort of treatment naive anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis to rigorously characterise gut dysbiosis and investigated the roles of immune function and behaviors. Methods : We first conducted a cross-sectional study of 40 MSA treatment-naive patients and 54 matched healthy controls (HCs). Then fecal microbiota transplantation (FMT) from anti-NMDAR encephalitis patients to microbiota-depleted mice was performed to investigate the immune function and behaviors. Findings : Relative to HCs, anti-NMDAR encephalitis patients had a decreased microbiome alpha-diversity index, marked disturbances of gut microbial composition and intestinal permeability damage. Disturbed microbiota in anti-NMDAR encephalitis patients might be linked with different clinical characteristics. Imputed KEGG analysis revealed perturbations of functional modules in the gut microbiomes of anti-NMDAR encephalitis. Compared to HCs, microbiota-depleted mice receiving FMT from anti-NMDAR encephalitis patients had hypersensitivity and cognitive impairment. Furthermore, anti-NMDAR encephalitis FMT mice showed altered T cells in the spleen and inguinal lymph node with an increased Th17 cells. Interpretation: Our study presents a comprehensive gut microbiota landscape in anti-NMDAR encephalitis. It suggests that the anti-NMDAR encephalitis microbiome itself can influence neurologic, immune and behavioral function. The gut microbiota is a potential therapeutic target for anti-NMDAR encephalitis. Funding Statement: This work was supported by grants from the National Natural Science Foundation of China (No. 81971141), the Israel Science Foundation and the National Natural Science Foundation of China joint programme (No. 813111290), the Natural Science Foundation of Guangdong Province (No. 2019A1515010201) and Tianhe District Technology Project (2018YT019). Declaration of Interests: All authors declare that there are no conflicts of interest. Ethics Approval Statement: This study was approved by the Medical Ethics Committee of the Third Affiliated Hospital of Sun Yat-sen University (approval ID: [2018]02-363-01). The subjects or the guardians of patients with severe cognitive impairment provided written informed consent for research and publication. Animal protocols were performed using C57BL/6J wild-type (WT) female mice in accordance with guidelines for animal care, according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Bioethics Committee of South China Agricultural University (approval ID: 2019-D063).

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

Jak Inhibition Prevents Bleomycin-Induced Fibrosis in Mice and Is Effective in Patients with Morphea

The Novel Antimicrobial Activities of Carrimycin Against Multidrug Resistant <i>Acinetobacter baumannii </i>and <i>Pseudomonas aeruginosa</i> Systemic Infection

Background: The multidrug-resistant(MDR) Acinetobacter baumannii and Pseudomonas aeruginosa have been listed as the top two critical pathogens to threat the public health by the World Health Organization (WHO) and regarded as a global priority for investment in new drugs. Carrimycin is a novel macrolide antibiotic used in therapy of community acquired respiratory infection. In this study, we want to further examine its antibacterial effect against the MDR A. baumannii and P. aeruginosa in vitro and in vivo. Methods: In vitro, we tested the minimal inhibitory concentration(MIC) of the strains of A. baumannii or P. aeruginosa isolated from clinic or ATCC. In vivo, using the wild-type(WT) Balb/c mice and T-cell-deficient Balb/c nude mice bacteremia model, we examined the count of A. baumannii and P. aeruginosa colony culture and histopathological damage in target organs tissue after carrimycin administration. Findings: Our results demonstrated that most strains of A. baumannii and P. aeruginosa were resistant to drugs and inhibited growth by carrimycin partly in vitro, but could be cleaned in infected Balb/c mice with the dose-dependent manner, but not in Balb/c nude mice. Inflammation relief in target organs also be observed in Balb/c mice but not in Balb/c nude mice. Interpretation: These findings indicated that carrimycin could not only clean pathogens directly, but also could improve the host's immunity to anti-inflammation and relieve damage against A. baumannii and P. aeruginosa infection. Funding: This work was funded by the National Emergency Response Program of China (Grant number 2020YFC0844900) and the National Natural Science Foundation of China (Grant number 81872852). Declaration of Interests: The authors declare no conflicts of interest. Ethics Approval Statement: The experimental procedures were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the local Animal Care and Use Committee.

Exosome-Transported circRNA_0000253 Increase Intervertebral Disc Degeneration and Its Potential Mechanism

Background: As society ages, intervertebral disc degeneration (IDD) causes great damage to humans. The pathogenesis of IDD is complex, and a better understanding of IDD pathogenesis at the molecular level may provide a better method for the treatment of IDD. Exosomes are 40-100 nm nano-sized vesicles that are released from many cell types into the extracellular space. We speculated that exosome-transported circular RNAs (circRNAs) could regulate IDD. Methods: Exosomes from samples of different degenerative grades were isolated and added to nucleus pulposus cells (NPCs), and indicators of NPC proliferation and apoptosis were detected. CircRNA microarray analysis was carried out, and the top 10 circRNAs were selected. PCR was performed to detect circRNA expression in normal and degenerated NPC exosomes, and the circRNA with the maximum upregulation was selected. Competing endogenous RNA (ceRNA) analysis was carried out. The function of the selected circRNA was verified in vivo and in vitro. Results: NPCs of different degenerative grades secreted exosomes, which could regulate IDD. CircRNA_0000253 was selected with the maximum upregulation in degenerative NPC exosomes. CeRNA analysis showed that circRNA_0000253 could adsorb microRNA-141-5p to up-regulate SIRT1. CircRNA_0000253 was also confirmed to increase IDD by competitively adsorbing microRNA-141-5p and up-regulating SIRT1 in vivo and in vitro. Conclusion: Exosomes secreted by NPCs could regulate IDD. CircRNA_0000253 was selected with the maximum upregulation in degenerative NPC exosomes and could promote IDD by competitively adsorbing microRNA-141-5p and down-regulating SIRT1. Funding Statement: Government funds were received in support of this work: National Natural Science Foundation of China (81972093, 81972109). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: The study protocol was approved by Huashan Hospital Ethics Committee Affiliated with Fudan University. Informed consent was obtained from each donor. All animal procedures were approved by the Animal Care and Use Committee of Fudan University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Ubiquitin-Specific Protease 10 Suppresses Cardiac Hypertrophy by Inhibiting Sirt6

Background and Purpose: Cardiac hypertrophy (CH) is a physiological response that compensates for blood pressure overload. Under pathological conditions, hypertrophy can evolve to heart failure as a consequence of the disorganized growth of cardiomyocytes and cardiac tissue. Ubiquitin-specific protease 10 (USP10) is a member of the ubiquitin-specific protease family of cysteine proteases, which are involved in viral infection, oxidative stress, lipid drop formation and heat shock. However, the role of USP10 in CH remains largely unclear. Here, we investigated the roles of USP10 in CH. Experimental Approach: In this study, by utilizing conditional cardiac-specific USP10 knockout (USP10-CKO) mice and USP10- transgenic(USP10-TG) mice, we examined the role of USP10 in hypertrophic hearts. We further examined the specific function of USP10 in cardiomyocytes. Key Results: We show that USP10 expression increased in murine hypertrophic hearts. Mice deficient in USP10 in the heart exhibited an aggravated aortic banding (AB)-induced CH phenotype. However, overexpression of USP10 protected against pressure overload-induced hypertrophy. At the molecular level, the prevention of CH in the absence of USP10 involves elevated Akt pathway components, which are key elements that regulate cell apoptosis and proliferation. Moreover, USP10 physically binds to Sirt6 and inhibits Akt phosphorylation. Conclusion and Implications: These observations demonstrate that USP10 functions as a positive switch in CH. Funding Statement: This work was supported by grants from National Natural Science Foundation of China (81770048;81970242); Cooperative Project of Academy training Foundation of Zhengzhou University(2016-BSTDJJ-13). Declaration of Interests: The authors stated: None. Ethics Approval Statement: All animal use protocols were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Zhengzhou University. The related procedures were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Quantitative Study on Self-Renewal of Vestibular Hair Cells in a Sox9-CreER Mouse Line

Background: Hair cells (HCs) are the mechanoreceptors for body position changes. Studies have suggested that HCs in mammals are terminally differentiated cells, which cannot regenerate after injury. Vestibular organs can generate new HCs, albeit in limited numbers. Methods: We carried out a study to analyse quantitatively and qualitatively spontaneous regeneration of vestibular HCs at different life stages. A Sox9-CreER transgenic mouse model specifically labelled with vestibular SCs was established. The proliferating cells, proliferative SCs, proliferative regenerated HCs and trans-differentiated HCs in mouse utricles of different ages were counted and analysed. Findings: We report that, from P1 to P30, the number of HCs in mouse utricles increased while the number of SCs decreased. The proliferation of SCs could be maintained until two weeks after birth. In the first week after birth, more than 30% of the total HCs were born. With an increase of age, the efficiency of HC regeneration decreased. In the adult stage, approximately 9% of new HCs still existed. Both types of HCs can be regenerated. Regenerated traced HCs are characterized by short, long, and absent bundles. Interpretation: Sox9 specifically labelled the vestibular SCs. HCs in the mouse vestibular apparatus undergo low-level turnover even in adulthood. These findings expand our understanding of sensorineural plasticity in adult vestibular organs in mammals after birth. Funding Statement: This work was supported by the National Natural Science Foundation of China (Nos. 81470687). Declaration of Interests: No authors have any professional or financial affiliations that may be regarded as a conflict of interest. Ethics Approval Statement: All animal procedures were approved by the Animal Care and Use Committee of Fudan University and were consistent with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

ROCK2 Confers Acquired Gemcitabine Resistance in Pancreatic Cancer Cells by Upregulating Transcription Factor ZEB1

Background: Gemcitabine resistance is a major clinical challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC). The increasing attention to Rho-associated coil protein kinase 2 (ROCK2)-mediated chemotherapy resistance in a variety of cancers reminded us to explore the effect of ROCK2 signalnaling in the acquired gemcitabine-resistant pancreatic cancer cells (GR cells). Methods: The relative expression of related proteins and mRNA were detected by western blot and real-time PCR, respectively. Short hairpin RNA (shRNA) was used to silencing related genes. The double luciferase reporter gene experiment was used to explore the activation of related gene promoter. Chromatin immunoprecipitation technique (CHIP) was used to detect the ability of transcription factor binding to the promoter. Many other cell based assays were adopted to explore the mechanism of ROCK2 on resistance of GR cells to gemcitabine. Findings: Pharmacological inhibition or gene silencing of ROCK2 markedly sensitized GR cells to gemcitabine by suppressing the expression of zinc-finger-enhancer binding protein 1 (ZEB1). Mechanically, ROCK2-induced sp1 phosphorylation at Thr-453 enhanced the ability of sp1 binding to ZEB1 promoter regions in a p38-dependent manner. Moreover, transcriptional activation of ZEB1 facilitated GR cells to repair gemcitabine-mediated DNA damage via ATM/p-CHK1 signaling pathway. Interpretation: ROCK2 contributed to EMT-induced gemcitabine resistance in pancreatic cancer cells and might provided strong evidence for the clinical application of fasudil, a ROCK2 inhibitor, in gemcitabine-refractory PDAC. Funding Statement: This work was supported by the National Natural Science Foundation of China (No.81372268, No.81672816 and No.81872337), the Program for Jiangsu Province Innovative Research (KYLX16_1120), the Natural Science Foundation for Distinguished Young Scholars of Jiangsu Province (No. BK20130026), the Project Program of State Key Laboratory of Natural Medicines, China Pharmaceutical University (No. ZJ11173). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and all animal experimental procedures were approved by Experimentation Ethics Review Committee of China Pharmaceutical.

Inhibition of miR-155-5p Exerts Anti-Fibrotic Effects in Silicotic Rats by Regulating Meprin α

Silicosis is a fatal profession-related disease linked to long term inhalation of silica. The present study aimed to determine whether meprin α, a master regulator of anti-fibrotic peptide N-acetyl-serylaspartyl-lysyl-proline (Ac-SDKP), is diminished by miR-155-5p in silicosis and control lung macrophages and fibroblasts upon activation. RAW 264.7 macrophages, primary lung fibroblasts, and mouse embryonic fibroblasts were used to evaluate the expression and function of meprin α and miR-155-5p. In vitro meprin α manipulation was performed by recombinant mouse meprin α protein, actinonin (its inhibitor), and small interfering RNA knockdown. Macrophage and fibroblast activation was assessed by western blotting, real-time PCR, matrix deposition, immunohistochemistry, and immunofluorescence staining. The roles of meprin α and miR-155-5p were also investigated in mice exposed to silica. We found that the meprin α level was stably repressed in silicotic rats.In vitro, silica decreased meprin α, and exogenous meprin α reduced activation of macrophages and fibroblasts induced by profibrotic factors. MiR-155-5p negatively regulated Mep1a by binding to the 3′ untranslated region. Treatment with anti-miR-155-5p elevated meprin α, ameliorated macrophage and fibroblast activation, and attenuated lung fibrosis in mice induced by silica. The sustained repression of meprin α and beneficial effects of its rescue by inhibition of miR-155-5p during silicosis indicate that miR-155-5p/meprin α are major regulators of silicosis. Funding Statement: This work was supported by the National Natural Science Foundation of China (No. 81472953), the Natural Science Foundation of Hebei Province (No. H20162091705), Science and Technology Research Project of Hebei Province universities (No. ZD2019077), and the Preeminence Youth Foundation of North China University of Science and Technology (JP201513). Declaration of Interests: The authors declare they have no competing interests. Ethics Approval Statement: All experimental and surgical procedures were approved (2013-038 and 2017-025) by the Ethics Committee for Animal Experimentation of North China University of Science and Technology, which complies with the US National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Bach1-Induced Suppression of Angiogenesis Is Dependent on the BTB Domain

The transcription factor BTB and CNC homology 1 (Bach1) impairs angiogenesis after ischemic injury by suppressing Wnt/β-catenin signaling; however, the specific domains responsible for the antiangiogenic effects of Bach1 remain unclear. This study determined the role of the BTB domain of Bach1 in ischemic angiogenesis. Bach1 is highly expressed in circulating endothelial cells from acute myocardial infarction patients, and is the early induction gene after ischemic inuced injury. Mice were treated with adenoviruses coding for GFP (AdGFP), Bach1 (AdBach1), or a Bach1 mutant lacking the BTB domain (AdBach1-ΔBTB) after surgically induced hind-limb ischemia. Measures of blood-flow recovery, capillary density, and the expression of vascular endothelial growth factor (VEGF) were significantly lower in the AdBach1 group, but not in AdBach1-ΔBTB animals. Furthermore, transfection with AdBach1, but not AdBach1-ΔBTB, in human umbilical-vein endothelial cells (HUVECs) was associated with significant declines in 1) capillary density and hemoglobin content in the Matrigel-plug assay, 2) proliferation, migration, tube formation, and VEGF transcriptional activity and mRNA expression in HUVECs. Bach1 binds directly with TCF4, and this interaction is mediated by residues 81-89 of the Bach1 BTB domain and the N-terminal CTNNB1 domain of TCF4. Bach1, but not Bach1-ΔBTB, also copreciptated with histone deacetylase 1 (HDAC1), while the full-length HDAC1 proteins, but not HDAC1 mutants lacking the protein-interaction domain, co-precipitated with Bach1. Collectively, these results demonstrate that the anti-angiogenic activity of Bach1 is crucially dependent on molecular interactions that are mediated by the protein's BTB domain, and this domain could be a drug target for angiogenic therapy. Funding Statement: This work was supported by the Great Program (91639103 to D. Meng), and General Programs (81670450 and 81873469 to D. Meng, 81570081 and 81770083 to S. Li, 81800234 to L. Jiang, 81572713 to X. Zhi, 81873536 to X. Wang) of the National Natural Science Foundation of China, the National Key Research and Development Program of China (2018YFC1313600 to S. Li). Declaration of Interests: The authors state: None. Ethics Approval Statement: HUVECs were isolated (12) from the fresh umbilical cord of normal parturients with informed consent. The procedure was approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College. All experimental protocols were approved by the Ethics Committee of Experimental Research at Fudan University Shanghai Medical College and performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH publication No 85-23).

SAT-188 EXPRESSION OF (PRO)RENIN RECEPTOR IN RAT RENAL CONGESTION MODEL

The physiological association between the kidney and heart has received significant attention. Reduction of cardiac output induces renal hypoperfusion, which causes renal ischemia. On the other hand, renal venous hypertension leads to high renal interstitial hydrostatic pressure and reduction in glomerular filtration rate (GFR). In patients with chronic congestive heart failure, central venous pressure and renal venous pressure are higher than in healthy subjects, causing renal congestion and dysfunction. We have recently developed a novel rat renal congestion model, and reported its pathophysiological and molecular aspects. In this model, renal congestion reduced renal blood flow and GFR, and increased renal interstitial hydrostatic pressure, resulting renal fibrosis together with podocyte and tubular injury. Molecules related to extracellular matrix expansion, tubular injury and focal adhesion were upregulated. (Pro)renin receptor [(P)RR] is initially identified as a member of renin angiotensin system. (P)RR also functions in intracellular acidification (a part of vacuolar type H+-ATPase), and cell proliferation through Wnt signaling pathway. We have reported the inhibition of (P)RR and V-ATPase suppresses cellular fibrosis induced by TGF-β1 in peritoneal mesothelial cells. No study, however, have investigated the association between (P)RR and renal congestion. Thus, we assessed (P)RR expression in our rat renal congestion model. All animal experiments were in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals, and were approved by animal experiment committees in Tohoku Medical and Pharmaceutical University and Tohoku University. The inferior vena cava (IVC) between the renal veins was ligated by suture in male Sprague-Dawley rats (7-9 weeks) to increase upstream IVC pressure and induce congestion in the left kidney only. Three days after surgery, both right control kidney and left congestive kidney were removed under anesthesia. The kidneys were immediately sectioned and fixed with 10% neutral-buffered formalin for histological analyses, and then divided into the cortex and medulla for RNA and protein analyses. mRNA expression of (P)RR and V-ATPase subunits was examined by quantitative real-time polymerase chain reaction. (P)RR protein expression was detected by western blot analysis and immunohistochemistry. mRNA expression levels of (P)RR and V-ATPase subunits (Atp6v0b, Atp6v0c, Atp6v1b1, Atp6v1b2 and Atp6v1c1) were significantly decreased in the congestive kidney, compared with the control kidney in both cortex and medulla. (P)RR protein was also decreased in the congestive kidney, especially in the medulla. By immunohistochemistry, (P)RR was highly expressed in the intercalated cells of the collecting ducts and weakly stained podocyte and other tubular segments in the control kidney, and the expression was abolished in the congestive kidney. Decrease of (P)RR and V-ATPase might relate to the progression of renal fibrosis in renal congestion.