NPC1 is required for postnatal islet ß cell differentiation by maintaining mitochondria turnover.

Rationale: NPC1 is a protein localized on the lysosome membrane regulating intracellular cholesterol transportation and maintaining normal lysosome function. GWAS studies have found that NPC1 variants in T2D was a pancreatic islet expression quantitative trait locus, suggesting a potential role of NPC1 in T2D islet pathophysiology. Methods: Two-week-old Npc1-/- mice and wild type littermates were employed to examine pancreatic ß cell morphology and functional changes induced by loss of Npc1. Single cell RNA sequencing was conducted on primary islets. Npc1-/- Min6 cell line was generated using CRISPR/Cas9 gene editing. Seahorse XF24 was used to analyze primary islet and Min6 cell mitochondria respiration. Ultra-high-resolution cell imaging with Lattice SIM2 and electron microscope imaging were used to observe mitochondria and lysosome in primary islet ß and Min6 cells. Mitophagy Dye and mt-Keima were used to measure ß cell mitophagy. Results: In Npc1-/- mice, we found that ß cell survival and pancreatic ß cell mass expansion as well as islet glucose induced insulin secretion in 2-week-old mice were reduced. Npc1 loss retarded postnatal ß cell differentiation and growth as well as impaired mitochondria oxidative phosphorylation (OXPHOS) function to increase mitochondrial superoxide production, which might be attributed to impaired autophagy flux particularly mitochondria autophagy (mitophagy) induced by dysfunctional lysosome in Npc1 null ß cells. Conclusion: Our study revealed that NPC1 played an important role in maintaining normal lysosome function and mitochondria turnover, which ensured establishment of sufficient mitochondria OXPHOS for islet ß cells differentiation and maturation.

Ferroptosis-related biomarkers for Alzheimer's disease: Identification by bioinformatic analysis in hippocampus.

Background: Globally, Alzheimer's Disease (AD) accounts for the majority of dementia, making it a public health concern. AD treatment is limited due to the limited understanding of its pathogenesis. Recently, more and more evidence shows that ferroptosis lead to cell death in the brain, especially in the regions of the brain related to dementia. Materials and methods: Three microarray datasets (GSE5281, GSE9770, GSE28146) related to AD were downloaded from Gene Expression Omnibus (GEO) datasets. Ferroptosis-related genes were extracted from FerrDb database. Data sets were separated into two groups. GSE5281 and GSE9770 were used to identify ferroptosis-related genes, and GSE28146 was used to verify results. During these processes, protein-protein interaction (PPI), the Gene Ontology (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were conducted. Finally, the differentiated values of ferroptosis-related genes were determined by receiver operator characteristic (ROC) monofactor analysis to judge their potential quality as biomarkers. Results: Twenty-four ferroptosis-related genes were obtained. Using STRING (https://cn.string-db.org/) and Cytoscape with CytoHubba, the top 10 genes (RB1, AGPAT3, SESN2, KLHL24, ALOX15B, CA9, GDF15, DPP4, PRDX1, UBC, FTH1, ASNS, GOT1, PGD, ATG16L1, SLC3A2, DDIT3, RPL8, VDAC2, GLS2, MTOR, HSF1, AKR1C3, NCF2) were identified as target genes. GO analysis revealed that response to carboxylic acid catabolic process, organic acid catabolic process, alpha-amino acid biosynthetic process and cellular amino acid biosynthetic process were the most highly enriched terms. KEGG analysis showed that these overlapped genes were enriched in p53 signaling pathways, longevity regulating pathway, mTOR signaling pathway, type 2 diabetes mellitus and ferroptosis. Box plots and violine plots were created and verified to confirm the significance of identified target genes. Moreover, ROC monofactor analysis was performed to determine the diagnostic value of identified genes. Two genes (ASNS, SESN2) were subsequently obtained. For the tow genes, STRING was used to obtain the five related genes and determined enriched GO terms and KEGG pathways for those genes. Conclusion: Our results suggest that ASNS and SENS2 may serve as potential diagnostic biomarkers for AD and provide additional evidence regarding the essential role of ferroptosis in AD.

Third dose of anti-SARS-CoV-2 inactivated vaccine for patients with RA: Focusing on immunogenicity and effects of RA drugs.

Objectives: To evaluate the immunogenicity of the third dose of inactivated SARS-CoV-2 vaccine in rheumatoid arthritis (RA) patients and explore the effect of RA drugs on vaccine immunogenicity. Methods: We recruited RA patients (n = 222) and healthy controls (HC, n = 177) who had been injected with a third dose of inactivated SARS-CoV-2 vaccine, and their neutralizing antibody (NAb) titer levels were assessed. Results: RA patients and HC were age- and gender-matched, and the mean interval between 3rd vaccination and sampling was comparable. The NAb titers were significantly lower in RA patients after the third immunization compared with HC. The positive rate of NAb in HC group was 90.4%, while that in RA patients was 80.18%, and the difference was significant. Furthermore, comparison of NAb titers between RA treatment subgroups and HC showed that the patients in the conventional synthetic (cs) disease-modifying anti-rheumatic drugs (DMARDs) group exhibited no significant change in NAb titers, while in those receiving the treatment of biological DMARDs (bDMARDs), Janus Kinase (JAK) inhibitors, and prednisone, the NAb titers were significantly lower. Spearman correlation analysis revealed that NAb responses to SARS-CoV-2 in HC did differ significantly according to the interval between 3rd vaccination and sampling, but this finding was not observed in RA patients. In addition, NAb titers were not significantly correlated with RA-related laboratory indicators, including RF-IgA, RF-IgG, RF-IgM, anti-CCP antibody; C-RP; ESR; NEUT% and LYMPH%. Conclusion: Serum antibody responses to the third dose of vaccine in RA patients were weaker than HC. Our study will help to evaluate the efficacy and safety of booster vaccination in RA patients and provide further guidance for adjusting vaccination strategies.

Immunogenicity of Inactivated SARS-CoV-2 Vaccines in Patients With Rheumatoid Arthritis: A Case Series.

Objectives: Attenuated humoral response to mRNA SARS-CoV-2 vaccines has been reported in some patients with autoimmune disease, e.g., rheumatoid arthritis (RA). However, data of immune responses to inactivated SARS-CoV-2 vaccine in the RA population are still unknown. Herein, the safety and immunogenicity of inactivated SARS-CoV-2 vaccines in RA patients were analyzed. Methods: Seventy five RA patients and 26 healthy controls (HC) were respectively recruited from Yunnan Provincial Hospital of Traditional Chinese Medicine and the community in Kunming city. Neutralizing Antibody (NAb) Test ELISA kit was used to measure the percentage of inhibition. AKA (anti-keratin antibody) positivity was detected using indirect immunofluorescence. Rheumatoid factor (RF)-IgA was detected by ELISA. RF-IgG, RF-IgM, and anti-cyclic citrullinated peptide (CCP) antibodies were measured by chemiluminescence. ESR (erythrocyte sedimentation rate) was detected by ESR analyzer. C-RP (c-reactive protein) was detected by immunoturbidimetry. NEUT% (percentage of neutrophils) and LYMPH% (percentage of percentage) were calculated by a calculation method. Results: Compared with the HC group, the percentage of inhibition was significantly lower in RA patients receiving two doses of vaccines. Vaccines-induced percentage of inhibition was the lowest in RA patients who had not been vaccinated. In total 80.77% of the HC group had a percentage of inhibition ≧20%, compared with 45.24% of vaccinated RA patients and 6.06% of unvaccinated RA patients. Spearman correlation analysis revealed that antibody responses to SARS-CoV-2 did not differ between RA patients according to their age and disease duration. Furthermore, the results showed that no correlation was found between the percentage of inhibition and indices for RA, including RF-IgA, IgG, IgM; anti-CCP antibody; ESR; C-RP; NEUT% and LYMPH%. Conclusion: Our study showed inactivated vaccine-induced SARS-COV-2 antibody responses differ in RA patients and healthy subjects, emphasizing the importance of a third or fourth vaccination in RA patients.

Gene editing in monogenic autism spectrum disorder: animal models and gene therapies.

Autism spectrum disorder (ASD) is a lifelong neurodevelopmental disease, and its diagnosis is dependent on behavioral manifestation, such as impaired reciprocal social interactions, stereotyped repetitive behaviors, as well as restricted interests. However, ASD etiology has eluded researchers to date. In the past decades, based on strong genetic evidence including mutations in a single gene, gene editing technology has become an essential tool for exploring the pathogenetic mechanisms of ASD via constructing genetically modified animal models which validates the casual relationship between genetic risk factors and the development of ASD, thus contributing to developing ideal candidates for gene therapies. The present review discusses the progress in gene editing techniques and genetic research, animal models established by gene editing, as well as gene therapies in ASD. Future research should focus on improving the validity of animal models, and reliable DNA diagnostics and accurate prediction of the functional effects of the mutation will likely be equally crucial for the safe application of gene therapies.

Enhancing Acsl4 in absence of mTORC2/Rictor drove ß-cell dedifferentiation via inhibiting FoxO1 and promoting ROS production.

Rapamycin insensitive companion of mechanistic target of Rapamycin (Rictor), the key component of mTOR complex 2 (mTORC2), controls both ß-cell proliferation and function. We sought to study whether long chain acyl-CoA synthetase 4 (Acsl4) worked downstream of Rictor/mTORC2 to maintain ß-cell functional mass. We found Acsl4 was positively regulated by Rictor at transcriptional and posttranslational levels in mouse ß-cell. Infecting adenovirus expressing Acsl4 in ß-cell-specific-Rictor-knockout (ßRicKO) islets and Min6 cells knocking down Rictor with lentivirus-expressing siRNA-oligos targeting Rictor(siRic), recovered the ß-cell dysplasia but not dysfunction. Cell bioenergetic experiment performed with Seahorse XF showed that Acsl4 could not rescue the dampened glucose oxidation in Rictor-lacking ß-cell, but further promoted lipid oxidation. Transposase-Accessible Chromatin (ATAC) and H3K27Ac chromatin immunoprecipitation (ChIP) sequencing studies reflected the epigenetic elevated molecular signature for ß-cell dedifferentiation and mitigated oxidative defense/response. These results were confirmed by the observations of elevated acetylation and ubiquitination of FoxO1, increased protein levels of Gpx1 and Hif1an, excessive reactive oxygen species (ROS) production and diminished MafA in Acsl4 overexpressed Rictor-lacking ß-cells. In these cells, antioxidant treatment significantly recovered MafA level and insulin content. Inducing lipid oxidation alone could not mimic the effect of Acsl4 in Rictor lacking ß-cell. Our study suggested that Acsl4 function in ß-cell was context dependent and might facilitate ß-cell dedifferentiation with attenuated Rictor/mTORC2 activity or insulin signaling via posttranslational inhibiting FoxO1 and epigenetically enhancing ROS induced MafA degradation.

The Intestinal Effect of Atorvastatin: Akkermansia muciniphila and Barrier Function.

Studies have shown that the cholesterol-lowering medicine statins alter the gut microbiome, induce chronic metabolic inflammation, and disrupt glycemic homeostasis. In this study, we aimed to investigate whether effects of atorvastatin (Ator) on gut microbiome and metabolic inflammation could be causally correlated. Mice at 8-week age were fed with high-fat diet (HFD) or HFD with Ator (HFD+Ator) for 16 weeks. 16S rRNA sequencing of stool and RNA sequencing of colon tissue were employed to analyze the intestinal alterations that could be induced by Ator. A human colon carcinoma cell line (Caco2) was used for in vitro experiments on barrier function. Compared to HFD, HFD+Ator induced more weight gain, impaired glucose tolerance, and led to gut microbiota dysbiosis, such as suppressing Akkermansia muciniphila in mice. The expressions of tight junction (TJ) proteins were attenuated in the colon, and the serum LPS-binding-protein (LBP) level was elevated in HFD+Ator mice, so as to transcriptionally activate the intestinal nuclear factor-k-gene binding (NF-κB) signaling pathway. Consistently, Ator impaired the barrier function of Caco2, and treatment of supernatant of A. Muciniphila culture could decrease the intestinal permeability and recover the attenuated expression of TJ proteins induced by Ator. In conclusion, long-term use of Ator with HFD may alter gut microbiota, induce intestinal barrier dysfunction, and hence promote chronic inflammation that contributes to disrupted glycemic homeostasis.

Sleep Disturbances and Depression Are Co-morbid Conditions: Insights From Animal Models, Especially Non-human Primate Model.

The incidence rates of depression are increasing year by year. As one of the main clinical manifestations of depression, sleep disorder is often the first complication. This complication may increase the severity of depression and lead to poor prognosis in patients. In the past decades, there have been many methods used to evaluate sleep disorders, such as polysomnography and electroencephalogram, actigraphy, and videography. A large number of rodents and non-human primate models have reproduced the symptoms of depression, which also show sleep disorders. The purpose of this review is to examine and discuss the relationship between sleep disorders and depression. To this end, we evaluated the prevalence, clinical features, phenotypic analysis, and pathophysiological brain mechanisms of depression-related sleep disturbances. We also emphasized the current situation, significance, and insights from animal models of depression, which would provide a better understanding for the pathophysiological mechanisms between sleep disturbance and depression.

lncRNA:mRNA expression profile in CD4+ T cells from patients with Graves' disease.

Graves' disease (GD) is a common autoimmune disease that affects the thyroid gland. As a new class of modulators of gene expression, long noncoding RNAs (lncRNAs) have been reported to play a vital role in immune functions and in the development of autoimmunity and autoimmune disease. The aim of this study is to identify lncRNAs in CD4+ T cells as potential biomarkers of GD. lncRNA and mRNA microarrays were performed to identify differentially expressed lncRNAs and mRNAs in GD CD4+ T cells compared with healthy control CD4+ T cells. Quantitative PCR (qPCR) was used to validate the results, and correlation analysis was used to analyze the relationship between these aberrantly expressed lncRNAs and clinical parameters. The microarray identified 164 lncRNAs and 93 mRNAs in GD CD4+ T cells differentially expressed compared to healthy control CD4+ T cells (fold change >2.0 and a P < 0.05). Further analysis consistently showed that the expression of HMlincRNA1474 (P < 0.01) and TCONS_00012608 (P < 0.01) was suppressed, while the expression of AK021954 (P < 0.01) and AB075506 (P < 0.01) was upregulated from initial GD patients. In addition, their expression levels were recovered in euthyroid GD patients and GD patients in remission. Moreover, these four aberrantly expressed lncRNAs were correlated with GD clinical parameters. Moreover, the areas under the ROC curve were 0.8046, 0.7579, 0.8115 for AK021954, AB075506, HMlincRNA1474, respectively. The present work revealed that differentially expressed lncRNAs were associated with GD, which might serve as novel biomarkers of GD and potential targets for GD treatment.

The glucose-lowering effects of α-glucosidase inhibitor require a bile acid signal in mice.

AIMS/HYPOTHESIS: Bile-acid (BA) signalling is crucial in metabolism homeostasis and has recently been found to mediate the therapeutic effects of glucose-lowering treatments, including α-glucosidase inhibitor (AGI). However, the underlying mechanisms are yet to be clarified. We hypothesised that BA signalling may be required for the glucose-lowering effects and metabolic benefits of AGI. METHODS: Leptin receptor (Lepr)-knockout (KO) db/db mice and high-fat high-sucrose (HFHS)-fed Fxr (also known as Nr1h4)-KO mice were treated with AGI. Metabolic phenotypes and BA signalling in different compartments, including the liver, gut and endocrine pancreas, were evaluated. BA pool profiles were analysed by mass spectrometry. The islet transcription profile was assayed by RNA sequencing. The gut microbiome were assayed by 16S ribosomal RNA gene sequencing. RESULTS: AGI lowered microbial BA levels in BA pools of different compartments in the body, and increased gut BA reabsorption in both db/db and HFHS-fed mouse models via altering the gut microbiome. The AGI-induced changes in BA signalling (including increased activation of farnesoid X receptor [FXR] in the liver and inhibition of FXR in the ileum) echoed the alterations in BA pool size and composition in different organs. In Fxr-KO mice, the glucose- and lipid-lowering effects of AGI were partially abrogated, possibly due to the Fxr-dependent effects of AGI on decelerating beta cell replication, alleviating insulin hypersecretion and improving hepatic lipid and glucose metabolism. CONCLUSIONS/INTERPRETATION: By regulating microbial BA metabolism, AGI elicited diverse changes in BA pool composition in different host compartments to orchestrate BA signalling in the whole body. The AGI-induced changes in BA signalling may be partly required for its glucose-lowering effects. Our study, hence, sheds light on the promising potential of regulating microbial BA and host FXR signalling for the treatment of type 2 diabetes. DATA AVAILABILITY: Sequencing data are available from the BioProject Database (accession no. PRJNA600345; www.ncbi.nlm.nih.gov/bioproject/600345).

Atorvastatin Targets the Islet Mevalonate Pathway to Dysregulate mTOR Signaling and Reduce ß-Cell Functional Mass.

Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic ß-cell size and ß-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and ß-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and ß-cell function. Rab5a knockdown mimicked the effect of ator treatment on ß-cells. Thus, ator impairs ß-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional ß-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.

LncRNA KB-1471A8.2 Overexpression Suppresses Cell Proliferation and Migration and Antagonizes the Paclitaxel Resistance of Ovarian Cancer Cells.

Objective: The aim was to investigate the lncRNA KB-1471A8.2 function in ovarian cancer progression and paclitaxel resistance. Methods: The expression and distribution of KB-1471A8.2 was detected by qRT-PCR. The cell proliferation, apoptosis, invasion, migration and chemoresistance were analyzed by CCK8 assay, flow cytometry and transwell assay. The expression of DEPTOR, whose sequence is reverse overlapped with KB-1471A8.2 was analyzed by qRT-PCR, western blot and immunofluorescence. The cell cycle and the cell cycle related gene expression were analyzed by flow cytometry and qRT-PCR, respectively. Results: KB-1471A8.2 was significantly downregulated in both ovarian cancer tissues and chemoresistant ovarian cancer cells. Overexpression of KB-1471A8.2 significantly inhibited the proliferation, invasion, migration, and paclitaxel resistance, and increased the apoptosis of ovarian cancer cells. KB-1471A8.2 was mainly distributed in the nucleus of ovarian cancer cells. KB-1471A8.2 overexpression significantly decreased the S phase cell ratio, increased the G0/G1 phase cell ratio, but not affected the expression and distribution of DEPTOR. However, cyclin-dependent kinase 4 (CDK4), which is an important regulator of G1/S transition, was significantly decreased in KB-1471A8.2-overexpressed ovarian cancer cells. Conclusion: KB-1471A8.2 could significantly inhibit the development and paclitaxel resistance of ovarian cancer cells, at least partly, by suppressing CDK4 expression.

Cancer-testis antigens in ovarian cancer: implication for biomarkers and therapeutic targets.

Ovarian cancer remains the most fatal gynecologic malignancy worldwide due to delayed diagnosis as well as recurrence and drug resistance. Thus, the development of new tumor-related molecules with high sensitivity and specificity to replace or supplement existing tools is urgently needed. Cancer-testis antigens (CTAs) are exclusively expressed in normal testis tissues but abundantly found in several types of cancers, including ovarian cancer. Numerous novel CTAs have been identified by high-throughput sequencing techniques, and some aberrantly expressed CTAs are associated with ovarian cancer initiation, clinical outcomes and chemotherapy resistance. More importantly, CTAs are immunogenic and may be novel targets for antigen-specific immunotherapy in ovarian cancer. In this review, we attempt to characterize the expression of candidate CTAs in ovarian cancer and their clinical significance as biomarkers, activation mechanisms, function in malignant phenotypes and applications in immunotherapy.

Multidrug resistant lncRNA profile in chemotherapeutic sensitive and resistant ovarian cancer cells.

Most ovarian cancer patients are chemosensitive initially, but finally relapse with acquired chemoresistance. Multidrug-resistance is the extremely terrible situation. The mechanism for the acquired chemoresistance of ovarian cancer patients is still not clear. LncRNAs have been recognized as the important regulator of a variety of biological processes, including the multidrug-resistant process. Here, we carried out the lncRNA sequencing of the ovarian cancer cell line A2780 and the paxitaxel resistant cell line A2780/PTX which is also cross resistant to the cisplatin and epirubicin. Through integrating the published data with the cisplatin resistant lncRNAs in ovarian cancer cell line or ovarian cancer patients, 5 up-regulated and 21 down-regulated lncRNAs are considered as the multidrug-resistant lncRNAs. By real-time PCR analysis, we confirmed the 5 up-regulated and 4 down-regulated multidrug resistant lncRNAs were similarly changed in both the multidrug resistant ovarian cancer cell lines and the multidrug resistant colon cancer cell lines. Furthermore, we conducted the lncRNA-mRNA co-expression network to predict the potential multidrug resistant lncRNAs' targets. Interestingly, the multidrug resistant genes ABCB1, ABCB4, ABCC3, and ABCG2 are all co-expressed with lncRNA CTD-2589M5.4. Our results provide the valuable information for the understanding of the lncRNA function in the multidrug resistant process.

Colour-opponent mechanisms are not affected by age-related chromatic sensitivity changes.

The purpose of this study was to assess whether age-related chromatic sensitivity changes are associated with corresponding changes in hue perception in a large sample of colour-normal observers over a wide age range (n = 185; age range: 18-75 years). In these observers we determined both the sensitivity along the protan, deutan and tritan line; and settings for the four unique hues, from which the characteristics of the higher-order colour mechanisms can be derived. We found a significant decrease in chromatic sensitivity due to ageing, in particular along the tritan line. From the unique hue settings we derived the cone weightings associated with the colour mechanisms that are at equilibrium for the four unique hues. We found that the relative cone weightings (w(L) /w(M) and w(L) /w(S)) associated with the unique hues were independent of age. Our results are consistent with previous findings that the unique hues are rather constant with age while chromatic sensitivity declines. They also provide evidence in favour of the hypothesis that higher-order colour mechanisms are equipped with flexible cone weightings, as opposed to fixed weights. The mechanism underlying this compensation is still poorly understood.