Rab26 restricts insulin secretion via sequestering Synaptotagmin-1.

Rab26 is known to regulate multiple membrane trafficking events, but its role in insulin secretion in pancreatic ß cells remains unclear despite it was first identified in the pancreas. In this study, we generated Rab26-/- mice through CRISPR/Cas9 technique. Surprisingly, insulin levels in the blood of the Rab26-/- mice do not decrease upon glucose stimulation but conversely increase. Deficiency of Rab26 promotes insulin secretion, which was independently verified by Rab26 knockdown in pancreatic insulinoma cells. Conversely, overexpression of Rab26 suppresses insulin secretion in both insulinoma cell lines and isolated mouse islets. Islets overexpressing Rab26, upon transplantation, also failed to restore glucose homeostasis in type 1 diabetic mice. Immunofluorescence microscopy revealed that overexpression of Rab26 results in clustering of insulin granules. GST-pulldown experiments reveal that Rab26 interacts with synaptotagmin-1 (Syt1) through directly binding to its C2A domain, which interfering with the interaction between Syt1 and SNAP25, and consequently inhibiting the exocytosis of newcomer insulin granules revealed by TIRF microscopy. Our results suggest that Rab26 serves as a negative regulator of insulin secretion, via suppressing insulin granule fusion with plasma membrane through sequestering Syt1.

MEN1 is a regulator of alternative splicing and prevents R-loop-induced genome instability through suppression of RNA polymerase II elongation.

The fidelity of alternative splicing (AS) patterns is essential for growth development and cell fate determination. However, the scope of the molecular switches that regulate AS remains largely unexplored. Here we show that MEN1 is a previously unknown splicing regulatory factor. MEN1 deletion resulted in reprogramming of AS patterns in mouse lung tissue and human lung cancer cells, suggesting that MEN1 has a general function in regulating alternative precursor mRNA splicing. MEN1 altered exon skipping and the abundance of mRNA splicing isoforms of certain genes with suboptimal splice sites. Chromatin immunoprecipitation and chromosome walking assays revealed that MEN1 favored the accumulation of RNA polymerase II (Pol II) in regions encoding variant exons. Our data suggest that MEN1 regulates AS by slowing the Pol II elongation rate and that defects in these processes trigger R-loop formation, DNA damage accumulation and genome instability. Furthermore, we identified 28 MEN1-regulated exon-skipping events in lung cancer cells that were closely correlated with survival in patients with lung adenocarcinoma, and MEN1 deficiency sensitized lung cancer cells to splicing inhibitors. Collectively, these findings led to the identification of a novel biological role for menin in maintaining AS homeostasis and link this role to the regulation of cancer cell behavior.

Deletion of Fbxw7 in oocytes causes follicle loss and premature ovarian insufficiency in mice.

Premature ovarian insufficiency (POI) is one of the important causes of female infertility. Yet the aetiology for POI is still elusive. FBXW7 (F-box with 7 tandem WD) is one of the important components of the Skp1-Cullin1-F-box (SCF) E3 ubiquitin ligase. FBXW7 can regulate cell growth, survival and pluripotency through mediating ubiquitylation and degradation of target proteins via triggering the ubiquitin-proteasome system, and is associated with tumorigenesis, haematopoiesis and testis development. However, evidence establishing the function of FBXW7 in ovary is still lacking. Here, we showed that FBXW7 protein level was significantly decreased in the ovaries of the cisplatin-induced POI mouse model. We further showed that mice with oocyte-specific deletion of Fbxw7 demonstrated POI, characterized with folliculogenic defects, early depletion of follicle reserve, disordered hormonal secretion, ovarian dysfunction and female infertility. Impaired oocyte-GCs communication, manifested as down-regulation of connexin 37, may contribute to follicular development failure in the Fbxw7-mutant mice. Furthermore, single-cell RNA sequencing and in situ hybridization results indicated an accumulation of Clu and Ccl2 transcripts, which may alter follicle microenvironment deleterious to oocyte development and accelerate POI. Our results establish the important role of Fbxw7 in folliculogenesis and ovarian function, and might provide valuable information for understanding POI and female infertility.

Reduced Mitochondrial Protein Translation Promotes Cardiomyocyte Proliferation and Heart Regeneration.

BACKGROUND: The importance of mitochondria in normal heart function are well recognized and recent studies have implicated changes in mitochondrial metabolism with some forms of heart disease. Previous studies demonstrated that knockdown of the mitochondrial ribosomal protein S5 (MRPS5) by small interfering RNA (siRNA) inhibits mitochondrial translation and thereby causes a mitonuclear protein imbalance. Therefore, we decided to examine the effects of MRPS5 loss and the role of these processes on cardiomyocyte proliferation. METHODS: We deleted a single allele of MRPS5 in mice and used left anterior descending coronary artery ligation surgery to induce myocardial damage in these animals. We examined cardiomyocyte proliferation and cardiac regeneration both in vivo and in vitro. Doxycycline treatment was used to inhibit protein translation. Heart function in mice was assessed by echocardiography. Quantitative real-time polymerase chain reaction and RNA sequencing were used to assess changes in transcription and chromatin immunoprecipitation (ChIP) and BioChIP were used to assess chromatin effects. Protein levels were assessed by Western blotting and cell proliferation or death by histology and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assays. Adeno-associated virus was used to overexpress genes. The luciferase reporter assay was used to assess promoter activity. Mitochondrial oxygen consumption rate, ATP levels, and reactive oxygen species were also analyzed. RESULTS: We determined that deletion of a single allele of MRPS5 in mice results in elevated cardiomyocyte proliferation and cardiac regeneration; this observation correlates with improved cardiac function after induction of myocardial infarction. We identified ATF4 (activating transcription factor 4) as a key regulator of the mitochondrial stress response in cardiomyocytes from Mrps5+/- mice; furthermore, ATF4 regulates Knl1 (kinetochore scaffold 1) leading to an increase in cytokinesis during cardiomyocyte proliferation. The increased cardiomyocyte proliferation observed in Mrps5+/- mice was attenuated when one allele of Atf4 was deleted genetically (Mrps5+/-/Atf4+/-), resulting in the loss in the capacity for cardiac regeneration. Either MRPS5 inhibition (or as we also demonstrate, doxycycline treatment) activate a conserved regulatory mechanism that increases the proliferation of human induced pluripotent stem cell-derived cardiomyocytes. CONCLUSIONS: These data highlight a critical role for MRPS5/ATF4 in cardiomyocytes and an exciting new avenue of study for therapies to treat myocardial injury.

miR-124-3p improves mitochondrial function of renal tubular epithelial cells in db/db mice.

Diabetic kidney disease (DKD) is one of the most serious complications of diabetes mellitus (DM) and the main cause of end-stage renal failure. However, the pathogenesis of DKD is complicated. In this study, we found that miR-124-3p plays a key role in regulating renal mitochondrial function and explored its possible mechanism in DKD progression by performing a series of in vitro and in vivo experiments. Decreased expression of miR-124-3p was found in db/db mice compared to db/m mice. Moreover, miR-124-3p down-regulated FOXQ1 by targeting FOXQ1 mRNA 3'-UTR in NRK-52E cells. Also, an increase in FOXQ1 and down-regulation of Sirt4 were found in db/db mouse kidney and renal tubular epithelial cells cultured with high glucose and high lipid. Overexpression of FOXQ1 could further down-regulate the expression of Sirt4 and aggravate the damage of mitochondria. Conversely, the knockdown of the FOXQ1 gene induced Sirt4 expression and partially restored mitochondrial function. To verify the effects of miR-124-3p on Sirt4 and mitochondria, we found that miR-124-3p mimics could up-regulate Sirt4 and inhibit ROS production and MitoSOX, thus restoring the number and morphology of mitochondria. These results showed that under high-glucose and high-lipid conditions, the down-regulation of miR-124-3p induces FOXQ1 in renal tubular epithelial cells, which in turn suppresses Sirt4 and leads to mitochondrial dysfunction, promoting the development of DKD.

New Loss-of-Function Mutations in PCSK9 Reduce Plasma LDL Cholesterol.

BACKGROUND: Lower plasma levels of LDL (low-density lipoprotein) cholesterol (LDL-C) can reduce the risk of atherosclerotic cardiovascular disease. The loss-of-function mutations in PCSK9 (proprotein convertase subtilisin/kexin type 9) have been known to associate with low LDL-C in many human populations. PCSK9 genetic variants in Chinese Uyghurs who are at high risk of atherosclerotic cardiovascular disease due to their dietary habits have not been reported. METHODS: The study involved the whole-exome and target sequencing of college students from Uyghur and other ethnic groups in Xinjiang, China, for the association of PCSK9 loss-of-function mutations with low plasma levels of LDL-C. The mechanisms by which the identified mutations affect the function of PCSK9 were investigated in cultured cells using biochemical and cell assays. The causal effects of the identified PCSK9 mutations on LDL-C levels were verified in mice injected with adeno-associated virus expressing different forms of PCSK9 and fed a high-cholesterol diet. RESULTS: We identified 2 PCSK9 mutations-E144K and C378W-in Chinese Uyghurs with low plasma levels of LDL-C. The E144K and C378W mutations impaired the maturation and secretion of the PCSK9 protein, respectively. Adeno-associated virus-mediated expression of E144K and C378W mutants in Pcsk9 KO (knockout) mice fed a high-cholesterol diet also hampered PCSK9 secretion into the serum, resulting in elevated levels of LDL receptor in the liver and reduced levels of LDL-C in the serum. CONCLUSIONS: Our study shows that E144K and C378W are PCSK9 loss-of-function mutations causing low LDL-C levels in mice and probably in humans as well.

Alpha lipoamide inhibits diabetic kidney fibrosis via improving mitochondrial function and regulating RXRα expression and activation.

Previous studies have shown mitochondrial dysfunction in various acute kidney injuries and chronic kidney diseases. Lipoic acid exerts potent effects on oxidant stress and modulation of mitochondrial function in damaged organ. In this study we investigated whether alpha lipoamide (ALM), a derivative of lipoic acid, exerted a renal protective effect in a type 2 diabetes mellitus mouse model. 9-week-old db/db mice were treated with ALM (50 mg·kg-1·d-1, i.g) for 8 weeks. We showed that ALM administration did not affect blood glucose levels in db/db mice, but restored renal function and significantly improved fibrosis of kidneys. We demonstrated that ALM administration significantly ameliorated mitochondrial dysfunction and tubulointerstitial fibrotic lesions, along with increased expression of CDX2 and CFTR and decreased expression of ß-catenin and Snail in kidneys of db/db mice. Similar protective effects were observed in rat renal tubular epithelial cell line NRK-52E cultured in high-glucose medium following treatment with ALM (200 µM). The protective mechanisms of ALM in diabetic kidney disease (DKD) were further explored: Autodock Vina software predicted that ALM could activate RXRα protein by forming stable hydrogen bonds. PROMO Database predicted that RXRα could bind the promoter sequences of CDX2 gene. Knockdown of RXRα expression in NRK-52E cells under normal glucose condition suppressed CDX2 expression and promoted phenotypic changes in renal tubular epithelial cells. However, RXRα overexpression increased CDX2 expression which in turn inhibited high glucose-mediated renal tubular epithelial cell injury. Therefore, we reveal the protective effect of ALM on DKD and its possible potential targets: ALM ameliorates mitochondrial dysfunction and regulates the CDX2/CFTR/ß-catenin signaling axis through upregulation and activation of RXRα. Schematic figure illustrating that ALM alleviates diabetic kidney disease by improving mitochondrial function and upregulation and activation of RXRα, which in turn upregulated CDX2 to exert an inhibitory effect on ß-catenin activation and nuclear translocation. RTEC renal tubular epithelial cell. ROS Reactive oxygen species. RXRα Retinoid X receptor-α. Mfn1 Mitofusin 1. Drp1 dynamic-related protein 1. MDA malondialdehyde. 4-HNE 4-hydroxynonenal. T-SOD Total-superoxide dismutase. CDX2 Caudal-type homeobox transcription factor 2. CFTR Cystic fibrosis transmembrane conductance regulator. EMT epithelial mesenchymal transition. α-SMA Alpha-smooth muscle actin. ECM extracellular matrix. DKD diabetic kidney disease. Schematic figure was drawn by Figdraw ( www.figdraw.com ).

EI24 alleviates renal interstitial fibrosis through inhibition of epithelial-mesenchymal transition and fibroblast activation.

Etoposide-induced 2.4 (EI24) exerts tumor suppressor activity through participating in cell apoptosis, autophagy, and inflammation. However, its role in renal diseases has not been elucidated. This study showed that the EI24 level decreased gradually in the kidneys of mice with unilateral ureteral obstruction (UUO) and in another fibrosis model induced by diabetic kidney disease. The overexpression of EI24 was used to investigate the possible role both in vivo and in vitro. The overexpression 1 day after UUO through tail vein injection alleviated the progression of renal interstitial fibrosis (RIF). EI24 inhibited epithelial-mesenchymal transition, excessive deposition of the extracellular matrix, and activation of fibroblasts. Furthermore, administration of EI24-overexpressing plasmids restrained the phosphorylation of nuclear factor-κB (NF-κB) and c-Jun kinase (JNK) through regulating the proteasome-dependent degradation of TRAF2, and then, inhibited the expression of downstream inflammation-associated cytokines (interleukin-6, tumor necrosis factor-α, and monocyte chemotactic protein-1) and infiltration of macrophages and neutrophils in mouse kidney after UUO. In conclusion, the data indicated that EI24, a novel anti-fibrosis regulator, was important in the progression of RIF.

SAA1 is transcriptionally activated by STAT3 and accelerates renal interstitial fibrosis by inducing endoplasmic reticulum stress.

Renal interstitial fibrosis (RIF) is the common irreversible pathway by which chronic kidney disease (CKD) progresses to the end stage. The transforming growth factor-ß (TGF-ß)/signal transducer and activator of transcription 3 (STAT3) signaling pathway is a common factor leading to inflammation-mediated RIF, but its downstream regulatory mechanism is still unclear. Bioinformatics analysis predicted that serum amyloid A protein 1 (SAA1) was one of the target genes for transcriptional activation of STAT3 signaling. As an acute phase reaction protein, SAA1 plays an important role in many inflammatory reactions, and research has suggested that SAA1 is significantly elevated in the serum of patients with CKD. In this research, multiple experiments were performed to investigate the role of SAA1 in the process of RIF. SAA1 was abnormally highly expressed in kidney tissue from individuals who underwent unilateral ureteral obstruction (UUO) and TGF-ß-induced HK2 cells, and the abnormal expression was directly related to the transcriptional activation of STAT3. Additionally, SAA1 can directly target and bind valosin-containing protein (VCP)-interacting membrane selenoprotein (VIMP) to inhibit the function of the Derlin-1/VCP/VIMP complex, preventing the transportation and degradation of the misfolded protein, resulting in endoplasmic reticulum (ER) stress characterized by an increase in glucose-regulated protein 78 (GRP78) levels and ultimately promoting the occurrence and development of RIF.

Identification of YAP1 as a novel downstream effector of the FGF2/STAT3 pathway in the pathogenesis of renal tubulointerstitial fibrosis.

Chronic kidney disease is a global health problem and eventually develops into an end-stage renal disease (ESRD). It is now widely believed that renal tubulointerstitial fibrosis (TIF) plays an important role in the progression of ESRD. Renal tubular epithelial-mesenchymal transition (EMT) is an important cause of TIF. Studies have shown that FGF2 is highly expressed in fibrotic renal tissue, although the mechanism remains unclear. We found that FGF2 can activate STAT3 and induce EMT in renal tubular epithelial cells. STAT3, an important transcription factor, was predicted by the JASPAR biological database to bind to the promoter region of YAP1. In this study, STAT3 was shown to promote the expression of the downstream target gene YAP1 through transcription, promote EMT of renal tubular epithelial cells, and mediate the occurrence of renal TIF. This study provides a theoretical basis for the involvement of the FGF2/STAT3/YAP1 signaling pathway in the process of renal interstitial fibrosis and provides a potential target for the treatment of renal fibrosis.

YAP1 Overexpression Is Associated with Kidney Dysfunction in Lupus Nephritis.

OBJECTIVE: The goal of the present study was to determine the expression of yes-associated protein 1 (YAP1) in renal tissues of mice with lupus nephritis (LN) and elucidate its role in the progression of renal fibrosis. METHODS: C57BL/6 mice and MRL/lpr mice were selected for experimental comparison. Mouse kidney tissues were removed and sectioned for hematoxylin and eosin staining, Masson's trichome staining, Sirius staining, and immunohistochemistry. The mRNA and protein levels of YAP1 in mouse kidney tissues were detected, and the correlation between YAP1 and fibronectin (FN) mRNA levels was analyzed. Mouse renal epithelial cells were used for in vitro experiments. After transfection and stimulation, the cells were divided into 4 groups, namely the C57BL/6 serum group (group 1), the MRL/lpr serum group (group 2), the MRL/lpr serum + siRNA-negative control group (group 3), and the MRL/lpr serum + siRNA-YAP1 group (group 4). Epithelial-mesenchymal transition (EMT) markers in each group were detected by Western blotting and immunofluorescence staining. Serum creatinine, blood urea nitrogen, and urinary protein levels were detected and assessed for their correlation with YAP1 mRNA levels by Spearman's analysis. RESULTS: Compared to C57BL/6 mice, MRL/lpr mice exhibited obvious changes in fibrosis in renal tissues. In addition, YAP1 expression was significantly higher in the renal tissues of MRL/lpr mice than in those of C57BL/6 mice, and YAP1 mRNA levels were positively correlated with those of FN. YAP1 silencing in lupus serum-stimulated cells could effectively relieve serum-induced EMT. Finally, we observed that YAP1 mRNA levels in mouse kidney tissue were significantly and positively correlated with the degree of renal function injury. CONCLUSION: YAP1 expression in the kidney tissues of LN mice was higher than that observed in normal mice, indicating that YAP1 may play an important role in the occurrence and development of LN.

IDOL G51S Variant Is Associated With High Blood Cholesterol and Increases Low-Density Lipoprotein Receptor Degradation.

OBJECTIVE: A high level of LDL-C (low-density lipoprotein cholesterol) is a major risk factor for cardiovascular disease. The E3 ubiquitin ligase named IDOL (inducible degrader of the LDLR [LDL receptor]; also known as MYLIP [myosin regulatory light chain interacting protein]) mediates degradation of LDLR through ubiquitinating its C-terminal tail. But the expression profile of IDOL differs greatly in the livers of mice and humans. Whether IDOL is able to regulate LDL-C levels in humans remains to be determined. Approach and Results: By using whole-exome sequencing, we identified a nonsynonymous variant rs149696224 in the IDOL gene that causes a G51S (Gly-to-Ser substitution at the amino acid site 51) from a Chinese Uygur family. Large cohort analysis revealed IDOL G51S carriers (+/G51S) displayed significantly higher LDL-C levels. Mechanistically, the G51S mutation stabilized IDOL protein by inhibiting its dimerization and preventing self-ubiquitination and subsequent proteasomal degradation. IDOL(G51S) exhibited a stronger ability to promote ubiquitination and degradation of LDLR. Adeno-associated virus-mediated expression of IDOL(G51S) in mouse liver decreased hepatic LDLR and increased serum levels of LDL-C, total cholesterol, and triglyceride. CONCLUSIONS: Our study demonstrates that IDOL(G51S) is a gain-of-function variant responsible for high LDL-C in both humans and mice. These results suggest that IDOL is a key player regulating cholesterol level in humans.

Alleviation of endoplasmic reticulum stress protects against cisplatin-induced ovarian damage.

BACKGROUND: Cisplatin (CDDP), a widely used chemotherapeutic agent, can induce excessive granulosa cell apoptosis, follicle loss and even premature ovarian insufficiency (POI). However, the mechanism remains elusive, although some studies have indicated the involvement of endoplasmic reticulum stress (ERS). The aim of our study was to investigate the possible mechanism ERS in CDDP-induced granulosa cell apoptosis and follicle loss. METHODS: A POI mouse model was generated by CDDP. The ovaries samples were collected and processed for isobaric tags for relative and absolute quantification analysis (iTRAQ) to screen out our interested proteins of HSPA5 and HSP90AB1, and the decline in their expression were verified by a real-time quantitative PCR and a western blotting assay. In vitro, human granulosa cells, KGN and COV434 cells were transfected with siRNA targeting HSPA5 and HSP90AB1 and then treated with CDDP, or treated with CDDP with/without CDDP+ 4-phenylbutyric acid (4-PBA) and 3-methyladenine (3-MA). The levels of ERS, autophagy and apoptosis were evaluated by western blotting, DALGreen staining and flow cytometry. In vivo, ovaries from mice that received intraperitoneal injections of saline, CDDP, CDDP+ 4-PBA and CDDP+ 3-MA were assayed by immunofluorescence, hematoxylin and eosin (H&E) staining for follicle counting, and terminal-deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining for cell apoptosis assay. The plasma hormone levels were measured by an enzyme-linked immunosorbent assay (ELISA) kit. RESULTS: We have clarified the relationships between ERS, autophagy, and apoptosis in CDDP-induced granulosa cell apoptosis, both in vitro and in vivo. Alleviating ERS by inhibiting HSPA5 and HSP90AB1 attenuated CDDP-induced autophagy and apoptosis. 4-PBA treatment significantly attenuated CDDP-induced cell autophagy and apoptosis in cultured KGN and COV434 cells. However, inhibiting cell autophagy with 3-MA negligibly restored the CDDP-induced changes in ERS and apoptosis. In vivo experiments also demonstrated that treatment with 4-PBA, but not 3-MA, prevented CDDP-induced ovarian damage and hormone dysregulation. CONCLUSIONS: CDDP-induced ERS could promote autophagy and apoptosis in granulosa cells, causing excessive follicle loss and endocrine disorders. Alleviation of ERS with 4-PBA, but not of autophagy with 3-MA, protect against CDDP-induced granulosa cell apoptosis and ovarian damage. Thus, 4-PBA can be used to protect the ovary during chemotherapy in women.

TSC1 deletion in fibroblasts alleviates lipopolysaccharide-induced acute kidney injury.

Mechanistic target of rapamycin complex 1 (mTORC1) signaling is active in inflammation, but its involvement in septic acute kidney injury (AKI) has not been shown. mTORC1 activation (p-S6) in renal fibroblasts was increased in a mouse AKI model induced by 1.5 mg/kg lipopolysaccharide (LPS). Deletion of tuberous sclerosis complex 1 (TSC1), an mTORC1 negative regulator, in fibroblasts (Fibro-TSC1-/-) inhibited the elevation of serum creatinine and blood urea nitrogen in AKI compared with that in TSC1fl/fl control mice. Endothelin-1 (EDN1) and phospho-Jun-amino-terminal kinase (p-JNK) were up-regulated in Fibro-TSC1-/- renal fibroblasts after LPS challenge. Rapamycin, an mTORC1 inhibitor, and bosentan, an EDN1 antagonist, eliminated the difference in renal function between TSC1fl/fl and Fibro-TSC1-/- mice after LPS injection. Rapamycin restored LPS-induced up-regulation of EDN1, endothelin converting enzyme-1 (ECE1), and p-JNK in TSC1-knockdown mouse embryonic fibroblasts (MEFs). SP600125, a Jun-amino-terminal kinase (JNK) inhibitor, attenuated LPS-induced enhancement of EDN1 and ECE1 in TSC1-knockdown MEFs without a change in phospho-S6 ribosomal protein (p-S6) level. The results indicate that mTORC1-JNK-dependent up-regulation of ECE1 elevated EDN1 in TSC1-knockout renal fibroblasts and contributed to improvement of renal function in Fibro-TSC1-/- mice with LPS-induced AKI. Renal fibroblast mTORC1 plays an important role in septic AKI.

Characterization of process-related impurities including forced degradation products of alogliptin benzoate and the development of the corresponding reversed-phase high-performance liquid chromatography method.

The characterization of process-related impurities and forced degradants of alogliptin benzoate (Alb) in bulk drugs and a stability-indicating HPLC method for the separation and quantification of all the impurities were investigated. Alb was found to be unstable under acid and alkali stress conditions and two major degradation products (Imp-F and Imp-G) were observed. The optimum separation was achieved on Kromasil C18 (250 × 4.6 mm, 5 µm) using 0.1% perchloric acid (pH adjusted to 3.0 with triethylamine) and acetonitrile as a mobile phase in gradient mode. The proposed method was found to be stability indicating, precise, linear (0.10-75.0 µg/mL), accurate, sensitive, and robust for the quantitation of Alb and its process-related substances and degradation products. The structures of 11 impurities were characterized and confirmed by NMR spectroscopy, MS, and IR spectroscopy, and the most probable formation mechanisms of all impurities were proposed according to the synthesis route.

Structural elucidation of potential impurities in Azilsartan bulk drug by HPLC.

During the synthesis of Azilsartan (AZS), it was speculated that 15 potential impurities would arise. This study investigated the possible mechanism for the formation of 14 of them, and their structures were characterized and confirmed by IR, NMR, and MS techniques. In addition, an efficient chromatographic method was developed to separate and quantify these impurities, using an Inertsil ODS-3 column (250 x 4.6 mm, 5 pm) in gradient mode with a mixture of acetonitrile and the potassium dihydrogen orthophosphate buffer (10 mM, pH adjusted to 3.0 with phosphoric acid). The HPLC method was validated for specificity, precision, accuracy, and sensitivity. LOQ of impurities were in the range of 1.04-2.20 ng. Correlation coefficient values of linearity were >0.9996 for AZS and its impurities. The mean recoveries of all impurities in AZS were between 93.0 and 109.7%. Thus, the validated HPLC method is suitable for the separation and quantification of all potential impurities in AZS.

Vaccines reduced hospital length of stay and fraction of inspired oxygen of COVID-19 patients: A retrospective cohort study.

Few studies have focused on the evaluation of vaccine effectiveness (VE) in mainland China. This study was to characterize the VE including the frequent symptoms, laboratory indices, along with endotracheal intubation, hospital length of stay (LoS), and survival status. This retrospective cohort study included patients with COVID-19 admitted to our hospital. Statistical comparisons of continuous variables were carried out with an independent Student's t-test or Mann-Whitney U test. For categorical variables, the Chi-square test and Fisher exact test were used. Multivariable regression analysis was performed to adjust the confounding factors such as age, gender, body mass index (BMI), residential area, smoking status, the Charlson comorbidity index (CCI) score, followed by investigating the effects of vaccination on critical ill prevention, reduced mortality and endotracheal intubation, LoS and inspired oxygen. This study included 549 hospitalized patients with COVID-19, including 222 (40.43 %) vaccinated participants and 327 (59.57 %) unvaccinated counterparts. There was no obvious difference between the two groups in typical clinical symptoms of COVID-19, clinical laboratory results and mortality. Multivariable analysis showed that COVID-19 vaccine obviously reduced LoS by 1.2 days (lnLoS = -0.14, 95 %CI[-0.24,-0.04]; P = 0.005) and decreased fraction of inspired oxygen by 40 % (OR: 0.60; 95 %CI[0.40,0.90]; P = 0.013) after adjusting age, gender, BMI, residential area, smoking status and CCI score. In contrast, vaccination induced reduction in the critically ill, mortality, and endotracheal intubation compared with the unvaccinated counterparts, but with no statistical differences. Vaccinated patients hospitalized with COVID-19 have a reduced LoS and fraction of inspired oxygen compared to unvaccinated cases in China.

Orchid2024: A cultivar-level dataset and methodology for fine-grained classification of Chinese Cymbidium Orchids.

BACKGROUND: Chinese Cymbidium orchids, cherished for their deep-rooted cultural significance and significant economic value in China, have spawned a rich tapestry of cultivars. However, these orchid cultivars are facing challenges from insufficient cultivation practices and antiquated techniques, including cultivar misclassification, complex identification, and the proliferation of counterfeit products. Current commercial techniques and academic research primarily emphasize species identification of orchids, rather than delving into that of orchid cultivars within species. RESULTS: To bridge this gap, the authors dedicated over a year to collecting a cultivar image dataset for Chinese Cymbidium orchids named Orchid2024. This dataset contains over 150,000 images spanning 1,275 different categories, involving visits to 20 cities across 12 provincial administrative regions in China to gather pertinent data. Subsequently, we introduced various visual parameter-efficient fine-tuning (PEFT) methods to expedite model development, achieving the highest top-1 accuracy of 86.14% and top-5 accuracy of 95.44%. CONCLUSION: Experimental results demonstrate the complexity of the dataset while highlighting the considerable promise of PEFT methods within flower image classification. We believe that our work not only provides a practical tool for orchid researchers, growers and market participants, but also provides a unique and valuable resource for further exploring fine-grained image classification tasks. The dataset and code are available at https://github.com/pengyingshu/Orchid2024 .

Analysis of age-specified and genotype distribution of HPV multiple infections in the Chinese population.

Multiple infections are a key component of HPV pathogenesis and have a direct impact on how an infection turns out. It's crucial to look at the associations between HPV multiple infections and both age and HPV genotypes in the Chinese population, searching for the causative factors of multiple infections with a view to providing new ideas for the treatment and prevention of multiple infections. In this study, we retrospectively analyzed the data of HPV infections among outpatients from the 2019 year to the 2021 year of Shandong Maternal and Child Health Hospital. Analyzed the correlation between HPV multiple infections and age using logistic regression. Differences in the percentage of multiple infections between age groups were compared using the chi-square test. The chi-square test compared the differences in the distribution of 15 common HPV genotypes in mono- versus multiple infections. A two-dimensional matrix presented the frequency of HPV genotype combinations. Logistics regression analysis showed that age was significantly associated with the occurrence of multiple infections, with a dominance ratio OR 1.026 (95% CI 1.02-1.04). Interestingly, the proportion of HPV multiple infections among HPV-positive individuals increases with age in people older than 30 years of age. The chi-square test showed there was a difference in the distribution of HPV genotypes between multiple infections and mono- HPV infection (χ2 = 76.4; p = 0.000), a difference in the composition of HPV genotypes for dual versus single infections (χ2 = 90.6; p = 0.000) and a difference in HPV genotypes for triple versus single infections (χ2 = 56.7; p = 0.000). A 2 × 2 matrix showed that the combination of HPV52/HPV58 (30; 6.4%) was the combination of the highest frequency of infection for dual infections; The HPV52/HPV58 (21; 4.8%) combination was the highest frequency of HPV triple infection combination. HPV multiple infections were positively correlated with age; increasing age was positively correlated with the proportion of HPV multiple infections in the total infected population; the distribution of the 15 common genotypes of HPV differed between multiple infections and single infections; and HPV52:58 was a common type of infection combination in the Shandong population.

Correlation Between MicroRNA by Extracellular Vesicle Mediated and Antiviral Effects of Interferon Omega in Feline Peripheral Blood.

Feline interferon omega (IFN-ω) has been proven to have high antiviral activity; however, its in-depth antiviral effects remain unknown. Extracellular vesicles (EVs) have been demonstrated to participate in the regulation of the immune response pathway for the body through various active substances, especially through the microRNA (miRNA) carried by them. In this study, we isolated EVs from feline peripheral blood by differential centrifugation, and further found that the content of IFN-ω in EVs increased continuously within 24 h after IFN-ω treatment, and a large number of miRNAs were significantly downregulated in EVs within 12 h after IFN-ω treatment. These significantly differentially expressed miRNAs were important for regulating changes in antiviral cytokines. This study reveals for the first time the correlation between EVs-mediated miRNA in feline peripheral blood and IFN-ω on antiviral immune response, which may provide strong data support for the development of novel antiviral nanomedicine and the research of the antiviral effects of IFN-ω.