Antidementia medication acetylcholinesterase inhibitors have therapeutic benefits on osteoporotic bone by attenuating osteoclastogenesis and bone resorption.

This study was designed to determine whether the use of acetylcholinesterase inhibitors (AChEIs), a group of drugs that stimulate acetylcholine receptors and are used to treat Alzheimer's disease (AD), is associated with osteoporosis protection and inhibition of osteoclast differentiation and function. Firstly, we examined the effects of AChEIs on RANKL-induced osteoclast differentiation and function with osteoclastogenesis and bone resorption assays. Next, we investigated the impacts of AChEIs on RANKL-induced nuclear factor κB and NFATc1 activation and expression of osteoclast marker proteins CA-2, CTSK and NFATc1, and dissected the MAPK signaling in osteoclasts in vitro by using luciferase assay and Western blot. Finally, we assessed the in vivo efficacy of AChEIs using an ovariectomy-induced osteoporosis mouse model, which was analyzed using microcomputed tomography, in vivo osteoclast and osteoblast parameters were assessed using histomorphometry. We found that Donepezil and Rivastigmine inhibited RANKL-induced osteoclastogenesis and impaired osteoclastic bone resorption. Moreover, AChEIs reduced the RANKL-induced transcription of Nfatc1, and expression of osteoclast marker genes to varying degrees (mainly Donepezil and Rivastigmine but not Galantamine). Furthermore, AChEIs variably inhibited RANKL-induced MAPK signaling accompanied by downregulation of AChE transcription. Finally, AChEIs protected against OVX-induced bone loss mainly by inhibiting osteoclast activity. Taken together, AChEIs (mainly Donepezil and Rivastigmine) exerted a positive effect on bone protection by inhibiting osteoclast function through MAPK and NFATc1 signaling pathways through downregulating AChE. Our findings have important clinical implications that elderly patients with dementia who are at risk of developing osteoporosis may potentially benefit from therapy with the AChEI drugs. Our study may influence drug choice in those patients with both AD and osteoporosis.

Elevated Insulin-Like Growth Factor-1-Induced Female Rats Perpetuate the Polycystic Ovary Syndrome Phenotype: Pathological Mechanism of Insulin-Like Growth Factor-1 in Polycystic Ovary Syndrome.

OBJECTIVES: This study was designated to establish a polycystic ovary syndrome (PCOS) rat model with recombinant human insulin-like growth factor-1 (RH-IGF-1). We made assessment on the characteristics of hyperinsulinemia and hyperandrogenism in the rat model. DESIGN: This study performed the characteristics of PCOS upon RH-IGF-1 injection and evaluated the disease process of PCOS syndrome caused by the insulin-resistant pathological condition of IGF-1 based on the comparative study of in vivo test. SETTING: The experiment was conducted in the experimental research center of Yinzhou NO.2 hospital, Ningbo, Zhejiang Province, China. MATERIALS AND METHODS: Thirty-four female Sprague Dawley immature rats aged 21 days were randomly divided into two groups. Those treated with RH-IGF-1 2 mg/100 g daily were in RH-IGF-1 group (n = 20), and those with 0.9% sodium chloride 0.2 mL/100 g daily were in the saline group (n = 14). The experiment was carried out in two stages. In stage I, rats were anesthetized upon the first estrous cycle in the saline group with tissue and blood samples collected (n = 7), and rats in the RH-IGF-1-treated group were anesthetized on the 5th day after vaginal opening (VO) (n = 10). In stage II, rats in the saline group were anesthetized after three complete cycles (n = 7), meanwhile, while on the 15th day after VO (n = 10) for those in the RH-IGF-1 group. RESULTS: We have found that compared with the control group, rats injected with RH-IGF-1 expressed an early VO, disordered estrous cycle, polycystic ovaries, and significantly increased ovarian weight/body weight ratio. And from the perspective of hormone secretion, their androgen increased significantly and the insulin resistance index also elevated distinctly, possessing main characteristics similar to PCOS. LIMITATIONS: In this study, we were limited by the inability to examine IGF-1 in hypothalamus. IGF-1 in hypothalamus and in vitro experiments would be taken into consideration for further study in the future. CONCLUSIONS: These findings suggest that IGF-1 may be a key factor in the pathogenesis of PCOS, and the increase of androgen may be the pathological result, not the cause of PCOS.

LncRNA HCG11 represses ovarian cancer cell growth via AKT signaling pathway.

AIM: Ovarian cancer is a main contributor of cancer-relevant deaths among women worldwide due to high incidence and mortality. Mounting evidence has unveiled that lncRNAs play critical roles in malignancies, including ovarian cancer. Although the tumor suppressor function of HCG11 in prostate cancer and glioma has been proved, investigations on HCG11 role in ovarian cancer are still scarce. METHODS: Gene or protein expression was quantified by RT-qPCR or western blot. HCG11 effects on ovarian cancer were assessed by functional assays. Bioinformatics analysis and mechanism experiments were implemented to identify the association among HCG11, miR-1270, and PTEN. RESULTS: HCG11 was weakly expressed in ovarian cancer and functioned as a tumor suppressor in ovarian cancer by retarding cell proliferation, migration, and EMT. Besides, HCG11 could bind to miR-1270 and PTEN was a target gene of miR-1270. Mechanically, HCG11 competitively bound with miR-1270 to upregulate PTEN. From rescue experiments, HCG11 impeded AKT/mTOR pathway to retard ovarian cancer cell growth by miR-1270/PTEN. CONCLUSIONS: HCG11 was a tumor suppressor in ovarian cancer cells and additionally, HCG11 regulated AKT/mTOR pathway to hinder ovarian cancer cell growth via modulating miR-1270/PTEN, indicating that HCG11 may represent a promising target for effective treatment of ovarian cancer patients.

MiR-182 inhibits kidney fibrosis by regulating transforming growth factor ß1/Smad3 pathway in autosomal dominant polycystic kidney disease.

The aim of the present study was to investigate the molecular mechanism of miR-182 in kidney fibrosis in polycystic kidney disease (PKD). We measured the expression of miR-182 in kidney tissue of autosomal dominant PKD. Additionally, we investigated the relationship between miR-182 and fibrotic protein by transfecting miR-182 mimics and miR-182 inhibitor into polycystic kidney cyst-lined epithelial cells, respectively. Furthermore, we observed the interaction between transforming growth factor ß1 (TGF-ß1) and miR-182 and fibrinogen factors of cyst-lined epithelial cells after TGF-ß1 intervention, and measured the expression of Smad2 and Smad3 protein. Results are presented as follows: (a) MiR-182 was positively correlated with fibrosis of cyst-lined epithelial cells; (b) TGF-ß1 could induce fibrosis of cyst-lined epithelial cells; (c) the expression of miR-182 had a remarkably impact on the fibrosis induced by TGF-ß1, but had little effect on the expression of TGF-ß1; (d) the expression of Smad3 protein in TGF-ß1 induce-cyst-lined epithelial cells was increased. TGF-ß1 and miR-182 promoting the fibrosis of polycystic kidney cyst-lined epithelial cells may be mediated by the TGF-ß1/Smad3 signaling pathway, of which Smad3 was an important regulator.

Spatial-Resolution Cell Type Proteome Profiling of Cancer Tissue by Fully Integrated Proteomics Technology.

Increasing attention has been focused on cell type proteome profiling for understanding the heterogeneous multicellular microenvironment in tissue samples. However, current cell type proteome profiling methods need large amounts of starting materials which preclude their application to clinical tumor specimens with limited access. Here, by seamlessly combining laser capture microdissection and integrated proteomics sample preparation technology SISPROT, specific cell types in tumor samples could be precisely dissected with single cell resolution and processed for high-sensitivity proteome profiling. Sample loss and contamination due to the multiple transfer steps are significantly reduced by the full integration and noncontact design. H&E staining dyes which are necessary for cell type investigation could be selectively removed by the unique two-stage design of the spintip device. This easy-to-use proteome profiling technology achieved high sensitivity with the identification of more than 500 proteins from only 0.1 mm2 and 10 µm thickness colon cancer tissue section. The first cell type proteome profiling of four cell types from one colon tumor and surrounding normal tissue, including cancer cells, enterocytes, lymphocytes, and smooth muscle cells, was obtained. 5271, 4691, 4876, and 2140 protein groups were identified, respectively, from tissue section of only 5 mm2 and 10 µm thickness. Furthermore, spatially resolved proteome distribution profiles of enterocytes, lymphocytes, and smooth muscle cells on the same tissue slices and across four consecutive sections with micrometer distance were successfully achieved. This fully integrated proteomics technology, termed LCM-SISPROT, is therefore promising for spatial-resolution cell type proteome profiling of tumor microenvironment with a minute amount of clinical starting materials.

TRAF3 delays cyst formation induced by NF-κB signaling.

This study aims to investigate the effects of TNF receptors associated factor 3 (TRAF3) on the signaling pathway and expression of downstream products of nuclear factor kappa B (NF-κB) in the epithelial cells of renal ducts in individuals with polycystic kidney disease (PKD). We observe the TRAF3 genic overexpression of the epithelial cells, which form a tubular branch structure, in polycystic kidneys and to explore the protective effect of TRAF3 on the cystogenesis and progression of PKD. Western blotting analysis was conducted to examine the signaling changes of NF-κB in PKD the epithelial cells and TRAF3 transgenic PKD epithelial cells. Changes in the downstream apoptosis factor and cell proliferation in PKD epithelial cells and TRAF3 transgenic PKD epithelial cells were detected. A three-dimensional matrigel culture experiment was performed to examine abnormal tubulomorphogenesis in vitro. The overexpression of TRAF3 significantly inhibited the signaling pathway of NF-κB in the PKD epithelial cells, downregulated the expression of downstream factors Bcl-2 and Bcl-xl, and significantly decreased cystic epithelial cell proliferation. Additional branch structures were observed in the PKD epithelial cells with a three-dimensional culture compared to wildtype cells. TRAF3 may likely induce apoptosis and resistance to proliferation and may be a new target to inhibit the cyst formation in PKD by regulating the NF-κB signaling pathway Bcl-2 and Bcl-xl activity. © 2017 IUBMB Life, 69(3):170-178, 2017.

Single-cell analyses reveal distinct expression patterns and roles of long non-coding RNAs during hESC differentiation into pancreatic progenitors.

BACKGROUND: Deep understanding the differentiation process of human embryonic stem cells (hESCs) is essential for developing cell-based therapeutic strategy. Substantial efforts have been made to investigate protein-coding genes, yet it remains lacking comprehensive characterization of long non-coding RNAs (lncRNAs) during this process. METHODS: hESCs were passaged every 5-6 days and had maintained stable karyotype even until the 50th generation. Pancreatic progenitor specification of in vitro differentiation from hESCs was performed and modified. The nuclei were stained with 4,6-Diamidino-2-phenylindole (DAPI). Droplet-based platform (10X Genomics) was applied to generate the single-cell RNA sequencing (scRNA-seq) data. The quality of the filtered read pairs was evaluated by using FastQC. Batch effects were removed using the size factor method. Dimension reduction and unsupervised clustering analyses were performed using Seurat R package. The Monocle 2 and MetaCell algorithms were used to order single cells on a pseudotime course and partition the scRNA-seq data into metacells, respectively. Co-expression network was constructed using WGCNA. Module- and hub-based methods were adopted to predict the functions of lncRNAs. RESULTS: A total of 77,382 cells during the differentiation process of hESCs toward pancreatic progenitors were sequenced. According to the single-cell map, the cells from different time points were authenticated to constitute a relatively homogeneous population, in which a total of 7382 lncRNAs could be detected. Through further analyzing the time course data, conserved and specific expression features of lncRNAs during hESC differentiation were revealed. Based upon pseudotime analysis, 52 pseudotime-associated lncRNAs that grouped into three distinct expression patterns were identified. We also implemented MetaCell algorithm and network-based methods to explore the functional mechanisms of these lncRNAs. Totally, 464 lncRNAs, including 49 pseudotime-associated lncRNAs were functionally annotated by either module-based or hub-based methods. Most importantly, we demonstrated that the lncRNA HOTAIRM1, which co-localized and co-expressed with several HOX genes, may play crucial role in the generation of pancreatic progenitors through regulation of exocytosis and retinoic acid receptor signaling pathway. CONCLUSIONS: Our single-cell analyses provide valuable data resources for biological researchers and novel insights into hESC differentiation processes, which will guide future endeavors to further elucidate the roles of lncRNAs.

Down-regulation of PKHD1 induces cell apoptosis through PI3K and NF-κB pathways.

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). Fibrocystin/polyductin (FPC), encoded by PKHD1, is a membrane-associated receptor-like protein. Although it is widely accepted that cystogenesis is mostly due to aberrant cell proliferation and apoptosis, it is still unclear how apoptosis is regulated. The aim of this study is to analyze the relationship among apoptosis, phosphatidylinositol 3-kinase (PI3K)/Akt and nuclear factor κB (NF-κB) in FPC knockdown kidney cells. We show that PKHD1-silenced HEK293 cells demonstrate a higher PI3K/Akt activity. Selective inhibition of PI3K/Akt using LY294002 or wortmannin in these cells increases serum starvation-induced HEK293 cell apoptosis with a concomitant decrease in cell proliferation and higher caspase-3 activity. PI3K/Akt inhibition also leads to increased NF-κB activity in these cells. We conclude that the PI3K/Akt pathway is involved in apoptotic function in PKHD1-silenced cells, and PI3K/Akt inhibition correlates with upregulation of NF-κB activity. These observations provide a potential platform for determining FPC function and therapeutic investigation of ARPKD.

Berberine protects against lipopolysaccharide-induced intestinal injury in mice via alpha 2 adrenoceptor-independent mechanisms.

AIM: To investigate the mechanisms responsible for the protective action of berberine (Ber) against gut damage in endotoxemic mice. METHODS: Male BALB/c mice were administered intragastrically with distilled water (0.1 mL/10 g), Ber (50 mg/kg) alone, yohimbine (2 mg/kg) alone, or Ber (50 mg/kg) in combination with yohimbine (2 mg/kg) for 3 d. On the third day, lipopolysaccharide (LPS, 18 mg/kg) or normal saline was intraperitoneally injected one hour after the intragastric administration. Following the treatment, intestinal injury in the ileum was histopathologically accessed; enterocyte apoptosis was examined using TUNEL method; Toll-like receptor 4 (TLR4) mRNA expression was measured using RT-PCR assay; inhibitor protein-κBα (I-κBα) phosphorylation and myeloperoxidase content were examined using Western blloting. The macrophage inflammatory protein-2 (MIP-2) production was measured using ELISA assay. RESULTS: Mice challenged with LPS caused extensive ileum injury, including a significantly increased injury score, decreased intestinal villus height, reduced gut mucosal weight and increased intestinal permeability. Furthermore, LPS significantly induced enterocyte apoptosis, increased TLR4 mRNA expression, I-κBα phosphorylation, MIP-2 production and myeloperoxidase content in the ileum. Pretreatment with Ber significantly alleviated all the alterations in the ileum in the endotoxemic mice. Pretreatment with the α2-adrenoceptor antagonist yohimbine did not block the protective action of Ber against LPS-induced intestinal injury. In addition, treatment with yohimbine alone did not prevent LPS-induced intestinal injury. CONCLUSION: Pretreatment with Ber provides significant protection against LPS-induced intestinal injury in mice, via reducing enterocyte apoptosis, inhibiting the TLR4-nuclear factor κB-MIP-2 pathway and decreasing neutrophil infiltration that are independent of α2-adrenoceptors.

Detection of HPV E6/E7 mRNA in the diagnosis of cervical cancer and precancerous lesions after kidney transplantation.

OBJECTIVE: To explore the value of human papillomavirus (HPV) E6/E7 mRNA detection in the diagnosis of cervical cancer and its precancerous lesions after kidney transplantation. METHODS: One hundred and sixty-six women who underwent cervical cancer screening after kidney transplantation were selected and received thinprep cytology test (TCT), HPV DNA and HPV E6/E7 mRNA tests. A biopsy under colposcopy was performed for those with suspicious test results. The positive rates of TCT, HPV DNA and HPV E6/E7 mRNA expressions in patients with different biopsy pathological grades, the positive rates of HPV E6/E7 mRNA in TCT and HPV DNA positive patients were compared. Besides, the relationship between the results of the three detections and the pathological results of cervical biopsy as well as the diagnostic efficacy of cervical cancer and its precancerous lesions were compared. RESULTS: Among the 166 women undergoing cervical cancer screening, 87 cases received histopathological biopsy, of which, the positive expression rates of HPV E6/E7 mRNA in the negative, cervical intraepithelial neoplasia (CIN) I, CIN II, CIN III and invasive carcinoma (ICC) patients were 51.43%, 54.55%, 66.67%, 81.82% and 100.00%, respectively. The positive expression rates of HPV E6/E7 mRNA in TCT and HPV DNA-positive patients were 47.50% and 51.96%, respectively; those rates for diagnosis of ≥CIN II were significantly greater than that of ≤CIN I (both P<0.01). Receiver operating characteristic curve revealed that the areas under the concentration-time curve of TCT, HPV DNA and HPV E6/E7 mRNA detection for cervical cancer and precancerous lesions were 0.723, 0.833, 0.929, respectively. Their sensibilities were 76.89%, 83.30% and 92.38%, and their specificities were 77.04%, 88.47% and 94.47%, respectively. CONCLUSION: HPV E6/E7 mRNA detection effectively improves the diagnostic sensitivity and specificity of cervical cancer and precancerous lesions, thereby avoiding over-examination and over-treatment.

The mitochondrial gene orfH79 plays a critical role in impairing both male gametophyte development and root growth in CMS-Honglian rice.

BACKGROUND: Cytoplasmic male sterility (CMS) has often been associated with abnormal mitochondrial open reading frames. The mitochondrial gene orfH79 is a candidate gene for causing the CMS trait in CMS-Honglian (CMS-HL) rice. However, whether the orfH79 expression can actually induce CMS in rice remains unclear. RESULTS: Western blot analysis revealed that the ORFH79 protein is mainly present in mitochondria of CMS-HL rice and is absent in the fertile line. To investigate the function of ORFH79 protein in mitochondria, this gene was fused to a mitochondrial transit peptide sequence and used to transform wild type rice, where its expression induced the gametophytic male sterile phenotype. In addition, excessive accumulation of reactive oxygen species (ROS) in the microspore, a reduced ATP/ADP ratio, decreased mitochondrial membrane potential and a lower respiration rate in the transgenic plants were found to be similar to those in CMS-HL rice. Moreover, retarded growth of primary and lateral roots accompanied by abnormal accumulation of ROS in the root tip was observed in both transgenic rice and CMS-HL rice (YTA). CONCLUSION: These results suggest that the expression of orfH79 in mitochondria impairs mitochondrial function, which affects the development of both male gametophytes and the roots of CMS-HL rice.

Regulation of cell proliferation and apoptosis through fibrocystin-prosaposin interaction.

Mutations in PKHD1 (polycystic kidney and hepatic disease gene 1) gene cause the autosomal recessive polycystic kidney disease (ARPKD). It is widely accepted that cystogenesis is owing to aberrant cell proliferation and apoptosis, increased fluid secretion, and extracellular matrix abnormality. Fibrocystin/polyductin (FPC), the encoded protein product by PKHD1, is a single transmembrane protein and believed to be a novel receptor-like molecule. FPC has been located mainly on the plasma membrane and cilium/basal body. However, its biological functions remain poorly understood. To investigate the roles of FPC in the pathogenesis of ARPKD, we searched for FPC-interacting proteins by yeast two-hybrid assay, and found a novel partner, prosaposin. Prosaposin is a glycoprotein with multiple functions. With GST pull-down assay and co-immunoprecipitation, we confirmed the interaction between FPC and prosaposin. In order to study the effects of FPC-prosaposin interaction on cell proliferation and apoptosis, we have made stable cell lines in which FPC was overexpressed or knocked down alone or in combination with prosaposin overexpression. By MTT assay, we found that FPC knockdown and prosaposin overexpression increased cell proliferation, respectively, while overexpression of FPC C-tail did the opposite. With apoptosis assay, we found that overexpression of FPC C-tail promoted cell apoptosis. However, overexpression of prosaposin significantly enhanced cell survival in FPC knockdown cells. All these findings indicated that FPC and prosaposin may play significant roles in regulation of cell proliferation and apoptosis. Taken together, we have disclosed a novel signaling pathway of FPC, which may be important for the pathogenesis of ARPKD.

Multi-Omics Integration Reveals a Competitive Endogenous RNAs Network for the Identification of Progression Biomarkers and the Stratification of Patients Diagnosed With Nephroblastoma.

Specific types of nephroblastoma (Wilms' tumor, WT) are known to associate with poor overall survival. Emerging experimental evidence has demonstrated that competitive endogenous RNA (ceRNA) networks have important roles in regulating cancer occurrence, but the roles of ceRNAs in regulating the WT progression and the patient outcomes remain unclear. Using the multi-omics data of 132 WT patients collected from TARGET database, an integration analysis pipeline was performed to construct a highly reliable ceRNA network. As results, a total of 147 nodes (116 mRNAs, 15 miRNAs, and 16 lncRNAs) were identified and used to explore the underlying mechanism for WT progression. WGCNA analysis further identified several prognostic molecules, including hsa-mir-93, LINC00087 and RP5-1086K13, that significantly associated with the overall survival rate. And, enrichment analysis verified the participation of these molecules in tumor-related pathways, such as those controlling autophagy and cadherin-mediated adhesion. Importantly, the WT patients were classified into three categories according to the ceRNA network, which significantly correlated with the overall survival. In conclusion, the ceRNA network could be a promising tool to further validate the prognostic biomarkers and categories of patients diagnosed with WT.

Expression levels of a gene signature in hiPSC associated with lung adenocarcinoma stem cells and its capability in eliciting specific antitumor immune-response in a humanized mice model.

BACKGROUND: Previous studies have reported that cancer stem cells (CSCs) play a key role in tumorigenesis, metastasis, and recurrence. CSC-based vaccination confers better protection in tumor cells. However, isolation and cultivation of CSCs are difficult. This study aimed to explore the similarities between CSCs and induced pluripotent stem cells (iPSCs). METHODS: ALDH1+ cancer stem cells were isolated from lung adenocarcinoma patients and their gene expression patterns compared with human induced pluripotent stem cells (hiPSCs). In addition, a tumor vaccine was developed using hiPSC and unmethylated cytosine-guanine (CpG). Finally, the antitumor properties of the vaccine were evaluated in a humanized mouse model. RESULTS: Preimmunization of iPSC+CpG elicited stronger antigen presentation and cytotoxic T cell response which suppressed the growth of tumors. Adoptive transfer of spleen T cells from the vaccine preimmunized mice inhibited tumor growth in unvaccinated recipients without any side effects. CONCLUSIONS: This study suggests a universal strategy for tumor therapy which simplifies future clinical procedures. Therefore, the application of hiPSC elicits tumor protective responses.

IFI16 promotes human embryonic stem cell trilineage specification through interaction with p53.

Transcriptional regulation plays an essential role in the self-renewal and differentiation of human embryonic stem cells (hESCs). However, how external signals disrupt the self-renewal regulatory network and further drive hESC differentiation remains largely unknown. Here, we found the immune regulative protein, gamma-interferon-inducible protein 16 (IFI16) was involved in the regulation of both self-renewal and differentiation gene expression during hESC trilineage specification through interaction with p53. IFI16 expression levels were upregulated through JNK activation. IFI16 knockdown delayed the downregulation of self-renewal gene expression and suppressed the upregulation of differentiation gene expression, while IFI16 overexpression accelerated trilineage specification. Furthermore, IFI16 stabilized p53-binding in the genome through IFI16-p53 interaction and differentially regulated self-renewal and differentiation gene expression. Together, our results suggest a particular role of IFI16 in differential gene expression regulation during trilineage specification of hESCs in a manner that is dependent on the genome-wide profile of p53-binding directed by IFI16-p53 interaction.

The Effects of Chloroquine and Hydroxychloroquine on ACE2-Related Coronavirus Pathology and the Cardiovascular System: An Evidence-Based Review.

The ongoing pandemic of coronavirus disease 2019 (COVID-19) caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a serious threat to global public health and there is currently no effective antiviral therapy. It has been suggested that chloroquine (CQ) and hydroxychloroquine (HCQ), which were primarily employed as prophylaxis and treatment for malaria, could be used to treat COVID-19. CQ and HCQ may be potential inhibitors of SARS-CoV-2 entry into host cells, which are mediated via the angiotensin-converting enzyme 2 (ACE2), and may also inhibit subsequent intracellular processes which lead to COVID-19, including damage to the cardiovascular (CV) system. However, paradoxically, CQ and HCQ have also been reported to cause damage to the CV system. In this review, we provide a critical examination of the published evidence. CQ and HCQ could potentially be useful drugs in the treatment of COVID-19 and other ACE2 involved virus infections, but the antiviral effects of CQ and HCQ need to be tested in more well-designed clinical randomized studies and their actions on the CV system need to be further elucidated. However, even if it were to turn out that CQ and HCQ are not useful drugs in practice, further studies of their mechanism of action could be helpful in improving our understanding of COVID-19 pathology.

Histone Deacetylase Inhibitors Reduce Cysts by Activating Autophagy in Polycystic Kidney Disease.

BACKGROUND: Histone deacetylase inhibitors (HDACi) have therapeutic effects on various models of renal diseases including autosomal dominant polycystic kidney disease (ADPKD), but the molecular mechanism is unclear. OBJECTIVES: Here, we studied the role of trichostatin A (TSA), a specific HDACi, in regulating cyst growth to test the possibility that HDACi might help manage ADPKD by enhancing autophagy. RESULTS: Autophagy protein expression was higher in cultured Pkd1 knockout (Pkd1-/-) cells, an in vitro model of cystogenesis, compared with control cells. TSA prevented cyst formation in Pkd1-/- cells. We further tested whether TSA could not reduce the size of an already established cyst after inhibition of autophagy by chloroquine in Pkd1-/- cells. In vivo, treatment with TSA significantly slowed cyst growth in Pkd1-/- mice. Moreover, TSA treatment stimulated AMPK and inactivated mTOR during cyst growth in Pkd1-/- cells and kidneys in mice. CONCLUSIONS: Our results suggest that HDACi may prevent cyst formation by activation of the AMPK pathway and autophagy. They also imply that HDACi could have therapeutic potential for ADPKD treatment.

[Construction of T7 phage display library from the anther of Honglian hybrid line of rice].

Phage display is a powerful method to study protein-protein interactions. In order to study the molecular mechanism of cytoplasmic male sterility and fertility restoration in Honglian rice, the mRNA was isolated with PolyA Tract mRNA Isolation Kit from the anther of F1 hybrid rice and the double strand (ds) cDNA was synthesized by reverse transcription. Then the directional EcoRI /Hind III linkers were ligated into the ends of ds cDNA and the ds cDNA was further digested with EcoR I and Hind, which resulted in ds cDNA with EcoR I and Hind III ends. The digested ds cDNA fragments longer than 300 bp in length were fractionated with Mini Column, then ligated into the T7 Select 10-3b vertor with EcoR I and Hind III ends. After packaging in vitro, the T7 Select 10-3b vertor was transformed into BL T5403 to construct the T7 phage display library. Analysis showed that the library contained 1.03 x 106 clones per microliter, and approximately 100% of the clones in library was recombinant. The titer of the amplied library was 2.14 x 1012 pfu/mL, and the insert length of the recombinants over 300 bp was about 97%.

PYNOD reduces microglial inflammation and consequent neurotoxicity upon lipopolysaccharides stimulation.

PYNOD, a nod-like receptors (NLR)-like protein, was indicated to inhibit NF-κB activation, caspase-1-mediated interleukin (IL)-1ß release and cell apoptosis in a dose-dependent manner. Exogenous addition of recombinant PYNOD to mixed glial cultures may suppress caspase-1 activation and IL-1ß secretion induced by Aß. However, to the best of our knowledge, there no study has focused on the immunoregulatory effects of PYNOD specifically in microglia. The present study aimed to explore the roles of PYNOD involved in the lipopolysaccharides (LPS)-induced microglial inflammation and consequent neurotoxicity. Murine microglial BV-2 cells were transfected with pEGFP-C2-PYNOD (0-5.0 µg/ml) for 24 h and incubated with or without LPS (1 µg/ml) for a further 24 h. Cell viability was determined using MTT assay and the secretion of nitric oxide (NO), IL-1ß and caspase-1 was measured using the Griess method or ELISA. Protein expression levels of NF-κB p65 and inducible nitric oxide synthase (iNOS) were detected by immunofluorescent staining and/or western blot analysis. Co-culture of BV-2 cells with human neuroblastoma cell line SK-N-SH was performed in Transwell plates and the cell viability and apoptosis (using flow cytometry) of SK-N-SH cells were determined. Results indicated that PYNOD overexpression inhibited NO secretion and iNOS protein expression induced by LPS in BV-2 cells, with no detectable cytotoxicity. PYNOD overexpression also reduced the secretion of IL-1ß and caspase-1 from BV-2 cells upon LPS stimulation. These effects were dose-dependent. Additionally, PYNOD overexpression prevented LPS-induced nuclear translocation of NF-κB p65 in BV-2 cells. The growth-inhibitory and apoptosis-promoting effects of BV-2 cells towards SK-N-SH cells were alleviated as a result of PYNOD overexpression. In conclusion, PYNOD may mitigate microglial inflammation and consequent neurotoxicity.

α2A-adrenergic blockade attenuates septic cardiomyopathy by increasing cardiac norepinephrine concentration and inhibiting cardiac endothelial activation.

Cardiomyopathy is a common complication associated with increased mortality in sepsis, but lacks specific therapy. Here, using genetic and pharmacological approaches, we explored the therapeutic effect of α2A-adrenergic receptor (AR) blockade on septic cardiomyopathy. CLP-induced septic rats were treated with BRL44408 (α2A-AR antagonist), prazosin (α1-AR antagonist) and/or reserpine. CLP-induced cardiomyopathy, indicated by reduced dP/dt and increased cardiac troponin I phosphorylation, was attenuated by BRL44408, this was associated with reduced cardiac TNF-α and endothelial VCAM-1 expression, cardiomyocyte apoptosis and related signal molecule phosphorylation. BRL44408 increased cardiac norepinephrine (NE) concentration in CLP rats. Pretreatment with reserpine that exhausts cardiac NE without affecting the circulating NE concentration or with prazosin partially abolished the cardioprotection of BRL44408 and reversed its inhibitory effects on myocardial TNF-α, apoptosis and related signal molecule phosphorylation, but not on VCAM-1 expression in septic rats. These effects of BRL44408 were confirmed by α2A-AR gene deletion in septic mice. Furthermore, α2-AR agonist not only enhanced LPS-induced TNF-α and VCAM-1 expression in cardiac endothelial cells that express α2A-AR, but also enhanced LPS-induced cardiac dysfunction in isolated rat hearts. Our data indicate that α2A-AR blockade attenuates septic cardiomyopathy by promoting cardiac NE release that activates myocardial α1-AR and suppressing cardiac endothelial activation.