Betacoronavirus ; isolation & purification ; pathogenicity ; Bile Acids and Salts ; metabolism ; Bile Ducts, Intrahepatic ; pathology ; virology ; Cell Culture Techniques ; Coronavirus Infections ; complications ; pathology ; Cytokine Release Syndrome ; etiology ; physiopathology ; Cytopathogenic Effect, Viral ; Epithelial Cells ; enzymology ; pathology ; virology ; Humans ; Hyperbilirubinemia ; etiology ; Liver ; pathology ; Organoids ; pathology ; virology ; Pandemics ; Peptidyl-Dipeptidase A ; analysis ; Pneumonia, Viral ; complications ; pathology ; Receptors, Virus ; analysis ; Serine Endopeptidases ; analysis ; Viral Load

With the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) all over the world, there is an increasing number of children with such infection. Angiotensin-converting enzyme 2 (ACE2), one of the binding sites for SARS-CoV-2 infection in humans, can bind to viral spike proteins, allowing transmembrane serine protease (TMPRSS2) to activate S-protein to trigger infection and induce the production of various inflammatory factors such as interleukin-1, interferon-l, and tumor necrosis factor. Compared with adults, children tend to have lower expression levels of ACE2 and TMPRSS2, which are presumed to be associated with milder symptoms and fewer cases in children. The article summarizes the research advances in the role of ACE2 during SARS-CoV-2 infection, in order to help understand the pathogenic mechanism of SARS-CoV-2 and provide a reference for better development of drugs and vaccines to prevent and treat coronavirus disease 2019 in children.
Angiotensin-Converting Enzyme 2/metabolism* ; COVID-19 ; Child ; Humans ; Receptors, Virus/metabolism* ; SARS-CoV-2 ; Serine Endopeptidases/metabolism*

Zfyve16 (a.k.a. endofin or endosome-associated FYVE-domain protein), a member of the FYVE-domain protein family, is involved in endosomal trafficking and in TGF-β, BMP, and EGFR signaling. The FYVE protein SARA regulates the TGF-β signaling pathway by recruiting Smad2/3 and accelerating their phosphorylation, thereby altering their susceptibility to TGF-β-mediated T cell suppression. Zfyve16 binds to Smad4 and their binding affects the formation of Smad2/3-Smad4 complex in TGF-β signaling. However, the in vivo function of Zfyve16 remains unknown. In this study, we generated a Zfyve16 knockout mouse strain (Zfyve16) and examined its hematopoietic phenotypes and hematopoietic reconstruction ability. The proportion of Tcells in the peripheral blood of Zfyve16 mice increases compared with that in wild-type mice. This finding is consistent with the role of Zfyve16 in facilitating TGF-β signaling. Unpredictably, B cell proliferation is inhibited in Zfyve16 mice. The proliferation potential of Zfyve16 B-lymphoid cells also significantly decreases in vitro. These results suggest that Zfyve16 inhibits the proliferation of T cells, possibly through the TGF-β signaling, but upregulates the proliferation of B-lymphoid cells.
Animals ; B-Lymphocytes ; metabolism ; CD4-Positive T-Lymphocytes ; metabolism ; Cell Movement ; Cell Proliferation ; genetics ; Intracellular Signaling Peptides and Proteins ; antagonists & inhibitors ; genetics ; metabolism ; Mice ; Mice, Knockout ; Serine Endopeptidases ; genetics ; metabolism ; Signal Transduction ; Smad Proteins, Receptor-Regulated ; metabolism ; Transforming Growth Factor beta ; metabolism ; Up-Regulation

OBJECTIVETo observe the expression of serine protease HtrA1 in human periodontal ligament tissue and to explore the effect of HtrA1 on the proliferation of human periodontal ligament cells (hPDLC).

METHODSSix human premolars and three human third molars(patient's ages ranging from 12 to 25, with intact root, without caries and/or periodontitis) were obtained in the Department of Maxillofacial Surgery of Wuhan University Hospital of Stomatology. Reverse transcription-PCR(RT-PCR) and immunohistochemistry analysis were applied to investigate the expression of HtrA1. Primary hPDLC were obtained by tissue-culture method in vitro. The proliferation of hPDLC was determined by methyl thiazolytetrazolium(MTT). Lentivirus-mediated over-expression and reduction of HtrA1 level was performed. An empty vector was used as negative control. On days 1, 3, 5, 7 and 9, the growth of hPDLC was characterized using cell counting kit-8(CCK-8) assay.

RESULTSRT-PCR data indicated that HtrA1 mRNA was expressed in human periodontal ligament tissue. Immunohistochemistry analysis showed HtrA1 was expressed in human periodontal ligament, mainly in the cytoplasm of hPDLC and the extracellular matrix. The MTT result suggested that the growth curve was consistent with the growth characteristics of hPDLC. The stable over-expression and knockdown cell lines was successfully established by lentivirus with more than 90% transfection efficiency. CCK-8 assay showed that HtrA1 over-expression inhibited the proliferation of hPDLC(0.897±0.060, 0.890±0.083, 1.631±0.038, 1.111±0.041, 1.110±0.189), while cell proliferation increased after down-regulation of HtrA1(0.329±0.021, 0.529±0.044, 0.973±0.056, 1.626±0.102, 2.344±0.198)(P<0.05).

CONCLUSIONSHtrA1 is expressed in human periodontal ligament tissue at both mRNA and protein levels, and may play an important role in regulating the proliferation of hPDLC.

Adolescent ; Adult ; Cell Count ; Cell Proliferation ; Cells, Cultured ; Child ; Down-Regulation ; Genetic Vectors ; High-Temperature Requirement A Serine Peptidase 1 ; Humans ; Lentivirus ; physiology ; Periodontal Ligament ; cytology ; metabolism ; RNA, Messenger ; metabolism ; Serine Endopeptidases ; genetics ; metabolism ; Transfection ; Young Adult

Animals ; Cell Differentiation ; Hepatocyte Growth Factor ; genetics ; metabolism ; Membrane Proteins ; genetics ; metabolism ; Mice ; Mouse Embryonic Stem Cells ; cytology ; metabolism ; Protein Precursors ; genetics ; metabolism ; Serine Endopeptidases ; genetics ; metabolism

OBJECTIVETo investigate the effect and molecular mechanisms of different doses of 8-hydroxy dihydroberberine (Hdber) for the treatment of hyperlipidemia in rats.

METHODSA rat model of hyperlipidemia was established by feeding rats a high-fat diet for 4 weeks in 70 rats of 80 animals, and 10 rats were randomly selected as control group. The hyperlipidemic rats were then randomly divided into the following groups: a model group (MOD); a berberine group [BBR, 156 mg/(kg day)]; Hdber groups, which were treated with different doses of Hdber [78, 39 and 19.5 mg/(kg day)]; and a simvastatin group [SIM, 4 mg/(kg day)]. The corresponding therapy was administered to the rats of each treatment via gastric tubes. Normal animals were used as a control group. The blood levels of various lipids, including total cholesterol, triglycerides, high-density lipoprotein cholesterol, low-density lipoprotein cholesterol, free fatty acid (FFA), apolipoprotein AI(Apo-AI) and apolipoprotein B (Apo-B) were examined. The protein expressions of low-density lipoprotein receptor (LDL-R), sterol regulatory element-binding protein 2 (SREBP-2), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) and proprotein convertase subtilisin/kexin type 9 (PCSK-9) in liver tissues were determined by Western blot analysis.

RESULTSCompared with the control group of rats, the model group demonstrated a deteriorated blood lipid profile and exhibited increased expression levels of PCSK-9 protein in their liver tissues (P<0.01). In addition, the high-fat diet decreased the expression levels of LDL-R, SREBP-2 and HMGCR proteins in murine liver tissues. However, the addition of berberine or Hdber reversed the blood lipid profile changes (P<0.05 or P<0.01), decreased the expression levels of PCSK-9 proteins (P<0.01), and increased the expression levels of LDL-R proteins in the hyperlipidemic rats (P<0.01). These compounds did not significantly influence the expression levels of SREBP-2 and HMGCR proteins in the hyperlipidemic rats.

CONCLUSIONSHdber is effective in the treatment of hyperlipidemia in rats. The therapeutic mechanisms of Hdber may be associated with increasing the expression of LDL-R protein and decreasing the expression of PCSK-9 protein in liver tissues.

Animals ; Apolipoprotein A-I ; blood ; Apolipoproteins B ; blood ; Berberine ; analogs & derivatives ; pharmacology ; therapeutic use ; Hydroxymethylglutaryl CoA Reductases ; metabolism ; Hyperlipidemias ; blood ; drug therapy ; Lipids ; blood ; Liver ; drug effects ; metabolism ; Male ; Proprotein Convertase 9 ; Rats, Wistar ; Receptors, LDL ; metabolism ; Serine Endopeptidases ; metabolism ; Sterol Regulatory Element Binding Protein 2 ; metabolism

The present study was aimed to investigate how the induced pluripotent stem cells (iPSCs) and bone marrow mesenchymal stem cells (BMSCs) differentiate into neuron-like cells under the induction of hippocampal microenvironments and Reelin's regulation. iPSCs or BMSCs were co-cultured with WT (wild type) or genotypic hippocampal slice and cerebral homogenate supernatant, then the stem cells' differentiation under the induction of hippocampal environment was observed by using immunofluorescence technique. In the meantime, stem cells were co-cultured with hippocampal slice and cerebral conditioned medium of reeler (Reelin deletion) mouse respectively. The results showed that both adhesive iPSCs and BMSCs on WT hippocampal slice exhibited lamination of double "C" shape with high density on granular and pyramidal layers. The stem cells could differentiate into neuron-like cells with obvious polarization on WT hippocampal slice. In pyramidal cell layer, the differentiated neuron-like cells were oriented vertically with similar shapes of pyramidal cell in vivo, and the cells within molecule layer were arranged horizontally. In addition, adhesive iPSCs and BMSCs could differentiate into Nestin positive neural stem cells and NeuN positive neurons, respectively, under WT hippocampal microenvironment. On the other hand, under induction of hippocampal microenvironment of reeler mouse, iPSCs and BMSCs differentiation could also be seen, but their lamination was in disorder, and cell polarization was irregular. Moreover, differentiation and polarization of the iPSCs and BMSCs were delayed. These results suggest both iPSCs and BMSCs can differentiate into neuron-like cells under the induction of hippocampal microenvironments. Reelin is involved in the regulation of neuronal differentiation and cell polarization. Without Reelin, the cellular lamination and polarization appear irregular, and the stem cells' differentiation is delayed.
Animals ; Cell Adhesion Molecules, Neuronal ; metabolism ; Cell Differentiation ; Cells, Cultured ; Coculture Techniques ; Culture Media, Conditioned ; Extracellular Matrix Proteins ; metabolism ; Hematopoietic Stem Cells ; cytology ; Hippocampus ; Induced Pluripotent Stem Cells ; cytology ; Mice ; Mice, Inbred C57BL ; Nerve Tissue Proteins ; metabolism ; Neural Stem Cells ; cytology ; Neurons ; cytology ; Serine Endopeptidases ; metabolism

PURPOSE: Proprotein convertase subtilisin/kexin type 9 (PCSK9) binds to the low density lipoprotein receptor (LDLR) and promotes degradation of the LDLR. Inhibition of PCSK9 either by reducing its expression or by blocking its activity results in the upregulation of the LDLR and subsequently lowers the plasma concentration of LDL-cholesterol. As a modality to inhibit PCSK9 action, we searched the chemical library for small molecules that block the binding of PCSK9 to the LDLR. MATERIALS AND METHODS: We selected 100 chemicals that bind to PCSK9 where the EGF-AB fragment of the LDLR binds via in silico screening of the ChemBridge chemical library, using the computational GOLD algorithm analysis. Effects of chemicals were evaluated using the PCSK9-LDLR binding assay, immunoblot analysis, and the LDL-cholesterol uptake assay in vitro, as well as the fast performance liquid chromatography assay for plasma lipoproteins in vivo. RESULTS: A set of chemicals were found that decreased the binding of PCSK9 to the EGF-AB fragment of the LDLR in a dose-dependent manner. They also increased the amount of the LDLR significantly and subsequently increased the uptake of fluorescence-labeled LDL in HepG2 cells. Additionally, one particular molecule lowered the plasma concentration of total cholesterol and LDL-cholesterol significantly in wild-type mice, while such an effect was not observed in Pcsk9 knockout mice. CONCLUSION: Our findings strongly suggest that in silico screening of small molecules that inhibit the protein-protein interaction between PCSK9 and the LDLR is a potential modality for developing hypercholesterolemia therapeutics.
Animals ; Cholesterol/*blood ; Cholesterol, LDL/blood ; Hep G2 Cells ; Humans ; Mice ; Mice, Knockout ; Proprotein Convertases/*metabolism ; Receptors, LDL/*metabolism ; Serine Endopeptidases/*metabolism ; *Small Molecule Libraries

Japanese encephalitis virus (JEV) is a single-stranded and positive-sense RNA, which has a single ORF (open reading frame), encoding a polyprotein precursor. Non-structural protein 3 (NS3) plays an important role in processing the polyprotein precursor and has become an important drug target of flavivirus. In this study, NS2BH-NS3 gene was amplified by PCR and subcloned to the prokaryotic expression plasmid, resulting pET30a-NS2BH-NS3. The fusion protein was expressed in Escherichia coli BL21 (DE3) in soluble form after induction by Isopropyl beta-D-1-Thiogalactopyranoside (IPTG). The recombinant protein was purified by Ni-NTA affinity column. Then a fluorescence resonance energy transfer (FRET) method was used to determine enzymatic activity and the assay conditions were optimized. After screening 113 compounds, we found two compounds inhibiting the activity of NS2BH-NS3. This study provides a convenient and cost-effective method for screening of JEV NS3 protease inhibitor.
Encephalitis Virus, Japanese ; enzymology ; Escherichia coli ; metabolism ; High-Throughput Screening Assays ; Protease Inhibitors ; chemistry ; RNA Helicases ; metabolism ; Recombinant Fusion Proteins ; metabolism ; Serine Endopeptidases ; metabolism ; Viral Nonstructural Proteins ; metabolism

OBJECTIVETo evaluate the immunophenotype conversion of fibroblasts and its clinical significance in the process of breast tumor stromal remodeling.

METHODSCD34, FAP-α, p63 and a-SMA were detected by immunohistochemistry in 273 breast biopsies, including 60 normal breast tissues, 46 atypical ductal hyperplasia (ADH), 60 ductal carcinoma in situ (DCIS), 47 DCIS microinvasive carcinoma (DCIS-MI) and 60 invasive ductal carcinoma (IDC).

RESULTSThe positive expression rates of CD34, FAP-α and α-SMA in the stromal fibroblasts of normal breast tissues were 93.3%, 6.7% and 18.3%, respectively. Those in the stromal fibroblasts of ADH tissues were 95.7%, 4.3% and 10.9%, respectively. Those in the stromal fibroblasts of DCIS tissues were 95.0%, 8.3% and 15.0%, respectively. Those in the IDC tissues were 35.0%, 85.0% and 93.3%, respectively. The expressions of CD34, α-SMA and FAP-α in the stromal fibroblasts of normal, ASH and DCIS breast tissues did not show significant differences (χ(2) = 1.142, P = 0.896). The main immunophenotype of stromal fibroblasts in the tumor-host interface at the invasive front of ADH and DCIS lesions was CD34(+)α-SMA(+)FAP-α(+). There were statistically significant differences in the expression of CD34, α-SMA and FAP-α between IDC and ADH, DCIS and normal breast tissues (χ(2) = 8.351, P < 0.001). The immunophenotype of stromal fibroblasts in the IDC and DCIS-MI breast tissues was CD34(-) α-SMA(+) FAP-α(+).

CONCLUSIONSImmunophenotype conversion from CD34(+) α-SMA(-) FAP-α(-) to CD34(-) α-SMA(+)FAP-α(+) may be a sensitive indicator to judge whether DCIS has microinvasion. Detection of the immunophenotype conversion of stromal fibroblasts may be helpful to determine the presence of microinvasion, and to improve the diagnostic accuracy rate of DCIS.

Breast ; Breast Neoplasms ; immunology ; pathology ; Carcinoma in Situ ; Carcinoma, Ductal, Breast ; Carcinoma, Intraductal, Noninfiltrating ; Fibroblasts ; immunology ; Gelatinases ; metabolism ; Humans ; Hyperplasia ; Immunohistochemistry ; Immunophenotyping ; Membrane Proteins ; metabolism ; Serine Endopeptidases ; metabolism