Could urinary ACE2 protein level help identify individuals susceptible to SARS-CoV-2 infection and complication?

Science China Life Sciences -     

Discovering novel chemical inhibitors of human cyclophilin A: virtual screening, synthesis, and bioassay

Cyclophilin A (CypA) is a member of cyclophilins, a family of the highly homologous peptidyl prolyl cis-trans isomerases (PPIases), which can bind to cyclosporin A (CsA). CypA plays critical roles in various biological processes, including protein folding, assembly, transportation, regulation of neuron growth, and HIV replication. The discovery of CypA inhibitor is now of a great special interest in the treatment of immunological disorders. In this study, a series of novel small molecular CypA inhibitors have been discovered by using structure-based virtual screening in conjunction with chemical synthesis and bioassay. The SPECS_1 database containing 85,000 small molecular compounds was searched by virtual screening against the crystal structure of human CypA. After SPR-based binding affinity assay, 15 compounds were found to show binding affinities to CypA at submicro-molar or micro-molar level (compounds 1-15). Seven compounds were selected as the starting point for the further structure modification in considering binding activity, synthesis difficulty, and structure similarity. We thus synthesized 40 new small molecular compounds (1-6, 15, 16a-q, 17a-d, and 18a-l), and four of which (compounds 16b, 16h, 16k, and 18g) showed high CypA PPIase inhibition activities with IC50s of 2.5-6.2 microM. Pharmacological assay indicated that these four compounds demonstrated somewhat inhibition activities against the proliferation of spleen cells.     

Site-directed mutagenesis to enable and improve crystallizability of Candida tropicalis (3R)-hydroxyacyl-CoA dehydrogenase

The N-terminal part of Candida tropicalis MFE-2 (MFE-2(h2Delta)) having two (3R)-hydroxyacyl-CoA dehydrogenases with different substrate specificities has been purified and crystallized as a recombinant protein. The expressed construct was modified so that a stabile, homogeneous protein could be obtained instead of an unstabile wild-type form with a large amount of cleavage products. Cubic crystals with unit cell parameters a=74.895, b=78.340, c=95.445, and alpha=beta=gamma=90 degrees were obtained by using PEG 4000 as a precipitant. The crystals exhibit the space group P2(1)2(1)2(1) and contain one molecule, consisting of two different (3R)-hydroxyacyl-CoA dehydrogenases, in the asymmetric unit. The crystals diffract to a resolution of 2.2A at a conventional X-ray source.     

Role of hepatic lipase and scavenger receptor BI in clearing phospholipid/free cholesterol-rich lipoproteins in PLTP-deficient mice

Phospholipid transfer protein knock-out (PLTP0) mice have defective transfer of phospholipids (PL) from triglyceride-rich lipoproteins (TRL) into high-density lipoproteins (HDL). In this study, we examined the role of diet, hepatic lipase (HL) and scavenger receptor BI (SRBI) in determining the accumulation of excess PL and free cholesterol (FC, "surface remnants") in plasma of PLTP0 mice. PL and FC accumulated in the very low-density lipoprotein (VLDL)-LDL region of PLTP0 mice on a highly saturated, coconut oil-based diet, but not on chow or milk-fat based Western diets. Accumulation of PL and FC was dramatically increased in PLTP0/HL0 mice, compared to PLTP0 mice, but only on the coconut oil diet. Turnover studies indicated that the coconut oil diet was associated with delayed catabolism of PL of PL/FC-rich particles. Incubation of these particles with primary hepatocytes in the presence of SRBI neutralizing antibody indicated that SRBI was primarily responsible for removal of FC and PL on the Western diet. In hepatocytes of coconut oil-fed mice, removal of FC and PL from these particles by SRBI was markedly reduced, even though SRBI protein expression levels were unchanged. These studies indicate that HL and SRBI both have major role in the clearance of PL and FC of surface remnants in PLTP0 mice. SRBI appears to be dysfunctional in coconut oil diet-fed animals, possibly related to changes in hepatocyte membrane fatty acid composition.     

Genome-wide microRNA expression analysis of clear cell renal cell carcinoma by next generation deep sequencing

MicroRNAs (miRNAs), non-coding RNAs regulating gene expression, are frequently aberrantly expressed in human cancers. Next-generation deep sequencing technology enables genome-wide expression profiling of known miRNAs and discovery of novel miRNAs at unprecedented quantitative and qualitative accuracy. Deep sequencing was performed on 11 fresh frozen clear cell renal cell carcinoma (ccRCC) and adjacent non-tumoral renal cortex (NRC) pairs, 11 additional frozen ccRCC tissues, and 2 ccRCC cell lines (n?=?35). The 22 ccRCCs patients belonged to 3 prognostic sub-groups, i.e. those without disease recurrence, with recurrence and with metastatic disease at diagnosis. Thirty-two consecutive samples (16 ccRCC/NRC pairs) were used for stem-loop PCR validation. Novel miRNAs were predicted using 2 distinct bioinformatic pipelines. In total, 463 known miRNAs (expression frequency 1-150,000/million) were identified. We found that 100 miRNA were significantly differentially expressed between ccRCC and NRC. Differential expression of 5 miRNAs was confirmed by stem-loop PCR in the 32 ccRCC/NRC samples. With respect to RCC subgroups, 5 miRNAs discriminated between non-recurrent versus recurrent and metastatic disease, whereas 12 uniquely distinguished non-recurrent versus metastatic disease. Blocking overexpressed miR-210 or miR-27a in cell line SKCR-7 by transfecting specific antagomirs did not result in significant changes in proliferation or apoptosis. Twenty-three previously unknown miRNAs were predicted in silico. Quantitative genome-wide miRNA profiling accurately separated ccRCC from (benign) NRC. Individual differentially expressed miRNAs may potentially serve as diagnostic or prognostic markers or future therapeutic targets in ccRCC. The biological relevance of candidate novel miRNAs is unknown at present.     

维生素E对体外培养的人脐静脉内皮细胞白介素-8表达的影响

目的探讨维生素E(α-生育酚)对人脐静脉内皮细胞(HUVEC)白介素-8(IL-8)表达的影响.方法体外培养HUVEC,将其随机分为正常对照组和实验组,实验组细胞在用激动剂脂多糖(LPS)刺激之前,分别给予0、10、20、30mg/Lα-生育酚,然后在6h、24h、48h三个时段,利用双抗体夹心酶联免疫吸附技术(ELISA)和原位杂交技术(ISH)检测各组细胞IL-8蛋白表达及mRNA表达水平.结果 (1)正常对照组IL-8有基础表达;(2)与正常对照组比较,ELISA和ISH的结果均显示LPS能够明显诱导IL-8高表达:蛋白表达增强约28倍(P<0.01),mRNA表达增强约4.3倍(P<0.01);(3)维生素E能够抑制LPS诱导的IL-8的表达:ELISA结果显示20mg/L-48hα-T处理组作用最强,IL-8蛋白表达减少72.7%(P<0.01);ISH结果显示20mg/L -24hα-T处理组作用最强,IL-8mRNA表达减少73.2%(P<0.01).结论维生素E可显著抑制LPS诱导的HUVEC高表达IL-8,表明维生素E可通过影响对动脉粥样硬化有重要作用的IL-8的表达来发挥其抗动脉粥样硬化作用.     

Inter- and intraclade neutralization of human immunodeficiency virus type 1: genetic clades do not correspond to neutralization serotypes but partially correspond to gp120 antigenic serotypes.

We have studied genetic variation among clades A through E of human immunodeficiency virus type 1 (HIV-1) at the levels of antibody binding to gp120 molecules and virus neutralization. We are unable to identify neutralization serotypes that correspond to the genetic clades. Instead, we observe that inter- and intraclade neutralization of primary isolates by HIV-1-positive sera is generally weak and sporadic; some sera show a reasonable degree of neutralization breadth and potency whereas others are relatively sensitive to neutralization, but no consistent pattern was found. However, a few sera were able to neutralize across clades with significant potency, an observation which may have implications for the feasibility of a broadly effective HIV-1 vaccine involving humoral immunity. Serological assays measuring anti-gp120 antibody binding also failed to identify serotypes that correspond precisely to the genetic clades, but some indications of clade-specific binding were observed, notably with sera from clades B and E. A representative protein for each clade (A through E) was selected on the basis of its specificity, defined as high seroreactivity with sera from individuals infected with virus of that clade and lower reactivity with sera from individuals infected with viruses from other clades. The seroreactivity patterns against these five proteins could be used to predict the genotype of the infecting virus with moderate success.