Systematic profiling of ACE2 expression in diverse physiological and pathological conditions for COVID‐19/SARS‐CoV‐2

Recent retrospective studies of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) disease (COVID‐19) revealed that the patients with common comorbidities of cancers and chronic diseases face significantly poorer clinical outcomes than those without. Since the expression profile of ACE2, a crucial cell entry receptor for SARS‐CoV‐2, could indicate the susceptibility to SARS‐CoV‐2 infection, here we systematically dissected ACE2 expression using large‐scale multi‐omics data from 30 organs/tissues, 33 cancer types and some common chronic diseases involving >28 000 samples. It was found that sex and age could be correlated with the susceptibility of SARS‐CoV‐2 infection for certain tissues. Strikingly, ACE2 was up‐regulated in cervical squamous cell carcinoma and endocervical adenocarcinoma, colon adenocarcinoma, oesophageal carcinoma, kidney renal papillary cell carcinoma, lung adenocarcinoma and uterine corpus endometrial carcinoma compared to controls. Furthermore, the patients with common chronic diseases regarding angiocardiopathy, type 2 diabetes, liver, pneumonia and hypertension were also with higher ACE2 expression compared to related controls, which were validated using independent data sets. Collectively, our study may reveal a novel important mechanism that the patients with certain cancers and chronic diseases may express higher ACE2 expression compared to the individuals without diseases, which could lead to their higher susceptibility to multi‐organ injury of SARS‐CoV‐2 infection.     

Identification and characterization of the zebrafish glutathione S‐transferase Pi‐1

Zebrafish has in recent years emerged as a popular vertebrate model for use in pharmacological and toxicological studies. While there have been sporadic studies on the zebrafish glutathione S‐transferases (GSTs), the zebrafish GST gene superfamily still awaits to be fully elucidated. We report here the identification of 15 zebrafish cytosolic GST genes in NCBI GenBank database and the expression, purification, and enzymatic characterization of the zebrafish cytosolic GST Pi‐1 (GSTP1). The cDNA encoding the zebrafish GSTP1 was cloned from a 3‐month‐old female zebrafish, expressed in Eschelichia coli host cells, and purified. Purified GSTP1 displayed glutathione‐conjugating activity toward 1‐chloro‐2,4‐dinitrobenzene as a representative substrate. The enzymatic characteristics of the zebrafish GSTP1, including pH‐dependency, effects of metal cations, and kinetic parameters, were studied. Moreover, the expression of zebrafish GSTP1 at different developmental stages during embryogenesis, throughout larval development, onto maturity was examined.     

Enhanced diastereoselective synthesis of t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate by using aldo‐keto reductase and glucose dehydrogenase co‐producing engineered Escherichia coli

t‐Butyl 6‐cyano‐(3R,5R)‐dihydroxyhexanoate ((3R,5R)‐ 2 ) is a key chiral diol precursor of atorvastatin calcium (Lipitor®). We have constructed a Kluyveromyces lactis aldo‐keto reductase mutant KlAKR‐Y295W/W296L (KlAKRm) by rational design in previous research, which displayed high activity and excellent diastereoselectivity (de_p > 99.5%) toward t‐butyl 6‐cyano‐(5R)‐hydroxy‐3‐oxohexanoate ((5R)‐ 1 ). To realize in situ cofactor regeneration, a robust KlAKRm and Exiguobacterium sibiricum glucose dehydrogenase (EsGDH) co‐producer E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm was constructed in this work. Under the optimized conditions, AKR and GDH activities of E. coli BL 21(DE3) pETDuet‐esgdh (MCS1)/pET‐28b (+)‐klakrm peaked at 249.9 U/g DCW (dry cellular weight) and 29100 U/g DCW, respectively. It completely converted (5R)‐ 1 at substrate loading size of up to 60.0 g/L (5R)‐ 1 in the absence of exogenous NADH, which was one‐fifth higher than that of the separately prepared KlAKRm and EsGDH under the same conditions. In this manner, a biocatalytic process for (3R,5R)‐ 2 with productivity of 243.2 kg/m³ d was developed. Compared with the combination of separate expressed KlAKRm with EsGDH, co‐expression of KlAKRm and EsGDH has the advantages of alleviating cell cultivation burden and elevating substrate load. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1235–1242, 2017     

Allelic imbalance metre (Allim), a new tool for measuring allele‐specific gene expression with RNA‐seq data

Estimating differences in gene expression among alleles is of high interest for many areas in biology and medicine. Here, we present a user‐friendly software tool, Allim, to estimate allele‐specific gene expression. Because mapping bias is a major problem for reliable estimates of allele‐specific gene expression using RNA‐seq, Allim combines two different strategies to account for the mapping biases. In order to reduce the mapping bias, Allim first generates a polymorphism‐aware reference genome that accounts for the sequence variation between the alleles. Then, a sequence‐specific simulation tool estimates the residual mapping bias. Statistical tests for allelic imbalance are provided that can be used with the bias corrected RNA‐seq data.     

Regulatory role of miR‐2909 in cell‐mediated immune response

Keeping in view the micromanagement of immune response by micro RNAs, the present study was directed to explore the role of miR‐2909 in the differentiation and maturation of T‐lymphocytes within the population of normal human peripheral blood mononuclear cells maintained in in vitro culture. The results of such a study revealed that miR‐2909 had the inherent capacity to significantly increase Treg (CD4+CD25+Foxp3+) cell population and dominant Th1‐type cytokine (especially with decrease in IL‐4 level and higher levels of INF‐β and INF‐γ) profile. Based upon these results, we propose that miR‐2909 may modulate native immunity in general and help in providing protective immunity against viral infections in particular. Copyright © 2012 John Wiley & Sons, Ltd.     

Heterologous expression and periplasmic secretion of an antifungal Bacillus amyloliquefaciensBLB369 endo‐β‐1,3‐1,4‐glucanase in Escherichia coli

The endo‐β‐1,3‐1,4‐glucanases are glycoside hydrolases involved in the enzymatic depolymerization of 1,3‐1,4 β‐glucans and showed an antifungal activity against some fungi. Bacillus amyloliquefaciensBLB369 has a high antagonistic activity against phytopathogenic fungi. Its glu369 full‐coding sequence of the endo‐β‐1,3‐1,4‐glucanase gene (732 bp) was sequenced, cloned and successfully expressed in Escherichia coli Top10. The encoded protein (243 amino acids) has a calculated molecular mass of 27.3 kDa. To simplify the purification procedure, the glu369 coding sequence was cloned into the vector pKJD4. The produced OmpA‐His‐Glu369 harboured OmpA signal sequence for E. coli periplasmic localization and followed by a 6His residues for its purification. The purified His‐tagged proteins revealed two bands on SDS‐PAGE analysis with molecular masses of about 30.5 (His‐Glu369) and 32.5 kDa (OmpA‐His‐Glu369). They had the ability to inhibit the growth of phytopathogenic fungus Alternaria alternata. These favourable properties make the endo‐β‐1,3‐1,4‐glucanase a good candidate for biotechnological applications.     

Quantitative DY‐maleimide‐based proteomic 2‐DE‐labeling strategies using human skin proteins

Sensitive differential proteomic analysis is challenging and often limited by distinct labeling or tagging strategies. In this study, we have examined the sensitivity, linearity, and photophysical properties of novel protein labeling DY‐maleimide dyes (DY‐505‐MAL, DY‐555‐MAL and DY‐635‐MAL). All MS compatible DY‐maleimide dyes exhibited excellent emission spectra, high sensitivity, and high linearity, when applied to standard 1‐DE protein analysis. Correspondingly, 2‐DE analysis of DY‐635‐MAL or DY‐505‐MAL maximal‐labeled human keratinocyte proteins displayed remarkably high sensitivity. Compared with a standard fluorescent protein stain, DY‐635‐MAL or DY‐505‐MAL 2‐DE analysis demonstrated equally high spot quality with an overall increase in the number of spots detectable (up to threefold higher;>1000 spots/gel). However, as determined with a FLA‐5100 imaging system, comparative MultiGauge, and Delta2D analysis, not all DY‐maleimide dyes possessed DIGE compatible fluorescent emission properties. However, DY‐505‐MAL and DY‐635‐MAL were found to be suitable for more complex, time and gel intensive, focused multiplexing analyses. Notably – as demonstrated with allergen‐stimulated human skin proteins – defined, singular DY‐maleimide dye protein labeling (SDPL) allows high quality, time saving, simple, and reliable differential proteomic examination.     

Efficient synthesis of polypeptide‐α‐thioester by the method combining polypeptide expression and chemical activation for the semi‐synthesis of interferon‐γ having oligosaccharides

In order to synthesize interferon‐γ glycoform having an oligosaccharide at the 97 position by a semi‐synthetic method, interferon‐γ‐polypeptide‐(1–94)‐α‐hydrazide was prepared by the specific Cys‐cyanylation of polypeptide‐(1–94)‐Cys‐His₆ expressed from E. coli and subsequent hydrazinolysis in 22% yield (two steps). This polypeptide‐α‐hydrazide was then converted into corresponding polypeptide‐α‐thioester under NaNO₂/acid conditions followed by thiolysis in 83% yield. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Small‐angle X‐ray and neutron scattering demonstrates that cell‐free expression produces properly formed disc‐shaped nanolipoprotein particles

Nanolipoprotein particles (NLPs), composed of membrane scaffold proteins and lipids, have been used to support membrane proteins in a native‐like bilayer environment for biochemical and structural studies. Traditionally, these NLPs have been prepared by the controlled removal of detergent from a detergent‐solubilized protein‐lipid mixture. Recently, an alternative method has been developed using direct cell‐free expression of the membrane scaffold protein in the presence of preformed lipid vesicles, which spontaneously produces NLPs without the need for detergent at any stage. Using SANS/SAXS, we show here that NLPs produced by this cell‐free expression method are structurally indistinguishable from those produced using detergent removal methodologies. This further supports the utility of single step cell‐free methods for the production of lipid binding proteins. In addition, detailed structural information describing these NLPs can be obtained by fitting a capped core‐shell cylinder type model to all SANS/SAXS data simultaneously.