Application of partial restriction procedure in both shotgun and non-random strategies for nucleotide sequencing

Partial digestion of a target DNA fragment with 4-bp-recognition restriction enzymes followed by a forced ligation to an M13 vector was employed for the construction of a subfragment library. The library can be used for either shotgun or non-random nucleotide sequencing. Application of the partial digests generated with the 4-bp recognition restriction enzymes instead of DNase I in the improved non-random strategy for nucleotide sequencing (Li and Wu, 1987) made the procedure as easy as that of the random strategy. The library can also be used in shotgun nucleotide sequencing directly, and few self-ligated subfragments were found. The usefulness of this procedure was demonstrated by the sequencing of a goat 6.5-kb EcoRI fragment, which is located 5' to the globin gene.     

CircRNA has_circ_0006427 suppresses the progression of lung adenocarcinoma by regulating miR-6783–3p/DKK1 axis and inactivating Wnt/β-catenin signaling pathway

Recently, circular RNAs (circRNAs) are identified as a novel class of noncoding RNAs playing important roles in human malignant tumors. However, the regulatory function of circRNA in lung adenocarcinoma (LUAD) is still largely unknown. Present study aimed to explore the role of circ_0006427 in LUAD progression. Firstly, the downregulation of circ_0006427 in LUAD tissues and cell lines was revealed by microarray analysis and qRT-PCR analysis. And we also confirmed the circ_0006427 as a prognostic target in LUAD patients. Functionally, overexpression of circ_0006427 effectively suppressed cell proliferation, migration and invasion. Mechanistically, circ_0006427 was found to be predominantly located in the cytoplasm of LUCA cell, and was further revealed to positively regulate DKK1 in LUAD by sponging miR-6783–3p. KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway analysis and western blot analysis revealed that circ_0006427 inactivated Wnt/β-catenin signaling pathway by upregulating DKK1. At last, rescue assays proved the function of circ_0006427/miR-6783–3p/DKK1 axis in LUAD progression. In conclusion, our study revealed that circ_0006427 suppressed lung adenocarcinoma progression through regulating miR-6783–3p/DKK1 axis.     

SARS-CoV-2 envelope protein causes acute respiratory distress syndrome (ARDS)-like pathological damages and constitutes an antiviral target

Cytokine storm and multi-organ failure are the main causes of SARS-CoV-2-related death. However, the origin of excessive damages caused by SARS-CoV-2 remains largely unknown. Here we show that the SARS-CoV-2 envelope (2-E) protein alone is able to cause acute respiratory distress syndrome (ARDS)-like damages in vitro and in vivo. 2-E proteins were found to form a type of pH-sensitive cation channels in bilayer lipid membranes. As observed in SARS-CoV-2-infected cells, heterologous expression of 2-E channels induced rapid cell death in various susceptible cell types and robust secretion of cytokines and chemokines in macrophages. Intravenous administration of purified 2-E protein into mice caused ARDS-like pathological damages in lung and spleen. A dominant negative mutation lowering 2-E channel activity attenuated cell death and SARS-CoV-2 production. Newly identified channel inhibitors exhibited potent anti-SARS-CoV-2 activity and excellent cell protective activity in vitro and these activities were positively correlated with inhibition of 2-E channel. Importantly, prophylactic and therapeutic administration of the channel inhibitor effectively reduced both the viral load and secretion of inflammation cytokines in lungs of SARS-CoV-2-infected transgenic mice expressing human angiotensin-converting enzyme 2 (hACE-2). Our study supports that 2-E is a promising drug target against SARS-CoV-2.Subject terms: Cell death, Molecular biology     

Influence of Plasma Polymerized Dielectric Buffer Layer and Gold Film on the Excitation of Long-Range Surface Plasmon Resonance

Recently, long-range surface plasmon resonance (LRSPR) sensor has attracted a great deal of attention as a potentially non-destructive and label-free technique for cellular studies in real time. Thus, much effort has been placed on the fabrication and optimization of multilayered structure required for the excitation of LRSPR. In this work, a detailed study about the influence of both plasma polymerized dielectric buffer layer (DBL) and thin gold film on the excitation of LRSPR was performed. The DBLs of different thicknesses were deposited directly onto SF11 glass slides by radio frequency plasma polymerization (pp) of perfluorooctyl ethylene (PFOE). Thereafter, Au films of different thicknesses were thermally evaporated onto the ppPFOE layers. Atomic force microscopy (AFM) results suggest that the resulting SF11/ppPFOE/Au structure has a smooth surface regardless of Au film’s thickness. LRSPR measurements indicate that the excitation of LRSPR relies not only on the thickness of the ppPFOE buffer layer, but also on the thickness and optical property of thin Au film. Theoretical simulation based on Fresnel’s equation allows for the determination of both the thickness and optical constant of each layer supporting the LRSPR, and also enables us to predict the optimum combination of ppPFOE and Au film in a LRSPR sensor. The performance of various LRSPR sensors to monitor the bulk refractive index variation has also been investigated.     

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a major pathogen of the economic insect silkworm, Bombyx mori. Virus‐encoded microRNAs (miRNAs) have been proven to play important roles in host–pathogen interactions. In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA, BmCPV‐miR‐1, from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR (qPCR) and investigated its functions with qPCR and lentiviral expression systems. Bombyx mori inhibitor of apoptosis protein (BmIAP) gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′ untranslated region. It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae. At the same time, it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics. Furthermore, BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm. In the midgut of BmCPV‐infected larvae, BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene. With the viral genomic RNA segments S1 and S10 as indicators, BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm. These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression, providing the virus with a better cell circumstance for its replication.     

Heterologous expression, purification, and biochemistry of the oligomycin sensitivity conferring protein (OSCP) from yeast.

Yeast Saccharomyces cerevisiae oligomycin sensitivity conferring proteins (OSCP) have been expressed in Escherichia coli. Heterologous expression results in production of a protein that is identical to yeast mature OSCP, including the absence of the initiating methionine residue. Yeast OSCP expressed in E. coli has been purified to homogeneity and it is able to reconstitute oligomycin-sensitive ATPase using purified F1- and F1/OSCP-depleted membranes (electron transport particles (ETP). Binding of F1 to ETP is dependent on the addition of OSCP. Binding studies using 35S-OSCP indicated that OSCP binds to ETP with a Kd of 200 nM and a capacity of 420 pmol/mg particle protein, whereas OSCP does not interact with F1 in the absence of ETP. These data indicate that yeast OSCP must first form a specific complex with F0, which then binds F1 forming the functional complex. To identify functional domains in yeast OSCP, two deletion mutants have been made. Antibodies directed to these deletion products do not inhibit OSCP-dependent binding of F1 to ETP. However, antibodies directed against the last one-third of OSCP greatly reduce the oligomycin sensitivity of the reconstituted ATPase. These data suggest that OSCP is involved in a functional role in energy transduction or proton translocation and serves a structural role in the yeast mitochondrial ATP synthase.     

Phospho-regulated ACAP4-Ezrin Interaction Is Essential for Histamine-stimulated Parietal Cell Secretion

The ezrin-radixin-moesin proteins provide a regulated linkage between membrane proteins and the cortical cytoskeleton and also participate in signal transduction pathways. Ezrin is localized to the apical membrane of parietal cells and couples the protein kinase A activation cascade to the regulated HCl secretion. Our recent proteomic study revealed a protein complex of ezrin-ACAP4-ARF6 essential for volatile membrane remodeling (Fang, Z., Miao, Y., Ding, X., Deng, H., Liu, S., Wang, F., Zhou, R., Watson, C., Fu, C., Hu, Q., Lillard, J. W., Jr., Powell, M., Chen, Y., Forte, J. G., and Yao, X. (2006) Mol. Cell Proteomics 5, 1437–1449). However, knowledge of whether ACAP4 physically interacts with ezrin and how their interaction is integrated into membrane-cytoskeletal remodeling has remained elusive. Here we provide the first evidence that ezrin interacts with ACAP4 in a protein kinase A-mediated phosphorylation-dependent manner through the N-terminal 400 amino acids of ACAP4. ACAP4 locates in the cytoplasmic membrane in resting parietal cells but translocates to the apical plasma membrane upon histamine stimulation. ACAP4 was precipitated with ezrin from secreting but not resting parietal cell lysates, suggesting a phospho-regulated interaction. Indeed, this interaction is abolished by phosphatase treatment and validated by an in vitro reconstitution assay using phospho-mimicking ezrin^S66D. Importantly, ezrin specifies the apical distribution of ACAP4 in secreting parietal cells because either suppression of ezrin or overexpression of non-phosphorylatable ezrin prevents the apical localization of ACAP4. In addition, overexpressing GTPase-activating protein-deficient ACAP4 results in an inhibition of apical membrane-cytoskeletal remodeling and gastric acid secretion. Taken together, these results define a novel molecular mechanism linking ACAP4-ezrin interaction to polarized epithelial secretion.