Necessary problems in re-emergence of COVID-19.

The ongoing pandemic of coronavirus disease 2019 poses a great threat to human beings. Although numerous patients have recovered, re-positive cases have been reported in several countries. Till now, we still know very little about the disease and its pathogen severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Therefore, more attention should be paid to the following aspects, such as post-discharge surveillance, asymptomatic infection, re-evaluation of influenza-like symptoms, and dynamic monitoring of genomic mutation of SARS-CoV-2.

Abnormality of intestinal cholesterol absorption in ApcMin/+ mice with colon cancer cachexia.

Colorectal cancer syndrome has been one of the greatest concerns in the world, particularly in developed countries. Several epidemiological studies have shown that dyslipidemia may be associated with the progression of intestinal cachexia, but there is little research on the function of the small intestine, which is involved in blood lipid metabolism, in dyslipidemia. In the present study, we aimed to explore the function of intestinal cholesterol absorption in the ApcMin/+ mouse model using an intestinal lipid absorption test. We found that both triglyceride (TG) and total cholesterol (TC) uptake were inhibited in the intestine of ApcMin/+ mice with age and the intestinal peroxisome proliferator-activated receptor α (PPARα) downregulated the processes of ß-oxidation, oxidative stress response, and cholesterol absorption in APC-deficient mice. In addition, reduced expression levels of farnesoid X receptor (FXR) and apical sodium-dependent bile acid transporter (ASBT) indicated that bile acid metabolism might be associated with intestinal cholesterol absorption in ApcMin/+ mice. Thus, our data suggested that the intestine plays an essential role in cholesterol uptake and that bile acid metabolism seems to cause a decrease in intestinal cholesterol uptake in ApcMin/+ mice.

Mouse models of porcine circovirus 2 infection.

PCV2 is considered the main pathogen of porcine circovirus diseases and porcine circovirus-associated diseases (PCVD/PCVAD). However, the exact mechanism underlying PCVD/PCVAD is currently unknown. Mouse models of PCV2 are valuable experimental tools that can shed light on the pathogenesis of infection and will enable the evaluation of antiviral agents and vaccine candidates. In this review, we discuss the current state of knowledge of mouse models used in PCV2 research that has been performed to date, highlighting their strengths and limitations, as well as prospects for future PCV2 studies.

Subculturing cells have no effect on CRISPR/Cas9-mediated cleavage of UL30 gene in pseudorabies virus.

CRISPR/Cas9-mediated genome editing can inhibit virus infection by targeting the conserved regions of the viral genomic DNA. Unexpectedly, we found previously that pseudorabies virus (PRV) could escape from CRISPR/Cas9-mediated inhibition. In order to elucidate whether the escape of PRV from Cas9-mediated inhibition was due to cell deficiencies, such as genetic instability of sgRNA or Cas9 protein, the positive cells were passaged ten times, and PRV infection in the sgRNA-expressing cells was evaluated in the present study. The results showed that subculturing cells has no effect on Cas9-mediated cleavage of PRV. Different passages of PX459-PRV cells can stably express sgRNA to facilitate Cas9/sgRNA cleavage on the UL30 gene of PRV, resulting in a pronounced inhibition of PRV infection. Studies to elucidate the mechanism of PRV escape are currently in progress.

Cirtical role for Salmonella effector SopB in regulating inflammasome activation.

OBJECTIVE: Salmonella is known to evolve many mechanisms to avoid or delay inflammasome activation which remain largely unknown. In this study, we investigated whether the SopB protein critical to bacteria virulence capacity was an effector that involved in the regulation of inflammasome activation. METHODS: BMDMs from NLRC4-, NLRP3-, caspase-1/-11-, IFI16- and AIM2-deficient mice were pretreated with LPS, and subsequently stimulated with a series of SopB-related strains of Salmonella, inflammasome induced cell death, IL-1ß secretion, cleaved caspase-1 production and ASC speckle formation were detected. RESULTS: We found that SopB could inhibit host IL-1ß secretion, caspase-1 activation and inflammasome induced cell death using a series of SopB-related strains of Salmonella; however the reduction of IL-1ß secretion was not dependent on sensor that contain PYD domain, such as NLRP3, AIM2 or IFI16, but dependent on NLRC4. Notably, SopB specifically prevented ASC oligomerization and the enzymatic activity of SopB was responsible for the inflammasome inhibition. Furthermore, inhibition of Akt signaling induced enhanced inflammasome activation. CONCLUSIONS: These results revealed a novel role in inhibition of NLRC4 inflammasome for Salmonella effector SopB.

Early lethality of shRNA-transgenic pigs due to saturation of microRNA pathways.

RNA interference (RNAi) is considered as a potential modality for clinical treatment and anti-virus animal breeding. Here, we investigate the feasibility of inhibiting classical swine fever virus (CSFV) replication by short hairpin RNA (shRNA) in vitro and in vivo. We generate four different shRNA-positive clonal cells and two types of shRNA-transgenic pigs. CSFV could be effectively inhibited in shRNA-positive clonal cells and tail tip fibroblasts of shRNA-transgenic pigs. Unexpectedly, an early lethality due to shRNA is observed in these shRNA-transgenic pigs. With further research on shRNA-positive clonal cells and transgenic pigs, we report a great induction of interferon (IFN)-responsive genes in shRNA-positive clonal cells, altered levels of endogenous microRNAs (miRNA), and their processing enzymes in shRNA-positive cells. What is more, abnormal expressions of miRNAs and their processing enzymes are also observed in the livers of shRNA-transgenic pigs, indicating saturation of miRNA/shRNA pathways induced by shRNA. In addition, we investigate the effects of shRNAs on the development of somatic cell nuclear transfer (SCNT) embryos. These results show that shRNA causes adverse effects in vitro and in vivo and shRNA-induced disruption of the endogenous miRNA pathway may lead to the early lethality of shRNA-transgenic pigs. We firstly report abnormalities of the miRNA pathway in shRNA-transgenic animals, which may explain the early lethality of shRNA-transgenic pigs and has important implications for shRNA-transgenic animal preparation.

Characterization of the structural and molecular defects in fibres and follicles of the Merino felting lustre mutant.

The felting lustre (FL) mutation found in Merino sheep results in a fleece that has a lustrous appearance and readily felts. This phenotype was described 50 years ago to result from the mutation of a single gene, but the molecular and cellular changes in the wool are not well understood. In this study, follicle and fibre material of FL mutant (n = 3) and normal control (n = 5) Merino ewes was compared using histological analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), real-time polymerase chain reaction (qPCR) and electron microscopy [scanning electron microscopy (SEM) and transmission electron microscopy (TEM)]. Histological examination suggested that follicle structure in FL mutants is essentially normal, while SDS-PAGE analysis found that some low molecular weight keratin-associated proteins (KAP) were present at much lower levels in FL wool. Examination of transcript prevalence revealed that the KAP6.1, KAP7 and KAP8 genes in FL mutant follicles are downregulated, while the KAP2.12 and KAP4.2 genes are upregulated. TEM analysis indicated that there is only one type of cortical cell, the paracortical cell, in the fibre of FL mutants, while there are paracortical and orthocortical cells in fibres of normal Merino sheep. In contrast, SEM suggested the surface topography of FL wool fibres is normal. The evidence presented here strongly suggests that the properties of FL wool can be ascribed, at least in part, to the lower content of high glycine/tyrosine proteins and the reduction in orthocortical cells in mutant wool fibres.

[The effect of basic fibroblast growth factor on the gene expression of decorin by periodontal ligament fibroblasts in culture].

OBJECTIVE: To study the effect of basic fibroblast growth factor (bFGF) on the gene expression of decorin by periodontal ligament fibroblasts (PLFs) in culture, and discuss the effect of bFGF in periodontal regeneration. METHODS: Human PLFs were cultured and stimulated by exogenous bFGF. Gene expression of decorin was assessed by semi-quantitive RT-PCR. RESULTS: The mRNA expression of decorin was suppressed by bFGF and the effect was dose-dependent. When the dose of bFGF increased, the inhibitive effect decreased. CONCLUSION: Decorin has many biological effects. The inhibitive effect may be one of important factors which participate in the healing process of periodontitis, and provide partly theoretical basis of bFGF in periodontal regeneration.

[Microsatellite analysis of genetic diversity of Aristichthys nobilis in China].

Seventeen microsatellite markers of Aristichthys nobilis previously discovered by our lab were selected to analyze the genetic diversity and characteristics of two populations of Aristichthys nobilis from Jiangxi and Sichuan provinces. The following parameters were calculated: heterozygosity, polymorphism information content (PIC), valid allele number, allele frequency, genetic distance, genetic similarity coefficient, Hardy-Weinberg balance deflection index and so on. Results show that there are 4 monomorphic and 13 polymorphic markers among the 17 selected microsatellite markers. The average of allele number in each microsatellite locus of the Jiangxi population and Sichuan populations is 3.325 and 3.882, respectively; the average valid allele number is 3.531 and 2.676, respectively; and the number of total alleles of these 17 microsatellite loci is 71. The PIC of polymorphic loci varies between 0.077-0.960, and the average PIC is 0.417. The average observed heterozygosity (Ho) of two populations is 0.385 and 0.360, respectively and the average expected heterzygosity (He) is 0.452 and 0.422, respectively. The genetic similarity coefficient of two populations of Aristichthys nobilis is 0.897 and the genetic distance of these populations is 0.109.

[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].

On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.

[Cloning and sequencing of the gp23 gene encoding a surface antigen on sporozoites of Cryptosporidium parvum].

OBJECTIVE: To clone and sequence the gp23 gene encoding a surface antigen on the sporozoites of Cryptosporidium parvum. METHODS: Genomic DNA was isolated from oocysts of Cryptosporidium parvum. The gp23 gene was amplified by polymerase chain reaction (PCR) and cloned into pMD18-T vector and sequenced by the methods of dideoxy-mediated chain termination. RESULTS AND CONCLUSION: The gp23 gene was 345 bp in length and included an open reading frame encoding a protein of 114 amino acid residues. The homology of the nucleotide and amino sequences of the gp23 gene was 97.3% and 98.2% to that in the GenBank, respectively. The gp23 gene cloned contained 6 nucleotides more than that in the GenBank.