Efficient assembly of a large fragment of monkeypox virus genome as a qPCR template using dual-selection based transformation-associated recombination

Transformation-associated recombination (TAR) has been widely used to assemble large DNA constructs. One of the significant obstacles hindering assembly efficiency is the presence of error-prone DNA repair pathways in yeast, which results in vector backbone recircularization or illegitimate recombination products. To increase TAR assembly efficiency, we prepared a dual-selective TAR vector, pGFCS, by adding a P_ADH1-URA3 cassette to a previously described yeast-bacteria shuttle vector, pGF, harboring a P_HIS3-HIS3 cassette as a positive selection marker. This new cassette works as a negative selection marker to ensure that yeast harboring a recircularized vector cannot propagate in the presence of 5-fluoroorotic acid. To prevent pGFCS bearing ura3 from recombining with endogenous ura3-52 in the yeast genome, a highly transformable Saccharomyces cerevisiae strain, VL6-48B, was prepared by chromosomal substitution of ura3-52 with a transgene conferring resistance to blasticidin. A 55-kb genomic fragment of monkeypox virus encompassing primary detection targets for quantitative PCR was assembled by TAR using pGFCS in VL6-48B. The pGFCS-mediated TAR assembly showed a zero rate of vector recircularization and an average correct assembly yield of 79% indicating that the dual-selection strategy provides an efficient approach to optimizing TAR assembly.     

Cytoplasmic domain and enzymatic activity of ACE2 are not required for PI4KB dependent endocytosis entry of SARS-CoV-2 into host cells

The recent COVID-19 pandemic poses a global health emergency. Cellular entry of the causative agent SARS-CoV-2 is mediated by its spike protein interacting with cellular receptor-human angiotensin converting enzyme 2 (ACE2). Here, by using lentivirus based pseudotypes bearing spike protein, we demonstrated that entry of SARS-CoV-2 into host cells was dependent on clathrin-mediated endocytosis, and phosphoinositides played essential roles during this process. In addition, we showed that the intracellular domain and the catalytic activity of ACE2 were not required for efficient virus entry. Finally, we showed that the current predominant Delta variant, although with high infectivity and high syncytium formation, also entered cells through clathrin-mediated endocytosis. These results provide new insights into SARS-CoV-2 cellular entry and present proof of principle that targeting viral entry could be an effective way to treat different variant infections.     

Molecular characterization and immunohistochemical localization of a mitogen-activated protein kinase, Accp38b, from Apis cerana cerana

The p38 mitogen-activated protein kinase (MAPK) is involved in various processes, including stress responses, development, and differentiation. However, little information on p38 MAPK in insects is available. In this study, a p38 MAPK gene, Accp38b, was isolated from Apis cerana cerana and characterized. The quantitative real-time PCR (Q-PCR) analysis revealed that Accp38b was induced by multiple stressors. Notably, the expression of Accp38b was relatively higher in the pupae phase than in other developmental phases. During the pupae phase, Accp38b expression was higher in the thorax than in the head and abdomen and higher in the fat body than in the muscle and midgut. Immunohistochemisty showed significant positive staining of Accp38b in sections from the brain, eyes, fat body, and midgut of A. cerana cerana. These results suggest that Accp38b may play a crucial role in stress responses and have multiple aspects function during development.     

Factors Influencing Formation of Sclerotia in Grifola umbellate （Pers.） Pilaet Under Artificial Conditions

The effects of substrate composition and temperature on myceilal growth and sclerotium production in Grlfola umbellate （Pers.） Pilaet were Investigated In the present study. The Induction of sclerotla of G. umbellate was affected greatly by the type of medium, as well as the type of carbon source. Malt-extract agar was able to induce the production of sclerotia. The production of sclerotia was also observed when the carbon source in the GPC agar medium （glucose 20 g/L, peptone 6 g/L, corn steep liquor 10 g/L, and agar 15 g/L） was replaced with glycerol or mannitol. Altering the composition of the GPC medium with milk powder, thiamine hydrochlorlde, extract of Armlllarla mellea, active clay, dlatomite, kaolin, or arginlne did not induce the production of sclerotla. A temperature range of 18-25 ℃ was suitable for both mycellai growth and sclerotium formation. Glycerol significantly Induced slerotium formation on nutrient supplemented with sawdust substrates In bottle culture. 24S-Polyporusterone A and polyporusterone B were assayed In samples of natural and cultured sclerotla. Both natural and cultured sclerotla contained 24S- polyporusterone A and polyporusterone B.     

Effects of sodium glucose cotransporter-2 inhibitors on serum uric acid in type 2 diabetes mellitus: A systematic review with an indirect comparison meta-analysis

To describe the effects of sodium-glucose co-transporter 2 (SGLT2) inhibitors on serum uric acid (SUA) in patients with type 2 diabetes mellitus (T2DM). PubMed, EMBASE, and CENTRAL were searched for randomized controlled trials of SGLT2 inhibitors in patients with T2DM up to Aug 10, 2017, without language or date restrictions. Thirty-one studies totaling 13,650 patients were included. SGLT2 inhibitors significantly decreased SUA levels compared with placebo, canagliflozin WMD –37.02?μmol/L, 95% CI [–38.41, –35.63], dapagliflozin WMD –38.05?μmol/L, 95% CI [–44.47, –31.62], empagliflozin WMD –42.07?μmol/L, 95% CI [–46.27, –37.86]. The drug class effect of SUA reduction suggesting SGLT2 inhibitors might be beneficial for diabetic patients with hyperuricemia.     

Modification of alternative splicing in bovine somatic cell nuclear transfer embryos using engineered CRISPR-Cas13d

Animal cloning can be achieved by somatic cell nuclear transfer(SCNT), but the resulting live birth rate is relatively low. We previously improved the efficiency of bovine SCNT by exogenous melatonin treatment or by overexpression of lysine-specific demethylase 4D(KDM4D) and 4E(KDM4E). In this study, we revealed abundant alternative splicing(AS) transitions during fertilization and embryonic genome activation, and demonstrated abnormal AS in bovine SCNT embryos compared with in vitro fertilized ...     

ITMSQ: A software tool for N‐ and C‐terminal fragment ion pairs based isobaric tandem mass spectrometry quantification

Tandem MS (MS2) quantification using the series of N‐ and C‐terminal fragment ion pairs generated from isobaric‐labelled peptides was recently considered an accurate strategy in quantitative proteomics. However, the presence of multiplexed terminal fragment ion in MS2 spectra may reduce the efficiency of peptide identification, resulting in lower identification scores or even incorrect assignments. To address this issue, we developed a quantitative software tool, denoted isobaric tandem MS quantification (ITMSQ), to improve N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantification. A spectrum splitting module was designed to separate the MS2 spectra from different samples, increasing the accuracy of both identification and quantification. ITMSQ offers a convenient interface through which parameters can be changed along with the labelling method, and the result files and all of the intermediate files can be exported. We performed an analysis of in vivo terminal amino acid labelling labelled HeLa samples and found that the numbers of quantified proteins and peptides increased by 13.64 and 27.52% after spectrum splitting, respectively. In conclusion, ITMSQ provides an accurate and reliable quantitative solutionfor N‐ and C‐terminal fragment ion pairs based isobaric MS2 quantitative methods.