Antibacterial activity and mechanism of sanguinarine against Staphylococcus aureus by interfering with the permeability of the cell wall and membrane and inducing bacterial ROS production

Staphylococcus aureus (SA) is representative of gram-positive bacteria. Sanguinarine chloride hydrate (SGCH) is the hydrochloride form of sanguinarine (SG), one of the main extracts of Macleaya cordata (M. cordata). There are few reports on its antibacterial mechanism against SA. Therefore, in this study, we investigated the in vitro antibacterial activity and mechanism of SGCH against SA. The inhibitory zone, minimum inhibitory concentration (MIC), and minimum bactericidal concentration (MBC) were measured, and the bactericidal activity curve was plotted. In addition, the micromorphology, alkaline phosphatase (AKP) activity, Na+K+, Ca2+Mg2+-adenosine triphosphate (ATP) activity, intracellular reactive oxygen species (ROS), and fluorescein diacetate (FDA) were observed and detected. The results showed that the inhibitory zone of SGCH against SA was judged as medium-sensitive; the MIC and MBC were 128 and 256 μg/mL, respectively; in the bactericidal activity curve, SGCH with 8 × MIC could completely kill SA within 24 h. SGCH was able to interfere with the integrity and permeability of the SA cell wall and membrane, as confirmed by the scanning electron microscopy (SEM) images, the increase in extracellular AKP and Na+ K+, Ca2+ Mg2+-ATP activities as well as the fluorescein diacetate (FDA) staining experiment results. Moreover, a high concentration of SGCH could induce SA to produce large amounts of ROS. In summary, these findings revealed that SGCH has a preferable antibacterial effect on SA, providing an experimental and theoretical basis for using SG as an antibiotic substitute in animal husbandry and for the clinical control and treatment of diseases caused by SA

Robust estimation of bacterial cell count from optical density

Optical density (OD) is widely used to estimate the density of cells in liquid culture, but cannot be compared between instruments without a standardized calibration protocol and is challenging to relate to actual cell count. We address this with an interlaboratory study comparing three simple, low-cost, and highly accessible OD calibration protocols across 244 laboratories, applied to eight strains of constitutive GFP-expressing E. coli. Based on our results, we recommend calibrating OD to estimated cell count using serial dilution of silica microspheres, which produces highly precise calibration (95.5% of residuals <1.2-fold), is easily assessed for quality control, also assesses instrument effective linear range, and can be combined with fluorescence calibration to obtain units of Molecules of Equivalent Fluorescein (MEFL) per cell, allowing direct comparison and data fusion with flow cytometry measurements: in our study, fluorescence per cell measurements showed only a 1.07-fold mean difference between plate reader and flow cytometry data

Establishment of a rabbit Oct4 promoter-based EGFP reporter system.

Rabbits are commonly used as laboratory animal models to investigate human diseases and phylogenetic development. However, pluripotent stem cells that contribute to germline transmission have yet to be established in rabbits. The transcription factor Oct4, also known as Pou5f1, is considered essential for the maintenance of the pluripotency of stem cells. Hence, pluripotent cells can be identified by monitoring Oct4 expression using a well-established Oct4 promoter-based reporter system. This study developed a rabbit Oct4 promoter-based enhanced green fluorescent protein (EGFP) reporter system by transfecting pROP2-EGFP into rabbit fetal fibroblasts (RFFs). The transgenic RFFs were used as donor cells for somatic cell nuclear transfer (SCNT). The EGFP expression was detected in the blastocysts and genital ridges of SCNT fetuses. Fibroblasts and neural stem cells (NSCs) were derived from the SCNT fetuses. EGFP was also reactivated in blastocysts after the second SCNT, and induced pluripotent stem cells (iPSCs) were obtained after reprogramming using Yamanaka's factors. The results above indicated that a rabbit reporter system used to monitor the differentiating status of cells was successfully developed

Current Knowledge on Epizootic Haemorrhagic Disease in China

Epizootic haemorrhagic disease (EHD) is an infectious, non-contagious viral disease of ruminants caused by epizootic haemorrhagic disease virus (EHDV) and is transmitted by insects of the genus Culicoides. In 2008, EHD was listed on the World Organization for Animal Health (WOAH) list of notifiable terrestrial and aquatic animal diseases. This article reviews the distribution of EHD in China and relevant studies and proposes several suggestions for the prevention and control of EHD. There have been reports of positivity for serum antibodies against EHDV-1, EHDV-2, EHDV-5, EHDV-6, EHDV-7, EHDV-8 and EHDV-10 in China. Strains of EHDV-1, -5, -6, -7, -8 and -10 have been isolated, among which the Seg-2, Seg-3 and Seg-6 sequences of serotypes -5, -6, -7 and -10 belong to the eastern topotype. The emergence of western topotype Seg-2 in EHDV-1 strains indicates that EHDV-1 strains in China are reassortant strains of the western and eastern topotypes. A novel serotype strain of EHDV named YNDH/V079/2018 was isolated in 2018. Chinese scholars have successfully expressed the EHDV VP7 protein and developed a variety of ELISA detection methods, including antigen capture ELISA and competitive ELISA. A variety of EHDV nucleic acid detection methods, including RT–PCR and qRT–PCR, have also been developed. LAMP and the liquid chip detection technique are also available. To prevent and control EHD, several suggestions for controlling EHD transmission have been proposed based on the actual situation in China, including controlling the number of Culicoides, reducing contact between Culicoides and hosts, continued monitoring of EHDV and Culicoides in different areas of China and further development and application of basic and pioneering research related to EHD prevention and control

Highly efficient generation of GGTA1 biallelic knockout inbred mini-pigs with TALENs.

Inbred mini-pigs are ideal organ donors for future human xenotransplantations because of their clear genetic background, high homozygosity, and high inbreeding endurance. In this study, we chose fibroblast cells from a highly inbred pig line called Banna mini-pig inbred line (BMI) as donor nuclei for nuclear transfer, combining with transcription activator-like effector nucleases (TALENs) and successfully generated α-1,3-galactosyltransferase (GGTA1) gene biallelic knockout (KO) pigs. To validate the efficiency of TALEN vectors, in vitro-transcribed TALEN mRNAs were microinjected into one-cell stage parthenogenetically activated porcine embryos. The efficiency of indel mutations at the GGTA1-targeting loci was as high as 73.1% (19/26) among the parthenogenetic blastocysts. TALENs were co-transfected into porcine fetal fibroblasts of BMI with a plasmid containing neomycin gene. The targeting efficiency reached 89.5% (187/209) among the survived cell clones after a 10 d selection. More remarkably 27.8% (58/209) of colonies were biallelic KO. Five fibroblast cell lines with biallelic KO were chosen as nuclear donors for somatic cell nuclear transfer (SCNT). Three miniature piglets with biallelic mutations of the GGTA1 gene were achieved. Gal epitopes on the surface of cells from all the three biallelic KO piglets were completely absent. The fibroblasts from the GGTA1 null piglets were more resistant to lysis by pooled complement-preserved normal human serum than those from wild-type pigs. These results indicate that a combination of TALENs technology with SCNT can generate biallelic KO pigs directly with high efficiency. The GGTA1 null piglets with inbred features created in this study can provide a new organ source for xenotransplantation research

sj-docx-1-npx-10.1177_1934578X241237657 - Supplemental material for <i>Macleaya cordata</i> Extract Inhibits LPS-Induced IPEC-J2 Inflammation Through TLR4-Mediated NF-κB and MAPK Signaling Pathways

Supplemental material, sj-docx-1-npx-10.1177_1934578X241237657 for Macleaya cordata Extract Inhibits LPS-Induced IPEC-J2 Inflammation Through TLR4-Mediated NF-κB and MAPK Signaling Pathways by Diangang Han, Hongqing Yang, Litao Che, Chong Zhang, Jing Li, Lingling Ye, Chunyong Zhang, Rongfu Guo and Jige Xin in Natural Product Communications</p

Efficient generation of GGTA1-null Diannan miniature pigs using TALENs combined with somatic cell nuclear transfer

Abstract Background α1,3-Galactosyltransferase (GGTA1) is essential for the biosynthesis of glycoproteins and therefore a simple and effective target for disrupting the expression of galactose α-1,3-galactose epitopes, which mediate hyperacute rejection (HAR) in xenotransplantation. Miniature pigs are considered to have the greatest potential as xenotransplantation donors. A GGTA1-knockout (GTKO) miniature pig might mitigate or prevent HAR in xenotransplantation. Methods Transcription activator-like effector nucleases (TALENs) were designed to target exon 6 of porcine GGTA1 gene. The targeting activity was evaluated using a luciferase SSA recombination assay. Biallelic GTKO cell lines were established from single-cell colonies of fetal fibroblasts derived from Diannan miniature pigs following transfection by electroporation with TALEN plasmids. One cell line was selected as donor cell line for somatic cell nuclear transfer (SCNT) for the generation of GTKO pigs. GTKO aborted fetuses, stillborn fetuses and live piglets were obtained. Genotyping of the collected cloned individuals was performed. The Gal expression in the fibroblasts and one piglet was analyzed by fluorescence activated cell sorting (FACS), confocal microscopy, immunohistochemical (IHC) staining and western blotting. Results The luciferase SSA recombination assay revealed that the targeting activities of the designed TALENs were 17.1-fold higher than those of the control. Three cell lines (3/126) showed GGTA1 biallelic knockout after modification by the TALENs. The GGTA1 biallelic modified C99# cell line enabled high-quality SCNT, as evidenced by the 22.3 % (458/2068) blastocyst developmental rate of the reconstructed embryos. The reconstructed GTKO embryos were subsequently transferred into 18 recipient gilts, of which 12 became pregnant, and six miscarried. Eight aborted fetuses were collected from the gilts that miscarried. One live fetus was obtained from one surrogate by caesarean after 33 d of gestation for genotyping. In total, 12 live and two stillborn piglets were collected from six surrogates by either caesarean or natural birth. Sequencing analyses of the target site confirmed the homozygous GGTA1-null mutation in all fetuses and piglets, consistent with the genotype of the donor cells. Furthermore, FACS, confocal microscopy, IHC and western blotting analyses demonstrated that Gal epitopes were completely absent from the fibroblasts, kidneys and pancreas of one GTKO piglet. Conclusions TALENs combined with SCNT were successfully used to generate GTKO Diannan miniature piglets

In vitro development of SCNT embryos.

<p>17#-RFF: 17# pROP2-EGFP transgenic rabbit fibroblasts; wt-RFF: wide type rabbit fibroblasts; PA: parthenogenetic embryos.</p><p>In vitro development of SCNT embryos.</p

Analysis of NT fetuses.

<p>(A) Two cloned fetuses (named 1## and 2##) derived from 17# transgenic clones by SCNT at 15 days after transplantation. (B) Genomic PCR analysis of fetuses 1## and 2## stably transfected with pROP2-EGFP. NC: negative control. (C) EGFP expressed in genital ridges isolated from 2## fetus but not in other tissues, such as intestinal tissues. Scale bars = 1 mm. (D) EGFP reactivated in morulae and blastocysts after the second SCNT was performed using Oct4-EGFP transgenic fibroblasts isolated from fetus 1## and 2## as donors. Parthenogenetic blastocysts did not express EGFP. Scale bars = 100 µm.</p

Construction of a rabbit Oct4 promoter-based EGFP vector.

<p>(A) Diagram of the pROP2-EGFP vector containing a 3.0 kb rabbit Oct4 promoter followed by EGFP in our experiments. (B) Partial EGFP expression in Rb-ESCs after transfection with pROP2-EGFP. (C) EGFP images after the transfection of CMV-GFP, PGK-EGFP, pROP2-EGFP and empty vector into murine ES cell line (R1) and rabbit fibroblasts (RFFs). Scale bars = 50 µm.</p