Optimized production of full-length PCV2d virus-like particles in Escherichia coli: A cost-effective and high-yield approach for potential vaccine antigen development.

Porcine circovirus type 2 (PCV2) is a globally prevalent infectious pathogen affecting swine, with its capsid protein (Cap) being the sole structural protein critical for vaccine development. Prior research has demonstrated that PCV2 Cap proteins produced in Escherichia coli (E. coli) can form virus-like particles (VLPs) in vitro, and nuclear localization signal peptides (NLS) play a pivotal role in stabilizing PCV2 VLPs. Recently, PCV2d has emerged as an important strain within the PCV2 epidemic. In this study, we systematically optimized the PCV2d Cap protein and successfully produced intact PCV2d VLPs containing NLS using E. coli. The recombinant PCV2d Cap protein was purified through affinity chromatography, yielding 7.5 mg of recombinant protein per 100 ml of bacterial culture. We augmented the conventional buffer system with various substances such as arginine, ß-mercaptoethanol, glycerol, polyethylene glycol, and glutathione to promote VLP assembly. The recombinant PCV2d Cap self-assembled into VLPs approximately 20 nm in diameter, featuring uniform distribution and exceptional stability in the optimized buffer. We developed the vaccine and immunized pigs and mice, evaluating the immunogenicity of the PCV2d VLPs vaccine by measuring PCV2-IgG, IL-4, TNF-α, and IFN-γ levels, comparing them to commercial vaccines utilizing truncated PCV2 Cap antigens. The HE staining and immunohistochemical tests confirmed that the PCV2 VLPs vaccine offered robust protection. The results revealed that animals vaccinated with the PCV2d VLPs vaccine exhibited high levels of PCV2 antibodies, with TNF-α and IFN-γ levels rapidly increasing at 14 days post-immunization, which were higher than those observed in commercially available vaccines, particularly in the mouse trial. This could be due to the fact that full-length Cap proteins can assemble into more stable PCV2d VLPs in the assembling buffer. In conclusion, our produced PCV2d VLPs vaccine elicited stronger immune responses in pigs and mice compared to commercial vaccines. The PCV2d VLPs from this study serve as an excellent candidate vaccine antigen, providing insights for PCV2d vaccine research.

Biological Characterization and Whole-Genome Analysis of Bacillus subtilis MG-1 Isolated from Mink Fecal Samples.

Bacillus subtilis is an important part of the gut microbiota and a commonly used probiotic. In the present study, to assess the biological characteristics and probiotic properties of B. subtilis derived from mink, we isolated B. subtilis MG-1 isolate from mink fecal samples, characterized its biological characteristics, optimized the hydrolysis of casein by its crude extract, and comprehensively analyzed its potential as a probiotic in combination with whole-genome sequencing. Biological characteristics indicate that, under low-pH conditions (pH 2), B. subtilis MG-1 can still maintain a survival rate of 64.75%; under the conditions of intestinal fluid, gastric acid, and a temperature of 70 °C, the survival rate was increased by 3, 1.15 and 1.17 times compared with the control group, respectively. This shows that it can tolerate severe environments. The results of hydrolyzed casein in vitro showed that the crude bacterial extract of isolate MG-1 exhibited casein hydrolyzing activity (21.56 U/mL); the enzyme activity increased to 32.04 U/mL under optimized reaction conditions. The complete genome sequencing of B. subtilis MG-1 was performed using the PacBio third-generation sequencing platform. Gene annotation analysis results revealed that B. subtilis MG-1 was enriched in several Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways, and most genes were related to Brite hierarchy pathways (1485-35.31%) and metabolism pathways (1395-33.17%). The egg-NOG annotation revealed that most genes were related to energy production and conversion (185-4.10%), amino acid transport and metabolism (288-6.38%), carbohydrate transport and metabolism (269-5.96%), transcription (294-6.52%), and cell wall/membrane/envelope biogenesis (231-5.12%). Gene Ontology (GO) annotation elucidated that most genes were related to biological processes (8230-45.62%), cellular processes (3582-19.86%), and molecular processes (6228-34.52%). Moreover, the genome of B. subtilis MG-1 was predicted to possess 77 transporter-related genes. This study demonstrates that B. subtilis MG-1 has potential for use as a probiotic, and further studies should be performed to develop it as a probiotic additive in animal feed to promote animal health.

16S rRNA Gene Amplicon Sequencing of Gut Microbiota Affected by Four Probiotic Strains in Mice.

Probiotics, also referred to as "living microorganisms," are mostly present in the genitals and the guts of animals. They can increase an animal's immunity, aid in digestion and absorption, control gut microbiota, protect against sickness, and even fight cancer. However, the differences in the effects of different types of probiotics on host gut microbiota composition are still unclear. In this study, 21-day-old specific pathogen-free (SPF) mice were gavaged with Lactobacillus acidophilus (La), Lactiplantibacillus plantarum (Lp), Bacillus subtilis (Bs), Enterococcus faecalis (Ef), LB broth medium, and MRS broth medium. We sequenced 16S rRNA from fecal samples from each group 14 d after gavaging. According to the results, there were significant differences among the six groups of samples in Firmicutes, Bacteroidetes, Proteobacteria, Bacteroidetes, Actinobacteria, and Desferribacter (p < 0.01) at the phylum level. Lactobacillus, Erysipelaceae Clostridium, Bacteroides, Brautella, Trichospiraceae Clostridium, Verummicroaceae Ruminococcus, Ruminococcus, Prevotella, Shigella, and Clostridium Clostridium differed significantly at the genus level (p < 0.01). Four kinds of probiotic changes in the composition and structure of the gut microbiota in mice were observed, but they did not cause changes in the diversity of the gut microbiota. In conclusion, the use of different probiotics resulted in different changes in the gut microbiota of the mice, including genera that some probiotics decreased and genera that some pathogens increased. According to the results of this study, different probiotic strains have different effects on the gut microbiota of mice, which may provide new ideas for the mechanism of action and application of microecological agents.

A retrospective analysis of preemptive pharmacogenomic testing in 22,918 individuals from China.

BACKGROUND: Pharmacogenomics (PGx) examines the influence of genetic variation on drug responses. With more and more Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines published, PGx is gradually shifting from the reactive testing of single gene toward the preemptive testing of multiple genes. But the profile of PGx genes, especially for the intra-country diversity, is not well understood in China. METHODS: We retrospectively collected preemptive PGx testing data of 22,918 participants from 20 provinces of China, analyzed frequencies of alleles, genotypes and phenotypes of pharmacogenes, predicted drug responses for each participant, and performed comparisons between different provinces. RESULTS AND CONCLUSION: After analyzing 15 pharmacogenes from CPIC guidelines of 31 drugs, we found that 99.97% of individuals may have an atypical response to at least one drug; the participants carry actionable genotypes leading to atypical dosage recommendation for a median of eight drugs. Over 99% of the participants were recommended a decreased warfarin dose based on genetic factors. There were 20 drugs with high-risk ratios from 0.18% to 58.25%, in which clopidogrel showed the highest high-risk ratio. In addition, the high-risk ratio of rasburicase in GUANGDONG (risk ratio (RR) = 13.17, 95%CI:4.06-33.22, p < 0.001) and GUANGXI (RR = 23.44, 95%CI:8.83-52.85, p < 0.001) were significantly higher than that in all provinces. Furthermore, the diversity we observed among 20 provinces suggests that preemptive PGx testing in different geographical regions in China may need to pay more attention to specific genes. These results emphasize the importance of preemptive PGx testing and provide essential evidence for promoting clinical implementation in China.

Clinical validation of visual LAMP and qLAMP assays for the rapid detection of Toxoplasma gondii.

Humans are exposed to Toxoplasma gondii infection as pet cats gradually become family members and represent an increasing public health risk worldwide. Toxoplasmosis diagnosis constitutes an important measure for disease prevention and control. In this study, real-time fluorescence quantitative loop-mediated isothermal amplification (qLAMP) and visual LAMP detection technologies were established to conduct tests of T. gondii based on the membrane DNA extraction method, and the optimal detection mix was determined by adding the protective reagent trehalose and screening the concentrations of Mg2+ and dNTPs. Paraffin and lyophilization were used to reduce and even remove aerosol pollution, constructing a detailed anti-contamination protocol. Based on the positive standard plasmid DNA, the LODs of qLAMP and visual LAMP were 92 copies/µL and 92 copies/µL, and the standard curve of qLAMP was Y=2.9503X+20.8992 with R2 = 0.99. The applicability of the qLAMP and visual LAMP assays in disease diagnosis was assessed by evaluating 200 clinical cat faeces samples. The assays showed good diagnostic consistency, with kappa values of 1.0 and 0.99 compared with TaqMan qPCR, respectively. Compared with TaqMan qPCR, the diagnostic specificity/sensitivity of qLAMP and visual LAMP were 100%/100% and 100%/80%, respectively. The qLAMP and visual LAMP assays reported here are rapid and simple tests without extensive sample preparation and have a short turnaround time within 60 min, making them suitable for point-of-care testing.

A Novel Cerium(IV)-Based Metal-Organic Framework for CO2 Chemical Fixation and Photocatalytic Overall Water Splitting.

Cerium (IV)-based metal-organic frameworks (MOFs) are highly desirable due to their unique potential in fields such as redox catalysis and photocatalysis. However, due to the high reduction potential of CeIV species in solution, it is still a great challenge to synthesize CeIV -MOFs with novel structures, which are extremely dominated by the hexanuclear Ce-O cluster inorganic building units (IBUs). Herein, a Ce-O IBU chain containing CeIV -MOF, CSUST-3 (CSUST: Changsha University of Science and Technology), was successfully prepared using the kinetic stabilization study of UiO-66(Ce)-NDC (H2 NDC=2,6-naphthalenedicarboxylic acid). Furthermore, owing to the superior redox activity, Lewis acidity and semiconductor-like behavior owing to Ce4+ , activated CSUST-3 was demonstrated to be an excellent catalyst for CO2 chemical fixation. One-pot synthesis of styrene carbonate from styrene and CO2 was achieved under mild conditions (1 atm CO2 , 80 °C, and solvent free). Moreover, activated CSUST-3 was shown to be a remarkable co-catalyst-free photocatalyst for overall water splitting (OWS), rendering 59 µmol g-1 h-1 of H2 and 22 µmol g-1 h-1 of O2 under simulated sunlight irradiation (Na2 S-Na2 SO3 as sacrificial agent).

Fabrication of Porous Aluminum Coating by Cored Wire Arc Spray for Anchoring Antifouling Hydrogel Layer.

Biofouling has been persisting as a worldwide problem due to the difficulties in finding efficient environment-friendly antifouling coatings for long-term applications. Developing novel coatings with desired antifouling properties has been one of the research goals for surface coating community. Recently hydrogel coating was proposed to serve as antifouling layer, for it offers the advantages of the ease of incorporating green biocides, and resisting attachment of microorganisms by its soft surface. Yet poor adhesion of the hydrogel on steel surfaces is a big concern. In this study, porous matrix aluminum coatings were fabricated by cored wire arc spray, and the sizes of the pores in the aluminum (Al) coatings were controlled by altering the size of the cored powder of sodium chloride. Silicone hydrogel was further deposited on the porous coating. The hydrogel penetrated into the open pores of the porous Al coatings, and the porous Al structure significantly enhanced the adhesion of the hydrogel. In addition, hydrogel coating exhibited very encouraging antifouling properties.

Oxygen Vacancy-Rich Mixed-Valence Cerium MOF: An Efficient Separator Coating to High-Performance Lithium-Sulfur Batteries.

Mixed-valence metal-organic frameworks (MOFs) have exhibited unique potential in fields such as catalysis and gas separation. However, it is still an open challenge to prepare mixed-valence MOFs with isolated Ce(IV, III) arrays due to the easy formation of CeIII under the synthetic conditions for MOFs. Meanwhile, the performance of Li-S batteries is greatly limited by the fatal shuttle effect and the slow transmission rate of Li+ caused by the inherent characteristics of sulfur species. Here, we report a mixed-valence cerium MOF, named CSUST-1 (CSUST stands for Changsha University of Science and Technology), with isolated Ce(IV, III) arrays and abundant oxygen vacancies (OVs), synthesized as guided by the facile and elaborate kinetic stability study of UiO-66(Ce), to work as an efficient separator coating for circumventing both issues at the same time. Benefiting from the synergistic function of the Ce(IV, III) arrays (redox couples), the abundant OVs, and the open Ce sites within CSUST-1, the CSUST-1/CNT composite, as a separator coating material in the Li-S battery, can remarkably accelerate the redox kinetics of the polysulfides and the Li+ transportation. Consequently, the Li-S cell with the CSUST-1/CNT-coated separator exhibited a high initial specific capacity of 1468 mA h/g at 0.1 C and maintained long-term stability for a capacity of 538 mA h/g after 1200 cycles at 2 C with a decay rate of only 0.037% per cycle. Even at a high sulfur loading of 8 mg/cm2, the cell with the CSUST/CNT-coated separator still demonstrated excellent performance with an initial areal capacity of 8.7 mA h/cm2 at 0.1 C and retained the areal capacity of 6.1 mA h/cm2 after 60 cycles.

Bioleaching of silicon in electrolytic manganese residue using single and mixed silicate bacteria.

Electrolytic manganese residue (EMR) is a type of industrial solid waste with a high silicon content. The silicon in EMR can be used as an essential nutrient for plant growth, but most of the silicon is found in silicate minerals with very low water solubility, that is, it is inactive silicon and cannot be absorbed and used by plants directly. Thus, developing a highly effective and environmentally friendly process for the activation of silicon in EMR is important both for reusing solid waste and environmental sustainability. The aim of this study was to investigate the desilication of EMR using cultures of Paenibacillus mucilaginosus (PM) and Bacillus circulans (BC). The results showed that the two types of silicate bacteria and a mixed strain of them were all able to extract silicon from EMR with a high efficiency, but the desilication performance of the mixed PM and BC was the best. Fourier transform infrared spectroscopy indicated that silicate bacteria can induce a suitable micro-environment near the EMR particles and release Si into the solution through their metabolism. X-ray diffraction analysis confirmed that layered crystal minerals, such as muscovite and diopside, were more likely to be destroyed by the bacterial action than quartz, which has a frame structure. Scanning electron microscopy-energy dispersive spectrometry proved that the silicate structures were destroyed and that Si in the residue was decreased, indicating the dissolution of silicon under the action of these microorganisms. This study suggests that bioleaching may be a promising method for the activation of silicon in EMR.

Genomic Analysis of a Highly Pathogenic Porcine Reproductive and Respiratory Syndrome Virus Circulating in Pig Farms in West China.

Porcine reproductive and respiratory syndrome virus (PRRSV), which leads to tremendous economic losses worldwide, is currently one of the most threatening viruses for the swine industry. However, PRRSV outbreaks in West China are rarely reported, even though the virus has remained active for a long time across the country. In this study, we report an outbreak of the highly pathogenic PRRSV strain QTX, isolated from a pig farm located in Ningxia, a province in West China.

Signature Gene Identification of Cancer Occurrence and Pattern Recognition.

To identify signature genes for the pathogenesis of cancer, which provides a theoretical support for prevention and early diagnosis of cancer. The pattern recognition method was used to analyze the genome-wide gene expression data, which was collected from the The Cancer Genome Atlas (TCGA) database. For the transcription of invasive breast carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, colon adenocarcinoma, renal clear-cell carcinoma, thyroid carcinoma, and hepatocellular carcinoma of the seven cancers, the signature genes were selected by means of a combination of statistical methods, such as correlation, t-test, confidence interval, etc. Modeling by artificial neural network model, the accuracy can be as high as 98% for the TCGA data and as high as 92% for the Gene Expression Omnibus (GEO) independent data, the recognition accuracy of stage I is more than 95%, which is higher compared with the previous study. The common genes emerging in five cancers were obtained from the signature genes of seven cancers, PID1, and SPTBN2. At the same time, we obtain three common pathways of cancer by using Kyoto Encyclopedia of Genes and Genomes' pathway analysis. A functional analysis of the pathways shows their close relationship at the level of gene regulation, which indicted that the identified signature genes play an important role in the pathogenesis of cancer and is very important for understanding the pathogenesis of cancer and the early diagnosis.

ATG16L1 phosphorylation is oppositely regulated by CSNK2/casein kinase 2 and PPP1/protein phosphatase 1 which determines the fate of cardiomyocytes during hypoxia/reoxygenation.

Recent studies have shown that the phosphorylation and dephosphorylation of ULK1 and ATG13 are related to autophagy activity. Although ATG16L1 is absolutely required for autophagy induction by affecting the formation of autophagosomes, the post-translational modification of ATG16L1 remains elusive. Here, we explored the regulatory mechanism and role of ATG16L1 phosphorylation for autophagy induction in cardiomyocytes. We showed that ATG16L1 was a phosphoprotein, because phosphorylation of ATG16L1 was detected in rat cardiomyocytes during hypoxia/reoxygenation (H/R). We not only demonstrated that CSNK2 (casein kinase 2) phosphorylated ATG16L1, but also identified the highly conserved Ser139 as the critical phosphorylation residue for CSNK2. We further established that ATG16L1 associated with the ATG12-ATG5 complex in a Ser139 phosphorylation-dependent manner. In agreement with this finding, CSNK2 inhibitor disrupted the ATG12-ATG5-ATG16L1 complex. Importantly, phosphorylation of ATG16L1 on Ser139 was responsible for H/R-induced autophagy in cardiomyocytes, which protects cardiomyocytes from apoptosis. Conversely, we determined that wild-type PPP1 (protein phosphatase 1), but not the inactive mutant, associated with ATG16L1 and antagonized CSNK2-mediated phosphorylation of ATG16L1. Interestingly, one RVxF consensus site for PPP1 binding in the C-terminal tail of ATG16L1 was identified; mutation of this site disrupted its association with ATG16L1. Notably, CSNK2 also associated with PPP1, but ATG16L1 depletion impaired the interaction between CSNK2 and PPP1. Collectively, these data identify ATG16L1 as a bona fide physiological CSNK2 and PPP1 substrate, which reveals a novel molecular link from CSNK2 to activation of the autophagy-specific ATG12-ATG5-ATG16L1 complex and autophagy induction.

Characterization of endogenous pyridoxal 5'-phosphate-dependent alanine racemase from Bacillus pseudofirmus OF4.

An open reading frame of 1100 bp in the partially sequenced genome sequence of alkaliphilic Bacillus pseudofirmus OF4 was identified as a putative alanine racemase gene (dadX(OF4)), which was cloned and expressed in Escherichia coli BL21 (DE3). The encoded protein DadX(OF4) was purified to homogeneity by His(6)-tag affinity column, gel filtration and ion-exchange chromatography. The amino acid sequence has highest identity with the known alanine racemase from Oceanobacillus iheyensis HTE831 (48%). The protein was a dimeric, endogenous PLP-dependent enzyme, which was demonstrated by absorption spectra and enzyme activity with or without PLP. The racemization temperature optimum was 40 degrees C and the optimal pH was 10.5. The kinetic parameters K(m) and V(max) at 40 degrees C of alanine racemase, determined by HPLC analysis, were 41.79 mM, 10,500 units/mg for L-alanine and 14.91 mM, 3708 units/mg for D-alanine, respectively.

Long terminal repeat sequences from virulent and attenuated equine infectious anemia virus demonstrate distinct promoter activities.

In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.