Construction of retrovirus vector with HBV Pol gene and its expression in vitro

Objective: To construct retroviral vector with HBV Pol gene and study its expression in vitro. Methods:The recombinant plasmid HBV2/pBR322 containing 2 copies of HBV full-length gene was provided as the amplification template. The PCR product was cloned into pMD 18-T and subcloned into pMSCVneo and pLNCX2 to construct the recombinant retroviral vectors with HBV Pol gene named by HBV P/pMSCVneo and HBV P/pLNCX2. HBV Pol gene was detected by RTPCR after transfecting the recombinant plasmids into SMMC7721 cells with liposome and G418 selection. Results: The retroviral vector with HBV Pol gene was successfully constructed and the expression of HBV Pol gene in vitro was detected by RTPCR. Conclusion: The retroviral vector with HBV Pol gene can be obtained, which will provide a new insight on the function of HBV Pol gene.

Simultaneous multiplex genome loci editing of Halomonas bluephagenesis using an engineered CRISPR-guided base editor

Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various products.However,the simultaneous editing of multiple loci in H.bluephagenesis TD remains a significant challenge.Herein,we report the development of a multiple loci genome editing system,named CRISPR-deaminase-assisted base editor(CRISPR-BE)in H.bluephagenesis TD.This system comprises two components:a cytidine(CRISPR-cBE)and an adenosine(CRISPR-aBE)deaminase-based base editor.CRISPR-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100%within a 7-nt editing window in H.bluephagenesis TD.Similarly,CRISPR-aBE demonstrates an efficiency of up to 100%in converting adenosine to guanosine mutation within a 7-nt editing window.CRISPR-cBE has been further validated and successfully employed for simultaneous multiplexed editing in H.bluephagenesis TD.Our findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop codon.This system achieved an editing efficiency of 100%and 41.67%in inactivating two genes and three genes,respectively.By substituting the Pcas promoter with the inducible promoter PMmp1,we optimized CRISPR-cBE system and ultimately achieved 100%editing efficiency in inactivating three genes.In conclusion,our research offers a robust and efficient method for concurrently modifying multiple loci in H.bluephagenesis TD,opening up vast possibilities for industrial applications in the future.

Effects of COVID-19 pandemic on human fertility:A scientometric and visualized evaluation

CovID-19,also known as coronavirus disease 2019,is a novel coronavirus disease with high infectivity,strong heterogeneity,and long incubation period(generally 3-14 days).Its main symptoms and signs include fever,dry cough,nasal congestion,fatigue,disorientation,lymphopenia,and dyspnea.The short-term and long-term impacts of covID-19 on human health,particularly its effects on human reproduction and offspring development,continue to receive significant concerns,as they may lead to potential sequelae for several decades or even centuries.

Optimizing a high-sensitivity NanoLuc-based bioluminescence system for in vivo evaluation of antimicrobial treatment

Focal and systemic infections are serious threats to human health.Preclinical models enable the development of new drugs and therapeutic regimens.In vivo,animal bioluminescence(BL)imaging has been used with bacterial reporter strains to evaluate antimicrobial treatment effects.However,high-sensitivity bioluminescent systems are required because of the limited tissue penetration and low brightness of the BL signals of existing approaches.Here,we report that NanoLuc(Nluc)showed better performance than LuxCDABE in bacteria.However,the retention rate of plasmid constructs in bacteria was low.To construct stable Staphylococcus aureus reporter strains,a partner protein enolase(Eno)was identified by screening of S.aureus strain USA300 for fusion expression of Nluc-based luciferases,including Nluc,Teluc,and Antares2.Different substrates,such as hydrofurimazine(HFZ),furimazine(FUR),and diphenylterazine(DTZ),were used to optimize a stable reporter strain/substrate pair for BL imaging.S.aureus USA300/Eno-Antares2/HFZ produced the highest number of photons of orange-red light in vitro and enabled sensitive BL tracking of S.aureus in vivo,with sensitivities of approximately 10 CFU from mouse skin and 750 CFU from mouse kidneys.USA300/Eno-Antares2/HFZ was a powerful combination based on the longitudinal evaluation of the therapeutic efficacy of antibiotics.The optimized S.aureus Eno-Antares2/HFZ pair provides a technological advancement for the in vivo evaluation of antimicrobial treatment.

Effectiveness of omega-3 polyunsaturated fatty acids against microbial pathogens

Microorganisms provide both beneficial and harmful effects to human beings. Beneficial effects come from the symbiotic relationship that exists between humans and microbiota, but then several human illnesses have turned some friendly microbes into opportunistic pathogens, causing several microbial-related diseases. Various efforts have been made to create and utilize antimicrobial agents in the treatment and prevention of these infections, but such efforts have been hampered by the emergence of antimicrobial resistance. Despite extensive studies on drug dis- covery to alleviate this problem, issues with the toxicity and tolerance of certain compounds and continuous microbial evolution have forced researchers to focus on screening various phytochemical dietary compounds for antimicrobial activity. Linolenic acid and its derivatives （eicosapentaenoic acid and docosahexaenoic acid） are omega-3 fatty acids that have been studied due to their role in human health, being important for the brain, the eye, the cardiovascular system, and general human growth. However, their utilization as antimicrobial agents has not been widely appreciated, perhaps due to a lack of understanding of antimicrobial mechanisms, toxicity, and route of administration. Therefore, this review focuses on the efficacy, mechanism, and toxicity of omega-3 fatty acids as alternative therapeutic agents for treating and preventing diseases associated with pathogenic microorganisms.

Antibodies against ribosomal protein S29(RPS29)fused with glutathione's transferase specially react with native RPS29 in mouse and human cells

The ribosomal protein S29 also known as RPS29, is not only a component of the 40S subunit of ribosome, but also involved in embryonic development, oncogenesis and other pathologic conditions. However, rare commercial antibody against RPS29 restricts the discovery of precise physiological and pathological function of this protein. In this study, the whole RPS29 gene was inserted into plasmid pGEX-6p-1 to express glutathione＇s transferase （GST） fusion proteins in Escherichia eoli （E. coli） strain BL21. High yields of soluble recombinant proteins were obtained. Mice were immunized with the recombinant RPS29 protein. The serum from the immunized mice could specially react with purified recombinant RPS29 proteins and native RPS29 proteins in CCE cells by western blotting, immunofluorescence staining and flow cytometric analysis. Further more the polyclonal antibodies also reacted specifically with human cell strain ECV304, which showed typical cytoplasmatic fluorescence. The polyclonal antibodies we prepared would be an available tool for studying the roles of RPS29 in embryonic development and human diseases.

Desulfovibrio desulfuricans aggravates atherosclerosis by enhancing intestinal permeability and endothelial TLR4/NF-kB pathway in Apoe^(-/-) mice

It is increasingly aware that gut microbiota is closely associated with atherosclerosis.However,which and how specific gut bacteria regulate the progression of atherosclerosis is still poorly understood.In this study,modified linear discriminant analysis was performed in comparing the gut microbiota structures of atherosclerotic and non-atherosclerotic mice,and Desulfovibrio desulfuricans(D.desulfuricans)was found to be associatedwith atherosclerosis.D.desulfuricans-treated Apoe^(-/-) mice showed significantly aggravated atherosclerosis.The proatherogenic effect of D.desulfuricans was attributed to its ability to increase intestinal permeability and subsequent raise in the transit of lipopolysaccharide(LPS)from the intestine to the bloodstream.Excessive LPS in the blood can elicit local and systemic inflammation and activate Toll-like receptor 4(TLR4)/nuclear factor-kB(NF-kB)signaling of endothelial cells.TAK-242,a specific inhibitor of TLR4,can ameliorate the development of D.desulfuricansinduced atherosclerosis by blocking the LPS-induced activation of TLR4/NF-kB signaling.

Beyond the classroom lecture： Liang Wang＇s personal war on tuberculosis in China

Tuberculosis, historically one of the deadliest and most prevalent infectious diseases, is caused by the bacterium Mycobacterium tuberculosis. It is estimated that one-third of the world＇s population is infected by this agent （Zumla et al., 2014）, and tuberculosis is therefore a major topic for medical students. M. tuberculosis is a small aerobic bacillus, with a remarkably high lipid-content cell wall that plays a critical role in its pathoeenicitv.