Modifying Escherichia coli to mimic Shigella for medical microbiology laboratory teaching: a new strategy to improve biosafety in class.

Introduction: Laboratory teaching of medical microbiology involves highly pathogenic microorganisms, thus posing potential biosafety risks to the students and the teacher. To address these risks, non/low-pathogenic microorganisms were modified to mimic highly pathogenic ones or highly pathogenic microorganisms were attenuated directly using the CRISPR/Cas9 technology. This study describes the modification of Escherichia coli DH5α to mimic Shigella and its evaluation as a safe alternative for medical laboratory teaching. Methods: To generate E. coli DH5α△FliC△tnaA2a, the tnaA and FliC genes in E. coli DH5α were knocked out using CRISPR/Cas9 technology; a plasmid bearing the O-antigen determinant of S. flexneri 2a was then constructed and transformed. Acid tolerance assays and guinea pig eye tests were used to assess the viability and pathogenicity, respectively. Questionnaires were used to analyze teaching effectiveness and the opinions of teachers and students. Results: The survey revealed that most teachers and students were inclined towards real-time laboratory classes than virtual classes or observation of plastic specimens. However, many students did not abide by the safety regulations, and most encountered potential biosafety hazards in the laboratory. E. coli DH5α△FliC△tnaA2a was biochemically and antigenically analogous to S. flexneri 2a and had lower resistance to acid than E. coli. There was no toxicity observed in guinea pigs. Most of teachers and students were unable to distinguish E. coli DH5α△FliC△tnaA2a from pure S. flexneri 2a in class. Students who used E. coli DH5α△FliC△tnaA2a in their practice had similar performance in simulated examinations compared to students who used real S. flexneri 2a, but significantly higher than the virtual experimental group. Discussion: This approach can be applied to other high-risk pathogenic microorganisms to reduce the potential biosafety risks in medical laboratory-based teaching and provide a new strategy for the development of experimental materials.

Desmethoxycurcumin aids IFNα's anti-HBV activity by antagonising CRYAB reduction and stabilising IFNAR1 protein.

The eradication of chronic hepatitis B (CHB) caused by hepatitis B virus (HBV) infection is a crucial goal in clinical practice. Enhancing the anti-HBV activity of interferon type I (IFNI) is a key strategy for achieving a functional cure for CHB. In this study, we investigated the effect of combined treatment with IFNα and Desmethoxycurcumin (DMC) on HBV replication in HepG2 cells and explored the underlying mechanism. Our results indicated IFNα alone was ineffective in completely inhibiting HBV replication, which was attributed to the virus-induced down-regulation of IFNI receptor 1 (IFNAR1) protein. However, the addition of a low dose of DMC significantly synergized with IFNα, leading to notable enhancement of IFNα anti-HBV activity. This effect was achieved by stabilising the IFNAR1 protein. Further investigation revealed that low dose DMC effectively blocked the ubiquitination-mediated degradation of IFNAR1, which was accomplished by rescuing the protein levels of alphaB-crystallin (CRYAB) and orchestrating the interaction between CRYAB and the E3 ubiquitin ligase, ß-Trcp. Importantly, over-expression of CRYAB was found to favour the antiviral activity of IFNα against HBV replication. In conclusion, our study demonstrates that low-dose DMC enhanced the anti-HBV activity of IFNα by counteracting the reduction of CRYAB and stabilising the IFNAR1 protein.

Heat shock protein: a double-edged sword linking innate immunity and hepatitis B virus infection.

Heat shock proteins (HSPs), which have a variety of functions, are one of the stress protein families. In recent years, They have been reported to play a dual role in hepatitis B virus (HBV) which as persistent infection which is associated with, cirrhosis and liver cancer. In this article, we have summarized the regulatory mechanisms between HSPs and viruses, especially HBV and associated diseases based on HSP biological functions of in response to viral infections. In view of their potential as broad-spectrum antiviral targets, we have also discuss current progress and challenges in drug development based on HSPs, as well as the potential applications of agents that have been evaluated clinically in HBV treatment.

Intramuscular injection of tetracycline decreased gut microbial diversity in mouse.

Antibiotics contribute a lot to human beings and can kill bacteria effectively. However, more and more studies show that antibiotics can disturb the intestinal microbial community. It has been widely reported that oral antibiotics can reduce the diversity of intestinal microflora, but the effect of intramuscular injection on intestinal microflora is less studied. In this study, we sequenced the intestinal microflora of mice treated with tetracycline by 16SrRNA method, and found that intramuscular injection of tetracycline (TET) can also reduce the intestinal microbial richness of mice. In addition, the results showed that within a certain range (3 mg), with the increase of TET injection concentration, the wind of intestinal microflora in mice decreased significantly. When the injection concentration reached saturation, although the amount of TET injection was increased, the degree of intestinal flora affected was not increased. The results showed that the degree of diversity decrease was in direct proportion to the amount of tetracycline injection in the saturated concentration, but not positively related to the high amount of TET injection after exceeding the saturated concentration.

Identification of the Functional Domain of HPIV3 Matrix Protein Interacting with Nucleocapsid Protein.

Human parainfluenza virus type 3 (HPIV3) is the main pathogen that causes respiratory infections in infants, young children, and the elderly. Currently, there are no vaccines and effective anti-infective drugs. Studying the replication and proliferation mechanism of HPIV3 is helpful for exploring the targets of anti-HPIV3 infection. Matrix protein (M) and nucleocapsid protein (N) are two key structural proteins of HPIV3 that exert important functions in HPIV3 proliferation. Herein, we aim to clarify the functional domains of M and N interaction. HPIV3 M and N expression plasmids of pCAGGS-HA-M and pCAGGS-N-Myc/Flag, M C-terminal truncation mutant plasmids of pCAGGSHA-MΔC120, MΔC170, MΔC190, and MΔC210, and M C-terminal plasmid of pCAGGS-HA-MC190 and C-terminal deletion mutant plasmid of pCAGGS-MΔN143-182 were constructed. By using immunoprecipitation, immunofluorescence, and virus-like particle (VLP) germination experiments, we found that N was encapsulated into M-mediated VLP through N and M interaction. Moreover, the C-terminus of the M played a key role in the interaction between M and N. The C-terminus of the M encapsulated the N into the VLP. We finally determined that the 143-182 amino acids in the M were the functional regions that encapsulated the N into the M-mediated VLP. Our findings confirmed the interaction between M and N and for the first time clarified that the 143-182 amino acid region in M was the functional region that interacted with N, which provides a molecular basis for exploring effective anti-HPIV3 targets.

Involvement of Actin-Regulating Factor Cofilin in the Inclusion Body Formation and RNA Synthesis of Human Parainfluenza Virus Type 3 via Interaction With the Nucleoprotein.

Human parainfluenza virus type 3 (HPIV3) is one of the primary pathogens that causing severe respiratory tract diseases in newborns and infants. It could induce inclusion bodies (IBs) in infected cells. Comprised of viral nucleoprotein (N) and phosphoprotein (P), as well as some cellular factors, HPIV3 IBs are unique platform for efficient viral synthesis. Although several studies have demonstrated the formation of IBs, little is known about cellular proteins involved in HPIV3 IBs formation. By quantitative real-time PCR assays after cytochalasin D treatment, we found actin microfilaments of the cytoskeleton were indispensible for HPIV3 RNA synthesis. Using co-immunoprecipitation and immunofluorescence assays, an actin-modulating protein, cofilin was found to involve in the IBs formation through interaction with the N protein in N-P induced IBs complex. Viral IBs formation reduced upon RNA interference knockdown of cellular cofilin, thus viral RNA synthesis and protein expression level were also suppressed. What's more, the inactive form of cofilin, p-cofilin was increased after HPIV3 infection, and phosphorylation of cofilin was required for interacting with N-P complex and IBs formation. We further identified that the regions in cofilin interacting with N protein lies in the C-terminus. Our findings for the first time to state that cellular cofilin involves in HPIV3 IBs and interaction with N is critical for cofilin to aid IBs formation and enhancing viral RNA synthesis.

[Extraction, Purification and Identification of a Dexamethasone-degrading Enzymes Generated by Pseudomonas Alcaligenes].

In this research a strain of isolated Pseudomonas alcaligenes which causes degradation of dexamethasone was acclimated further and its proteins of every position in the bacterium were separated by the osmotic shock method. The separated intracellular proteins which had the highest enzyme activity were extracted by the salting out with ammonium sulfate and were purified with the cation exchange chromatography and gel chromatography. The purified proteins which was active to cause degradation of dexamethasone had been detected were cut with enzyme and were analyzed with mass spectrometry. The results showed that the degradation rate to dexamethasone by acclimated Pseudomonas alcaligenes were increased from 23.63% to 52.84%. The degrading enzymes were located mainly in the intracellular of the bacteria and its molecular weight was about 41 kD. The specific activity of the purified degrading enzymes were achieved to 1.02 U x mg(-1). Its 5-peptide amino acid sequences were consistent with some sequences of the isovaleryl-CoA dehydrogenase. The protein enzyme may be a new kind degrading enzyme of steroidal compounds. Our experimental results provided new strategies for cleanup of dexamethasone in water environment with microbial bioremediation technique.