The hepatitis B virus (HBV) persistently infects roughly 300 million individuals worldwide, and the permanent suppression of the transcription within the covalently closed circular DNA (cccDNA), the episomal viral DNA reservoir, is a significant therapeutic focus for hepatitis B. However, the underlying mechanisms involved in the transcription of cccDNA are not entirely clear. Through investigation of cccDNA in wild-type HBV (HBV-WT) and transcriptionally inactive HBV with a defective HBV X gene (HBV-X), we discovered a statistically significant difference in their association with promyelocytic leukemia (PML) bodies. HBV-X cccDNA exhibited more frequent colocalization with PML bodies than HBV-WT cccDNA. A siRNA screen targeting 91 PML body-related proteins, identified SMC5-SMC6 localization factor 2 (SLF2) as a host restriction factor of cccDNA transcription. Subsequent investigations demonstrated that SLF2 facilitates HBV cccDNA entrapment within PML bodies through interaction with the SMC5/6 complex. Subsequently, our investigation revealed that the portion of SLF2 encompassing residues 590 to 710 interacts with and brings in the SMC5/6 complex to PML bodies, and the C-terminal domain of SLF2 containing this region is indispensable for the suppression of cccDNA transcription. PHHs primary human hepatocytes Research on cellular mechanisms that impede HBV infection provides novel perspectives, strengthening the rationale for targeting the HBx pathway to restrain HBV activity. The pervasive issue of chronic hepatitis B infection demonstrates its enduring global health impact. Despite their widespread use, current antiviral treatments often fall short of eradicating the infection because they cannot eliminate the viral reservoir, cccDNA, located in the nucleus of infected cells. Thus, the complete and lasting inhibition of HBV cccDNA transcription offers a compelling strategy for curing HBV. We discovered new details on cellular mechanisms that obstruct HBV infection, showcasing SLF2's activity in guiding HBV cccDNA to PML bodies for transcriptional repression. These observations are highly pertinent to the ongoing effort in creating antiviral agents to treat hepatitis B.
The critical functions of gut microbiota in severe acute pancreatitis-associated acute lung injury (SAP-ALI) are being extensively explored, and recent advancements in the gut-lung axis have offered promising therapeutic strategies for SAP-ALI. Qingyi decoction (QYD), a time-tested traditional Chinese medicine (TCM) approach, is commonly used in clinical settings for the care of SAP-ALI patients. Although this is the case, the fundamental mechanisms remain to be fully deciphered. In an attempt to clarify the roles of the gut microbiota, we employed a caerulein plus lipopolysaccharide (LPS)-induced SAP-ALI mouse model and an antibiotics (Abx) cocktail-induced pseudogermfree mouse model, along with QYD administration, to investigate its underlying mechanisms. The immunohistochemical assessment showed a possible correlation between a decrease in the intestinal bacterial population and the severity of SAP-ALI and the performance of the intestinal barrier. QYD treatment facilitated a partial recovery of gut microbiota composition, evidenced by a lower Firmicutes/Bacteroidetes ratio and a greater prevalence of bacteria producing short-chain fatty acids (SCFAs). A rise in the levels of short-chain fatty acids (SCFAs), predominantly propionate and butyrate, was observed in feces, intestinal contents, blood serum, and lung tissue, which, overall, matched changes within the gut microbial community. Biochemical analyses using Western blotting and RT-qPCR techniques revealed activation of the AMPK/NF-κB/NLRP3 signaling pathway subsequent to oral QYD administration. This activation may be correlated with QYD's influence on short-chain fatty acids (SCFAs) within the intestine and lungs. Our research, in its final analysis, presents novel understanding of treating SAP-ALI through adjustments to the gut microbiota, promising future clinical implications. Gut microbiota plays a pivotal role in determining the severity of SAP-ALI and the integrity of the intestinal barrier. Significant increases in the relative abundance of gut pathogens, including Escherichia, Enterococcus, Enterobacter, Peptostreptococcus, and Helicobacter, were observed following participation in the SAP program. Concurrently, QYD treatment diminished pathogenic bacteria while augmenting the relative abundance of SCFA-producing bacteria, including Bacteroides, Roseburia, Parabacteroides, Prevotella, and Akkermansia. Furthermore, the AMPK/NF-κB/NLRP3 pathway, facilitated by short-chain fatty acids (SCFAs) along the gut-lung axis, is crucial in mitigating the development of SAP-ALI, thereby reducing systemic inflammation and restoring the integrity of the intestinal barrier.
High-alcohol-producing K. pneumoniae (HiAlc Kpn) strains, in individuals afflicted with NAFLD, generate excess endogenous alcohol in the intestinal tract, glucose being the principal carbon resource, thereby potentially causing non-alcoholic fatty liver disease. The effect of glucose on the HiAlc Kpn's stress response, particularly when subjected to antibiotics, is not completely understood. Glucose was found to contribute to heightened polymyxin resistance in HiAlc Kpn strains, as evidenced in this investigation. In HiAlc Kpn cells, glucose's effect was to inhibit crp expression. This correlated with increased synthesis of capsular polysaccharide (CPS). The consequential buildup of CPS then strengthened drug resistance in HiAlc Kpn cells. Polymyxins' pressure on HiAlc Kpn cells was mitigated by glucose-induced high ATP levels, culminating in enhanced resistance to the cytotoxic effects of antibiotics. The observation that the inhibition of CPS formation and the reduction in intracellular ATP levels effectively reversed glucose-induced polymyxins resistance is noteworthy. Our research elucidated the pathway through which glucose fosters polymyxin resistance in HiAlc Kpn cells, thus establishing a basis for the development of effective treatments for NAFLD stemming from HiAlc Kpn. The Kpn system, in conditions of elevated alcohol concentration (HiAlc), utilizes glucose to create an excess of endogenous alcohol, potentially driving the development of non-alcoholic fatty liver disease (NAFLD). In instances of infections due to carbapenem-resistant K. pneumoniae, polymyxins are typically deployed as the last available antibiotic option. Our research indicated that glucose boosts bacterial resistance to polymyxins through the augmentation of capsular polysaccharide and the preservation of intracellular ATP. This potentiated resistance increases the risk of treatment failure in patients with NAFLD due to multidrug-resistant HiAlc Kpn infections. Further exploration revealed the significance of glucose and the global regulator, CRP, in bacterial resistance mechanisms, and demonstrated that hindering CPS synthesis and lowering intracellular ATP levels effectively reversed glucose-mediated polymyxin resistance. Teniposide price Our research uncovers a correlation between glucose and the regulatory factor CRP and their effect on bacterial resistance to polymyxins, offering a basis for treating multidrug-resistant bacterial infections.
The peptidoglycan-targeting action of phage-encoded endolysins shows promise as a new antibacterial agent against Gram-positive bacteria, although the envelope structure of Gram-negative bacteria restricts their broader application. By engineering modifications, the effectiveness of endolysins in penetrating and combating bacteria can be enhanced. This research effort produced a screening platform designed to discover engineered Artificial-Bp7e (Art-Bp7e) endolysins possessing extracellular antibacterial activity against Escherichia coli. Upstream of the Bp7e endolysin gene, within the pColdTF vector, a chimeric endolysin library was generated by incorporating an oligonucleotide sequence consisting of 20 repeated NNK codons. E. coli BL21 cells were transformed with the Art-Bp7e plasmid library to express chimeric proteins. These proteins were then recovered through chloroform fumigation. The activity of these proteins was subsequently evaluated utilizing a spotting and colony-counting assay to identify potentially promising proteins. Examination of protein sequences demonstrated that every screened protein exhibiting extracellular activity possessed a chimeric peptide, featuring a positive charge and an alpha-helical structure. In addition, the protein Art-Bp7e6 was subject to further characterization. Broad-spectrum antibacterial activity was observed, effectively targeting E. coli (7 of 21), Salmonella enterica serovar Enteritidis (4 out of 10 isolates), Pseudomonas aeruginosa (3 of 10 isolates), and even Staphylococcus aureus (1 of 10 isolates). Childhood infections During transmembrane action, the chimeric Art-Bp7e6 peptide induced depolarization of the host cell envelope, enhanced its permeability, and enabled the Art-Bp7e6 peptide to traverse the envelope, thereby hydrolyzing the peptidoglycan. The screening platform demonstrated a successful identification of chimeric endolysins with the ability to combat Gram-negative bacteria externally, thereby providing a valuable framework for the continued search for engineered endolysins showcasing strong external activity against Gram-negative bacteria. The established platform's demonstrated adaptability and broad utility include the ability to screen a large variety of proteins. The Gram-negative bacterial envelope restricts the application of phage endolysins, motivating the creation of engineered forms to improve both antibacterial and penetrative properties. For the purpose of endolysin engineering and evaluation, a platform was created by us. A chimeric endolysin library, generated by fusing a random peptide to the phage endolysin Bp7e, was screened, resulting in the identification of engineered Art-Bp7e endolysins with extracellular activity effective against Gram-negative bacteria. The meticulously crafted Art-Bp7e exhibited a chimeric peptide possessing a substantial positive charge and an alpha-helical conformation, enabling Bp7e to effectively lyse Gram-negative bacteria across a broad spectrum of species. Unfettered by the limitations of cataloged proteins and peptides, the platform provides a substantial library capacity.