The cyclin-dependent kinase inhibitor 1A (CDKN1A), also known as p21 (WAF1/CIP1) modulates cell cycle, apoptosis, senescence and differentiation via specific protein–protein interactions with the cyclins, cyclin-dependent kinase (Cdk), and many others. Expression of the p21 gene is mainly regulated at the transcriptional level. By conducting both ligation-mediated PCR (LMPCR) and chromatin immunoprecipitation (ChIP) in vivo, we identified a functional target site for the transcription factor, nuclear factor I (NFI), in the basal promoter from the p21 gene. Transfection of recombinant constructs bearing mutations in the p21 NFI site demonstrated that NFI acts as a repressor of p21 gene expression in various types of cultured cells. Inhibition of NFI in human skin fibroblasts through RNAi considerably increased p21 promoter activity suggesting that NFI is a key repressor of p21 transcription. Overexpression of each of the four NFI isoforms in HCT116 cells established that each of them contribute to various extend to the repression of the p21 gene. Most of all, over-expression of NFI-B in doxorubicin, growth-arrested HCT116 increased the proportion of cells in the S-phase of the cell cycle whereas NFI-A and NFI-X reduced it, thereby establishing a role for NFI in the cell cycle dependent expression of p21.

Transcriptional regulation of the cyclin-dependent kinase inhibitor 1A (p21) gene by NFI in proliferating human cells

Hepatitis B virus (HBV) X protein (HBx) plays diverse roles in both viral life cycle and HBV-related carcinogenesis. Its interaction with DNA damage-binding protein 1 (DDB1) was shown to be essential for engendering cellular conditions favorable for optimal viral transcription and replication. Previously, we described a mouse monoclonal antibody against HBx (anti-HBx 2A7) recognizing HBx encoded by representative strains from 7 of 8 known HBV genotypes. In this work, we further characterized 2A7 in order to explore its potential usefulness in HBx-targeting applications. We demonstrated that 2A7 recognizes a linear epitope mapped to L89PKVLHKR96 on HBx, a segment that is highly conserved across genotypes and coincidentally overlaps with the DDB1-interacting segment. HBx-DDB1 binding could be inhibited by 2A7 in vitro, suggesting therapeutic potential. Nucleic acid and amino acid sequences of 2A7 were then obtained, which allowed construction of recombinant antibody and single chain variable fragments (scFv). 2A7-derived recombinant antibody and scFv recapitulate 2A7’s HBx-binding capacity and epitope specificity. We also reported preliminary results using cell-penetrating peptide for delivering 2A7 antibody across cell membrane to target intracellular HBx. Anti-HBx 2A7 and 2A7-derived scFv characterized here may give rise to novel HBx-targeting diagnostics and therapeutics for HBV- and HBx-related pathologies.

/pdf/characterization-and-engineering-of-broadly-reactive-12kvvntp0b.pdf

Characterization and engineering of broadly reactive monoclonal antibody against hepatitis B virus X protein that blocks its interaction with DDB1

Abstract Traditional high‐dose antibiotic treatments of intracellular methicillin‐resistant staphylococcus aureus (MRSA) are highly inefficient and associated with a high rate of infection relapse. As an effective antibacterial technology, sonodynamic therapy (SDT) may be able to break the dilemma. However, indiscriminate reactive oxygen species (ROS) release leads to potential side effects. This study incorporates Staphylococcal Protein A antibody‐modified Cu 2+ /tetracarboxyphenylporphyrin nanoparticles (Cu(II)NS‐SPA) into hydrogel microspheres (HAMA@Cu(II)NS‐SPA) to achieve precise eradication of intracellular bacteria. This eradication is under bioorthogonal activation mediated by bacillithiol (BSH) (internally) and ultrasound (US) (externally). To specify, the US responsiveness of Cu(II)NS‐SPA is restored when it is reduced to Cu(I)NS‐SPA by the BSH secreted characteristically by intracellular MRSA, thus forming a bioorthogonal activation with the external US, which confines ROS production within the infected M Φ . Under external US activation at 2 W cm −2 , over 95% of intracellular MRSA can be cleared. In vivo, a single injection of HAMA@Cu(II)NS‐SPA achieves up to two weeks of antibacterial sonodynamic therapy, reducing pro‐inflammatory factor expression by 90%, and peri‐implant bone trabeculae numbers exceed the control group by five times. In summary, these micro/nano hydrogel microspheres mediated by internal and external bioorthogonal activation can precisely eliminate intracellular MRSA, effectively treating multi‐drug resistant intracellular bacterial infections. 

Precise Clearance of Intracellular MRSA via Internally and Externally Mediated Bioorthogonal Activation of Micro/Nano Hydrogel Microspheres

Hepatitis B virus (HBV) is a global health problem. The HBV-mediated pathogenesis is a multistep, multi-factorial event involving crucial interplay between the chronically persisting viral factors and the host system. This triggers an immune-elicited regenerative process that eventually leads to chronic liver inflammation, scarring and formation of new tissue. Further, expression of viral antigens modulates cellular signaling, nuclear transactivation and host stress-responsive pathways that produce a cumulative effect in favor of viral survival and replication. Chronic HBV infection in India affects a sizeable population and warrants more epidemiological studies in this area. There is also an urgent need to adopt new measures to limit and manage the infection in view of our low socioeconomic status and ignorance about the disease. This review highlights the role of host and viral factors in pathogenesis and outlines their underlying mechanisms. A better understanding of the viral life cycle, mode of pathogenesis and outcome of the infection should be helpful in devising new strategies to control such wide range of HBV-associated disease and developing better interventional protocols.

Hepatitis B Virus: A Molecular Perspective

Bacteria coordinate how they respond to translation and osmotic stress by using antisense RNA crosstalk between the genes involved in the respective stress response pathways. Excretion of cytoplasmic protein (ECP) is a commonly observed phenomenon in bacteria, and this partial extracellular localisation of the intracellular proteome has been implicated in a variety of stress response mechanisms. In response to hypoosmotic shock and ribosome stalling in Escherichia coli, ECP is dependent upon the presence of the large-conductance mechanosensitive channel and the alternative ribosome–rescue factor A gene products. However, it is not known if a mechanistic link exists between the corresponding genes and the respective stress response pathways. Here, we report that the corresponding mscL and arfA genes are commonly co-located on the genomes of Gammaproteobacteria and display overlap in their respective 3′ UTR and 3′ CDS. We show this unusual genomic arrangement permits an antisense RNA–mediated regulatory control between mscL and arfA, and this modulates MscL excretory activity in E. coli. These findings highlight a mechanistic link between osmotic, translational stress responses and ECP in E. coli, further elucidating the previously unknown regulatory function of arfA sRNA.

/pdf/arfa-antisense-rna-regulates-mscl-excretory-activity-1wctqp5z.pdf

arfA antisense RNA regulates MscL excretory activity

Abstract Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus secretory effectors within host cells is unclear. Here, we report that Staphylococcus aureus secretes extracellular fibrinogen-binding protein (Efb) into the cytoplasm of macrophages to suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of tumor necrosis factor receptor associated factor (TRAF) 3. The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 (cellular-inhibitor-of-apoptosis-1) complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of Staphylococcus aureus to suppress host defense, which may be a promising target for developing effective anti- Staphylococcus aureus immunomodulators. 

/pdf/extracellular-fibrinogen-binding-protein-released-by-10oelg95.pdf

Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

The protein-DNA interactions in the enhancer II (ENII) and core promoter regions of hepatitis B virus (HBV) were investigated by the ligation-mediated polymerase chain reaction (LMPCR) in vivo footprinting in HepG2.2.15 cell line. Major footprints are evident at the enhancer II and core promoter regions. In particular, proteins appear to interact with the sites for Sp1, C/EBP, HNF4, HNF3, HNF1 and hB1F, as well as the TATA-like element. It confirmed that these protein-DNA interactions defined in vitro play important roles in the regulation of 3.5 kb RNAs transcription of HBV in vivo. Notably, three novel sites nt 1774-1789, nt 1801-1818 and nt 1835-1843 were found to be protected or hypersensitive, indicating that they were also involved in protein-DNA interactions in vivo. Functional analysis using CAT reporter gene demonstrated that the site from nt 1801 to 1818 was important for the function of HBV core promoter.

Investigation of Protein-DNA Interactions in Enhancer II and Core Promoter of HBV by in vivo Footprinting.

To determine the potential functions of different regions in the HBV core promoter, several mutants containing B3 and/or C deletion (p3.6IIdeltaB3, p3.5IIdeltaB3, p3.6IIdeltaC, p3.5IIdeltaB3C) have been constructed based on the two HBV transcription units (p3.6II and p3.5II) which contained ENII/Cp and Cp immediate upstream of the linear HBV genome, respectively. The functional effects of deletions were assessed with respect to antigen production, viral mRNA synthesis, and viral DNA replication. When HepG2 cells were transfected with C deletion mutants, HBeAg became undetectable in the supernatant while the expression of HBcAg in the cell lysate was retained. Consistently, precore RNA disappeared but the synthesis of pregenomic RNA and viral DNA replication could still be observed. On the other hand, deletions of B3 fragment reduced the expression of HBeAg and HBcAg significantly, while maintained the synthesis of precore RNA, pregenomic RNA and viral progeny DNA. The results strongly suggest that both fragment C and fragment D serve as minimal promoter elements for the transcription initiation of 3.5 kb RNAs. Notably, the D fragment which contains a novel nuclear factor binding site close to the start site of pregenomic RNA, is critical for the basal transcription of pregenomic RNA, which has not been reported previously. B3 plays an important role in the efficient transcription of precore and pregenomic RNAs, but is not indispensable for their basal transcription.

Roles of Various Regions of the HBV Core Promoter in the Transcription of Precore and Pregenomic RNA.


 Many pathogens secrete effectors to hijack intracellular signaling regulators in host immune cells to promote pathogenesis. However, the pathogenesis of Staphylococcus aureus (S. aureus) secretory effectors within host cells is unclear. Here, we report that intracellular S. aureus secretes fibrinogen binding protein (Efb) into the cytoplasm of macrophages to inhibit proinflammatory cytokine expression and suppress host immunity. Mechanistically, RING finger protein 114, a host E3 ligase, mediates K27-linked ubiquitination of Efb at lysine 71, which facilitates the recruitment of TNF receptor associated factor 3 (TRAF3). The binding of Efb to TRAF3 disrupts the formation of the TRAF3/TRAF2/cIAP1 complex, which mediates K48-ubiquitination of TRAF3 to promote degradation, resulting in suppression of the inflammatory signaling cascade. Additionally, the Efb K71R mutant loses the ability to inhibit inflammation and exhibits decreased pathogenicity. Therefore, our findings identify an unrecognized mechanism of S. aureus to suppress host defense, which may be a promising target for developing effective anti-S. aureus immunomodulators.

Efb released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

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Extracellular fibrinogen-binding protein released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3

Investigation of Protein-DNA Interactions in Enhancer II and Core Promoter of HBV by in vivo Footprinting.

Roles of Various Regions of the HBV Core Promoter in the Transcription of Precore and Pregenomic RNA.

Efb released by intracellular Staphylococcus aureus suppresses host immunity by targeting TRAF3