Structure modeling and docking study of HCV NS5B-3a RNA polymerase for the identification of potent inhibitors

Homology modeling in combination with molecular docking studies has been applied to predict the binding modes of the Hepatitis C virus (HCV) NS5B-3a inhibitors within the pocket of NS5B polymerase of HCV genotype 3a. Structure modeling and docking using Genetic Optimization for Ligand Docking (GOLD) were carried out to understand the ligand binding properties of NS5B-3a and to identify a potent inhibitor against it. The three-dimensional homology model of Pakistani strain is not available and was built based on the HCV-J4 NS5B polymerase structure. Compound 1 possessing high GOLD fitness score of 62.17 with IC50 value of 0.04 μM was selected as the potent inhibitor. The docking analysis depicted that hydrogen bonding, ionic and hydrophobic interactions contributed significantly for its ligand binding and the amino acid residues Arg442 and His403 seemed to be the anchor residues for receptor recognition. For developing a connection between the experimental bioactivities and GOLD fitness scores, a squared correlation coefficient was calculated and found as acceptable with the value of r ² = 0.67. These findings may act as potent lead in designing effective NS5B-3a inhibitors.     

HNH proteins are a widespread component of phage DNA packaging machines

The genome packaging reactions of tailed bacteriophages and herpes viruses require the activity of a terminase enzyme, which is comprised of large and small subunits. Phage genomes are replicated as linear concatemers composed of multiple copies of the genome joined end to end. As the terminase enzyme packages the genome into the phage capsid, it cleaves the DNA into single genome-length units. In this work, we show that the phage HK97 HNH protein, gp74, is required for the specific endonuclease activity of HK97 terminase and is essential for phage head morphogenesis. HNH proteins are a very common family of proteins generally associated with nuclease activity that are found in all kingdoms of life. We show that the activity of gp74 in terminase-mediated cleavage of the phage cos site relies on the presence of an HNH motif active-site residue, and that the large subunit of HK97 terminase physically interacts with gp74. Bioinformatic analysis reveals that the role of HNH proteins in terminase function is widespread among long-tailed phages and is uniquely required for the activity of the Terminase_1 family of large terminase proteins.Tailed bacteriophages and herpes viruses package their large double-stranded DNA genomes into a preformed protein shell, known as the “prohead,” using terminase enzymes. In both types of viruses, the genome is synthesized as concatemers composed of multiple copies of the genome joined end to end. This concatemeric DNA is packaged into the prohead and cleaved into genome-length units by terminase in an ATP-dependent reaction. Phage terminases are composed of two proteins: the large subunit harbors an endonuclease domain and an ATPase that powers the DNA packaging reaction, and the small subunit mediates specific DNA-binding required for recognition of packaging sites in the phage genome. A variety of elegant structural and biophysical studies have recently provided insight into the molecular mechanisms of terminase function (1, 2). However, the factors that affect the action of terminase enzymes in vivo have been less well characterized.Terminase enzymes perform several functions. They specifically recognize and bind the viral genome, interact with the prohead, then drive the DNA into the head through the narrow entry channel formed by the portal protein that is positioned at a single vertex of the head. During this process terminases also cleave the viral DNA, either nonspecifically upon head filling or at a specific site known as “cos.” The efficient packaging of a phage genome in vivo may require phage-encoded cofactors in addition to the terminase enzyme. For example, Escherichia coli phage λ gpFI facilitates interaction of the terminase–DNA complex with proheads (3–6). A wide variety of phages appear to encode proteins with a function similar to λ gpFI (7). Additionally, the activity of Bacillus subtilis phage phi29 terminase requires a phage-encoded RNA molecule bound to its portal protein (8), and in vivo packaging of the E. coli phage T4 genome can only be completed with the participation of the phage-encoded endonuclease, gp49 (9). The general prevalence and importance of terminase cofactors is difficult to evaluate because few studies have addressed this issue.We recently reported that phage genomes often encode proteins possessing an HNH motif near their terminase genes (10). The HNH motif is ∼35 aa long, and is characterized by the presence of two highly conserved His residues and one Asn residue. These HNH motifs, as defined by the large (∼7,400-member) HNH Pfam (11) protein sequence family (PF01844), are often found in proteins that possess endonuclease activity, such as site-specific homing endonucleases (12, 13), colicins (14, 15), S pyocins (16), and restriction enzymes (17–19). HNH motif-containing proteins comprised of primarily an HNH motif as found in E. coli colicins, usually possess nonspecific endonuclease activity. Conversely, HNH motif-containing proteins may contain DNA-recognition domains in addition to the HNH motif and thus possess high sequence specificity, as found in the homing endonucleases.The frequent juxtaposition of HNH and phage terminase genes (10, 20) suggests a unique role for HNH proteins in the endonuclease and/or packaging activities of the terminases. To address this issue, we investigated the function of E. coli phage HK97 gp74, a 119-residue protein containing an HNH motif. The gene encoding gp74 is located at the extreme 3′ end of the mature linear HK97 genome, adjacent to the cos site. In both the lysogen and replicative form of the HK97 genome gene 74 is immediately adjacent to genes 1 and 2, which encode the small and large subunits of terminase (TerS and TerL), respectively. Whereas gp74 was previously found to possess endonuclease activity (10), its role in the HK97 replication cycle remained uncharacterized. In this study we used functional and bioinformatic analyses to investigate its function.     

Apigenin as an anticancer agent

Apigenin is an edible plant‐derived flavonoid that has been reported as an anticancer agent in several experimental and biological studies. It exhibits cell growth arrest and apoptosis in different types of tumors such as breast, lung, liver, skin, blood, colon, prostate, pancreatic, cervical, oral, and stomach, by modulating several signaling pathways. Apigenin induces apoptosis by the activation of extrinsic caspase‐dependent pathway by upregulating the mRNA expressions of caspase‐3, caspase‐8, and TNF‐α. It induces intrinsic apoptosis pathway as evidenced by the induction of cytochrome c, Bax, and caspase‐3, while caspase‐8, TNF‐α, and B‐cell lymphoma 2 levels remained unchanged in human prostate cancer PC‐3 cells. Apigenin treatment leads to significant downregulation of matrix metallopeptidases‐2, ?9, Snail, and Slug, suppressing invasion. The expressions of NF‐κB p105/p50, PI3K, Akt, and the phosphorylation of p‐Akt decreases after treatment with apigenin. However, apigenin‐mediated treatment significantly reduces pluripotency marker Oct3/4 protein expression which might be associated with the downregulation of PI3K/Akt/NF‐κB signaling.     

A Combination of Constitutive Damage Model and Artificial Neural Networks to Characterize the Mechanical Properties of the Healthy and Atherosclerotic Human Coronary Arteries

It has been indicated that the content and structure of the elastin and collagen of the arterial wall can subject to a significant alteration due to the atherosclerosis. Consequently, a high tissue stiffness, stress, and even damage/rupture are triggered in the arterial wall. Although many studies so far have been conducted to quantify the mechanical properties of the coronary arteries, none of them consider the role of collagen damage of the healthy and atherosclerotic human coronary arterial walls. Recently, a fiber family‐based constitutive equation was proposed to capture the anisotropic mechanical response of the healthy and atherosclerotic human coronary arteries via both the histostructural and uniaxial data. In this study, experimental mechanical measurements along with histological data of the healthy and atherosclerotic arterial walls were employed to determine the constitutive damage parameters and remodeling of the collagen fibers. To do this, the preconditioned arterial tissues were excised from human cadavers within 5‐h postmortem, and the mean angle of their collagen fibers was precisely determined. Thereafter, a group of quasistatic axial and circumferential loadings were applied to the arterial walls, and the constrained nonlinear minimization method was employed to identify the arterial parameters according to the axial and circumferential extension data. The remodeling of the collagen fibers during the tensile test was also predicted via Artificial Neural Networks algorithm. Regardless of loading direction, the results presented a noteworthy load‐bearing capability and stiffness of the atherosclerotic arteries compared to the healthy ones (P < 0.005). Theoretical fiber angles were found to be consistent with the experimental histological data with less than 2 and 5° difference for the healthy and atherosclerotic arterial walls, respectively. The pseudoelastic damage model data were also compared with that of the experimental data, and interestingly, the arterial mechanical behavior for both the primary loading (up to the elastic region) and the discontinuous softening (up to the ultimate stress) was well addressed. The proposed model predicted well the mechanical response of the arterial tissue considering the damage of collagen fibers for both the healthy and atherosclerotic arterial walls.     

Management of simultaneously ingested and aspirated foreign bodies in an infant

This report describes the management of a 7-month-old baby who aspirated one and ingested two paper pins at a time. Bronchoscopic removal was done for aspirated pin and ingested pins were observed for spontaneous passage in stool, which occurred in 48 hours without any untoward incidence.