Inhibition of respiratory syncytial virus infection with intranasal siRNA nanoparticles targeting the viral NS1 gene

Respiratory syncytial virus (RSV) infection is one of the major causes of respiratory tract infection for which no vaccine or antiviral treatment is available. The RSV NS1 protein seems to antagonize the host interferon (IFN) response; however, its mechanism is unknown. Here, we used a plasmid-borne small interfering RNA targeting the NS1 gene (siNS1) to examine the role of NS1 in modulating RSV infection. RSV replication was reduced in A549 cells, but not IFN-deficient Vero cells, transfected with siNS1. siNS1 induced upregulated expression of IFN-beta and IFN-inducible genes in A549 cells. siNS1-transfected human dendritic cells, upon RSV infection, produced elevated type-1 IFN and induced differentiation of naive CD4+ T cells to T helper type 1 (TH1) cells. Mice treated intranasally with siNS1 nanoparticles before or after infection with RSV showed substantially decreased virus titers in the lung and decreased inflammation and airway reactivity compared to controls. Thus, siNS1 nanoparticles may provide an effective inhibition of RSV infection in humans.     

Characterization of the HslU chaperone affinity for HslV protease

The HslVU complex is a bacterial two-component ATP-dependent protease, consisting of HslU chaperone and HslV peptidase. Investigation of protein-protein interactions using SPR in Escherichia coli HslVU and the protein substrates demonstrates that HslU and HslV have moderate affinity (Kd = 1 microM) for each other. However, the affinity of HslU for HslV fivefold increased (Kd approximately 0.2 microM) after binding with the MBP approximately SulA protein indicating the formation of a "ternary complex" of HslV-HslU-MBP approximately SulA. The molecular interaction studies also revealed that HslU strongly binds to MBP approximately SulA with 10(-9) M affinity but does not associate with nonstructured casein. Conversely, HslV does not interact with the MBP-SulA whereas it strongly binds with casein (Kd = 0.2 microM) requiring an intact active site of HslV. These findings provide evidence for "substrate-induced" stable HslVU complex formation. Presumably, the binding of HslU to MBP approximately SulA stimulates a conformational change in HslU to a high-affinity form for HslV.     

Females with paired occurrence of cancers in the UADT and genital region have a higher frequency of either Glutathione S-transferase M1/T1 null genotype

Upper Aero digestive Tract (UADT) is the commonest site for the development of second cancer in females after primary cervical cancer. Glutathione S-transferase (GSTM1 and / or T1) null genotype modulates the risk of developing UADT cancer (primary as well as second cancer). The aim of this study was to evaluate the difference in GST null genotype frequencies in females with paired cancers in the UADT and genital region as compared to females with paired cancers in the UADT and non-genital region. Forty-nine females with a cancer in the UADT and another cancer (at all sites-genital and non-genital) were identified from a database of patients with multiple primary neoplasms and were analyzed for the GSTM1 and T1 genotype in addition to known factors such as age, tobacco habits, alcohol habits and family history of cancer. Frequencies of GSTM1 null, GSTT1 null, and either GSTM1/T1 null were higher in females with paired occurrence of cancer in the UADT and genital site (54%, 33% and 75% respectively) in comparison to females with paired occurrence of cancer in the UADT and non-genital sites (22%, 6% and 24% respectively). The significantly higher inherited frequency of either GSTM1/T1 null genotype in females with a paired occurrence of cancers in UADT and genital region (p = 0.01), suggests that these females are more susceptible to damage by carcinogens as compared to females who have UADT cancers in association with cancers at non-genital sites.     

NAD+-dependent DNA Ligase (Rv3014c) from Mycobacterium tuberculosis. Crystal structure of the adenylation domain and identification of novel inhibitors

DNA ligases utilize either ATP or NAD+ as cofactors to catalyze the formation of phosphodiester bonds in nicked DNA. Those utilizing NAD+ are attractive drug targets because of the unique cofactor requirement for ligase activity. We report here the crystal structure of the adenylation domain of the Mycobacterium tuberculosis NAD+-dependent ligase with bound AMP. The adenosine nucleoside moiety of AMP adopts a syn-conformation. The structure also captures a new spatial disposition between the two subdomains of the adenylation domain. Based on the crystal structure and an in-house compound library, we have identified a novel class of inhibitors for the enzyme using in silico docking calculations. The glycosyl ureide-based inhibitors were able to distinguish between NAD+- and ATP-dependent ligases as evidenced by in vitro assays using T4 ligase and human DNA ligase I. Moreover, assays involving an Escherichia coli strain harboring a temperature-sensitive ligase mutant and a ligase-deficient Salmonella typhimurium strain suggested that the bactericidal activity of the inhibitors is due to inhibition of the essential ligase enzyme. The results can be used as the basis for rational design of novel antibacterial agents.     

Role of cell surface heparan sulfate proteoglycans in endothelial cell migration and mechanotransduction

Endothelial cell (EC) migration is critical in wound healing and angiogenesis. Fluid shear stress due to blood flow plays an important role in EC migration. However, the role of EC surface heparan sulfate proteoglycans (HSPGs) in EC adhesion, migration, and mechanotransduction is not well understood. Here, we investigated the effects of HSPG disruption on the adhesion, migration, and mechanotransduction of ECs cultured on fibronectin. We showed that disruption of HSPGs with heparinase decreased EC adhesion rate by 40% and adhesion strength by 33%. At the molecular level, HSPG disruption decreased stress fibers and the size of focal adhesions (FAs), increased filopodia formation, and enhanced EC migration. Under flow condition, heparinase treatment increased EC migration speed, but inhibited shear stress-induced directionality of EC migration and the recruitment of phosphorylated focal adhesion kinase in the flow direction, suggesting that HSPGs are important for sensing the direction of shear stress. In addition, decreasing cell adhesion by lowering fibronectin density enhanced EC migration under static and flow condition, but did not affect the directional migration of ECs under flow. Based on our results, we propose that HSPGs play dual roles as mechanotransducer on the EC surface: (1) HSPGs-matrix interaction on the abluminal surface regulates EC migration speed through an adhesion-dependent manner, and (2) HSPGs without binding to matrix (e.g., on the luminal surface) are involved in sensing the direction of flow through an adhesion-independent manner.     

Retinoid metabolism during development of liver cirrhosis

The changes in retinoid metabolism have been documented in liver cirrhosis. However, the dynamic alterations in levels of this vitamin between circulation and liver during development of the liver cirrhosis are not well understood. The aim of this study was to measure retinoids in the liver and circulation in parallel, during and after development of cirrhosis induced by carbon tetrachloride and thioacetamide. Retinoid levels were measured by HPLC. A decrease in retinaldehyde and total retinol, together with an increase in retinoic acid was evident in liver from both carbon tetrachloride or thioacetamide treated rats within a month after initiation of treatment. Activity of enzymes involved in retinoid metabolism such as retinaldehyde oxidase, retinaldehyde dehydrogenase, and retinaldehyde reductase were decreased in the liver. In parallel, levels of retinol and retinaldehyde in the serum were increased while retinoic acid was decreased. This study indicates that during development of cirrhosis, there is reciprocal transfer of retinoid metabolites between the circulation and the liver.     

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.     

AID and Igh switch region-Myc chromosomal translocations

Chromosomal translocations involving Ig heavy chain switch regions and an oncogene, like Myc, represent early initiating events in the development of many B cell malignancies. These translocations are widely believed to result from aberrant class switch recombination (CSR). Recent reports have produced conflicting models for the role of activation-induced cytidine deaminase (AID) in this process. Here, we discuss possible roles of AID, CSR, and somatic hypermutation in generating chromosomal translocations and in tumor progression.     

Can urine lamivudine be used to monitor antiretroviral treatment adherence?

Patient adherence to treatment is an important factor in the effectiveness of antiretroviral regimens. Adherence to treatment could be monitored by estimation of antiretroviral drugs in biological fluids. We aimed to obtain information on the quantity and duration of excretion of lamivudine in urine following oral administration of a single dose of 300 mg and to assess its suitability for adherence monitoring purposes. Spot urine samples were collected before dosing and at 4, 8, 12, 24, 28, 32, 48, 72, and 96 hours post dosing from 10 healthy subjects, and lamivudine was estimated by high-pressure liquid chromatography (HPLC). Lamivudine values were expressed as a ratio of urine creatinine. About 91% of the ingested drug was excreted by 24 hours, and the concentration thereafter in urine was very negligible. A lamivudine value of 0.035 mg/mg creatinine or less at 48 hours is suggestive of a missed dose in the last 24 hours. The study findings showed that estimation of urine lamivudine in spot specimens could be useful in monitoring patient adherence to antiretroviral treatment. However, this needs to be confirmed on a larger sample size and among patients on once-daily and twice-daily treatment regimens.