Hypoxia-inducible factor-1 α/platelet derived growth factor axis in HIV-associated pulmonary vascular remodeling

Background

Human immunodeficiency virus (HIV) infected patients are at increased risk for the development of pulmonary arterial hypertension (PAH). Recent reports have demonstrated that HIV associated viral proteins induce reactive oxygen species (ROS) with resultant endothelial cell dysfunction and related vascular injury. In this study, we explored the impact of HIV protein induced oxidative stress on production of hypoxia inducible factor (HIF)-1α and platelet-derived growth factor (PDGF), critical mediators implicated in the pathogenesis of HIV-PAH.

Methods

The lungs from 4-5 months old HIV-1 transgenic (Tg) rats were assessed for the presence of pulmonary vascular remodeling and HIF-1α/PDGF-BB expression in comparison with wild type controls. Human primary pulmonary arterial endothelial cells (HPAEC) were treated with HIV-associated proteins in the presence or absence of pretreatment with antioxidants, for 24 hrs followed by estimation of ROS levels and western blot analysis of HIF-1α or PDGF-BB.

Results

HIV-Tg rats, a model with marked viral protein induced vascular oxidative stress in the absence of active HIV-1 replication demonstrated significant medial thickening of pulmonary vessels and increased right ventricular mass compared to wild-type controls, with increased expression of HIF-1α and PDGF-BB in HIV-Tg rats. The up-regulation of both HIF-1α and PDGF-B chain mRNA in each HIV-Tg rat was directly correlated with an increase in right ventricular/left ventricular+septum ratio. Supporting our in-vivo findings, HPAECs treated with HIV-proteins: Tat and gp120, demonstrated increased ROS and parallel increase of PDGF-BB expression with the maximum induction observed on treatment with R5 type gp-120_CM. Pre-treatment of endothelial cells with antioxidants or transfection of cells with HIF-1α small interfering RNA resulted in abrogation of gp-120_CM mediated induction of PDGF-BB, therefore, confirming that ROS generation and activation of HIF-1α plays critical role in gp120 mediated up-regulation of PDGF-BB.

Conclusion

In summary, these findings indicate that viral protein induced oxidative stress results in HIF-1α dependent up-regulation of PDGF-BB and suggests the possible involvement of this pathway in the development of HIV-PAH.     

Dog bites in humans and estimating human rabies mortality in rabies endemic areas of Bhutan

Background

Dog bites in humans are a public health problem worldwide. The issues of increasing stray dog populations, rabies outbreaks, and the risk of dogs biting humans have been frequently reported by the media in Bhutan. This study aimed to estimate the bite incidence and identify the risk factors for dog bites in humans, and to estimate human deaths from rabies in rabies endemic south Bhutan.

Methods

A hospital-based questionnaire survey was conducted during 2009–2010 among dog bites victims who visited three hospitals in Bhutan for anti-rabies vaccine injection. Decision tree modeling was used to estimate human deaths from rabies following dog bite injuries in two rabies endemic areas of south Bhutan.

Results

Three hundred and twenty four dog bite victims were interviewed. The annual incidence of dog bites differed between the hospital catchment areas: 869.8 (95% CI: 722.8–1022.5), 293.8 (240–358.2) and 284.8 (251.2–323) per 100,000 people in Gelephu, Phuentsholing and Thimphu, respectively. Males (62%) were more at risk than females (P<0.001). Children aged 5–9 years were bitten more than other age groups. The majority of victims (71%) were bitten by stray dogs. No direct fatal injury was reported. In two hospital areas (Gelephu and Phuentsholing) in south Bhutan the annual incidence of death from rabies was 3.14 (95% CI: 1.57–6.29) per 100,000 population. The decision tree model predicted an equivalent annual incidence of 4.67 (95% CI: 2.53–7.53) deaths/100,000 population at risk. In the absence of post exposure prophylaxis, the model predicted 19.24 (95% CI: 13.69–25.14) deaths/year in these two areas.

Conclusions

Increased educational awareness of people about the risk of dog bites and rabies is necessary, particularly for children in rabies endemic areas of Bhutan.     

The influence of meteorology on the spread of influenza: survival analysis of an equine influenza (A/H3N8) outbreak

The influences of relative humidity and ambient temperature on the transmission of influenza A viruses have recently been established under controlled laboratory conditions. The interplay of meteorological factors during an actual influenza epidemic is less clear, and research into the contribution of wind to epidemic spread is scarce. By applying geostatistics and survival analysis to data from a large outbreak of equine influenza (A/H3N8), we quantified the association between hazard of infection and air temperature, relative humidity, rainfall, and wind velocity, whilst controlling for premises-level covariates. The pattern of disease spread in space and time was described using extraction mapping and instantaneous hazard curves. Meteorological conditions at each premises location were estimated by kriging daily meteorological data and analysed as time-lagged time-varying predictors using generalised Cox regression. Meteorological covariates time-lagged by three days were strongly associated with hazard of influenza infection, corresponding closely with the incubation period of equine influenza. Hazard of equine influenza infection was higher when relative humidity was <60% and lowest on days when daily maximum air temperature was 20-25°C. Wind speeds >30 km hour(-1) from the direction of nearby infected premises were associated with increased hazard of infection. Through combining detailed influenza outbreak and meteorological data, we provide empirical evidence for the underlying environmental mechanisms that influenced the local spread of an outbreak of influenza A. Our analysis supports, and extends, the findings of studies into influenza A transmission conducted under laboratory conditions. The relationships described are of direct importance for managing disease risk during influenza outbreaks in horses, and more generally, advance our understanding of the transmission of influenza A viruses under field conditions.     

Sulfonamide bearing oligonucleotides: simple synthesis and efficient RNA recognition

Four pyrimidine nucleosides wherein a benzensulfonamide group is linked to the C-5 position of the uracil nucleobase through a triazolyl or an alkynyl linker were prepared by Cu(I)-assisted azide-alkyne cycloadditions (CuAAC) or Sonogashira reactions, respectively, and incorporated into oligonucleotides. Efficient π-π-stacking between two or more phenyltriazoles in the major groove was found to increase the thermal stability of a DNA:RNA duplex significantly. On the other hand, the alkynyl group was not as efficient in stacking as the triazolyl group. No effect of positional orientation of the sulfonamide group on the stacking efficiency was observed, and the most stable DNA:RNA duplex contained four consecutive sulfonamide substituted phenyltriazole moieties in the major groove.     

A single-residue mutation destabilizes Vibrio harveyi flavin reductase FRP dimer

Our earlier studies have shown that the Vibrio harveyi flavin reductase FRP undergoes a monomer-dimer equilibrium, and luciferase forms a functional complex with the FRP monomer but not significantly with the dimer. This work is aimed at further investigating the nature and regulation of FRP subunit interactions by computation and site-directed mutagenesis approaches. In silico mutations of a number of residues were performed, and energetic analyses led us to target residue E99, which interacts directly with R113 and R225 from the second subunit of the FRP homodimer, for detailed investigation. E99 was found non-essential to the binding of either the FMN cofactor or the substrates. However, in comparison with the native enzyme, the E99K variant was shown to have an enhanced subunit dissociation as evident from a 44-fold higher K_d for the monomer-dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated.     

Mitochondrial NAD+-linked State 3 respiration and complex-I activity are compromised in the cerebral cortex of 3-nitropropionic acid-induced rat model of Huntington's disease

Mitochondrial complex-I dysfunction has been observed in patients of Huntington's disease (HD). We assessed whether such a defect is present in the 3-nitropropionic acid (3-NP) model of HD. Rats treated with 3-NP (10–20 mg/kg i.p., for 4 days) exhibited weight loss, gait abnormalities, and striatal lesions with increased glial fibrillary acidic protein immunostaining on fifth and ninth days, while increase in striatal dopamine and loss of tyrosine hydroxylase immunoreactivity were observed on fifth day following treatment. We report for the first time a dose-dependent reduction in complex-I activity in the cerebral cortex when analyzed spectrophotometrically and by blue native-polyacrylamide gel electrophoresis following 3-NP treatment. The citrate synthase normalized activities of mitochondrial complex-I, -II, -(I + III) and -IV were decreased in the cortex of 3-NP treated rats. In addition, succinate driven State 3 respiration was also significantly inhibited in vivo and in the isolated mitochondria. These findings taken together with the observation of a significant decrease in vivo but not in vitro of State 3 respiration with NAD⁺-linked substrates, suggest complex-I dysfunction in addition to irreversible inhibition of complex-II and succinate dehydrogenase activity as a contributing factor in 3-NP-induced cortico-striatal lesion.     

Introducing metallocene into a triazole peptide conjugate reduces its off-rate and enhances its affinity and antiviral potency for HIV-1 gp120

In this work, we identified a high affinity and potency metallocene-containing triazole peptide conjugate that suppresses the interactions of HIV-1 envelope gp120 at both its CD4 and co-receptor binding sites. The ferrocene-peptide conjugate, HNG-156, was formed by an on-resin copper-catalysed [2+3] cycloaddition reaction. Surface plasmon resonance interaction analysis revealed that, compared to a previously reported phenyl-containing triazole conjugate HNG-105 (105), peptide 156 had a higher direct binding affinity for several subtypes of HIV-1 gp120 due mainly to the decreased dissociation rate of the conjugate-gp120 complex. The ferrocene triazole conjugate bound to gp120 of both clade A (92UG037-08) and clade B (YU-2 and SF162) virus subtypes with nanomolar KD in direct binding and inhibited the binding of gp120 to soluble CD4 and to antibodies that bind to HIV-1YU-2 gp120 at both the CD4 binding site and CD4-induced binding sites. HNG-156 showed a close-to nanomolar IC50 for inhibiting cell infection by HIV-1BaL whole virus. The dual receptor site antagonist activity and potency of HNG-156 make it a promising viral envelope inhibitor lead for developing anti-HIV-1 treatments.     

Preparation of l-proline based aeruginosin 298-A analogs: Optimization of the P1-moiety

Aeruginosins are a family of naturally occurring oligopeptides that share a common bicyclic amino acid core structure. Many compounds in the family are inhibitors of serine proteases, such as thrombin and trypsin. Thrombin is an important enzyme in the blood coagulation cascade, and is a promising target for anticoagulant drug development. In order to understand the structure–activity relationship (SAR) and to find selective thrombin inhibitors, we synthesized a series of aeruginosin 298-A analogs, in which the P₂ bicyclic amino acid was replaced by a l-proline residue. The structure optimization was focused on modification of the P₁ position. In choosing the P₁ group, an effort was made to avoid using the highly basic guanidine groups present in nearly all naturally occurring aeruginosins. The synthesis and enzyme assays of these aeruginosin analogs against thrombin and trypsin are reported. We found that several compounds with neutral P₁ groups exhibit excellent selectivity over trypsin and good potency against thrombin. The SAR data of the P₁ groups obtained here can be used in preparing other thrombin inhibitors with better selectivity against trypsin.     

Synthesis of photoaffinity probes [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside and [(4(')-benzoyl)phenoxy]-D-glucopyranoside for the identification of sugar-binding and phlorizin-binding sites in the sodium/D-glucose cotransporter protein

In this paper we describe the synthesis and photochemical and biochemical properties of two new photoaffinity probes designed for studies on the structure-function relationship of the sodium D-glucose cotransporter (SGLT1). The two probes are [2(')-iodo-4(')-(3(")-trifluoromethyldiazirinyl)phenoxy]-D-glucopyranoside (TIPDG), a mimic for the phenyl glucopyranoside arbutin which is transported by SGLT1 with a very high affinity, and [(4(')-benzoyl)phenoxy]-D-glucopyranoside (BzG), a model compound for phlorizin, the most potent competitive inhibitor of sugar translocation by SGLT1. Both photoaffinity probes TIPDG (lambda(max)=358 nm) and BzG (lambda(max)=293 nm) can be activated at 350-360 nm, avoiding protein-damaging wavelengths. In inhibitor studies on sodium-dependent D-glucose uptake into rabbit intestinal brush border membrane vesicles TIPDG and BzG showed a fully competitive inhibition with regard to the sugar with respective K(i) values of 22+/-5 microM for TIPDG and 12+/-2 microM for BzG. These K(i) values are comparable to those of their parent compounds arbutin (25+/-6 microM) and phlorizin (8+/-1 microM). To further test the potential of TIPDG and BzG as photoaffinity probes, truncated loop 13 protein, supposed to be part of the substrate recognition site of SGLT1, was exposed to TIPDG and BzG in solution. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis demonstrated that TIPDG and BzG successfully labeled the protein. These preliminary results suggest that both photoaffinity probes are promising tools for the study of the structure-function relationship of SGLT1 and other SGLT1 family transporter proteins.