Antibacterial and antilarval-settlement potential and metabolite profiles of novel sponge-associated marine bacteria

In this study, we screened seven novel sponge-associated marine bacteria for their antibacterial and antilarval-settlement activity in order to find possible new sources of non-toxic or less toxic bioactive antifoulants. The anti-bacterial-growth activity of crude extracts of each bacterium was evaluated by the disk-diffusion assay. Extracts of four potent bacteria with high and broad spectra of antibacterial activity were further separated with solvents of different polarities (hexane and ethyl acetate). To evaluate their indirect inhibitive effect on larval settlement, we tested for their antibiofilm formation activity against two of the test bacteria (Vibrio halioticoli and Loktanella hongkongensis) inductive to Hydroides elegans larval settlement. About 60 and 87% of the extracts inhibited biofilm formation by V. halioticoli and by L. hongkongensis respectively. The extracts were also tested for their direct antilarval-settlement activity against the barnacle Balanus amphitrite and the polychaete H. elegans; 87% of the extracts had a strong inhibitive effect on larval settlement of both species. Extracts of two of the isolates completely inhibited larval settlement of B. amphitrite at 70 μg ml⁻¹ and H. elegans at 60 μg ml⁻¹. The organic extracts of Winogradskyella poriferorum effectively inhibited the larval settlement of both H. elegans and B. amphitrite and the biofilm formation of the two bacterial species. The metabolites present in the active crude extracts were profiled using GC MS, and the most prevalent metabolites present in all extracts were identified. This study successfully identified potential new sources of antifouling compounds.

Polymorphisms in the pituitary growth hormone gene and its receptor associated with coronary artery disease in a predisposed cohort from India

We investigated the promoter polymorphisms of the pituitary growth hormone gene (GH1) and exon 3 deletion polymorphism (GHRd3) in its receptor gene (GHR) in 299 angiographically proven patients with coronary artery disease (CAD) and 231 asymptomatic controls enrolled in the ongoing Indian Atherosclerosis Research Study. Real time PCR based analysis of the GHR variant showed significant association of the GHRd3 deletion allele with CAD (OR 0.48, 95% CI: 0.30–0.76, P = 0.0014) and a dominant model of inheritance (Akaike information criterion = 482). The deletion allele showed significant association with high plasma HDL-c levels (P = 0.001). Sequencing of the proximal promoter region of GH1 revealed 12 novel polymorphisms and a TAGA haplotype constituted by the functional SNPs rs2005171, rs11568828, rs2005172 and rs6171, that showed significant association with CAD alone (adjusted OR of 3.31 (95% CI = 1.33–8.29, P = 0.011) and in CAD patients with diabetes (P = 0.019). Mean standardized height was associated with three of the four haplotype-tagging SNPs in the cohort (P ≤ 0.03). Eleven of the 12 polymorphic promoter SNPs contributed to 14.7% of variation in height in females in the whole dataset (P = 0.029). CAD patients with history of stroke exhibited marginally significantly lower mean height as compared to rest of the cohort (P < 0.006). In conclusion, genetic polymorphisms in the GHR gene and its ligand, GH1, may modulate the risk of CAD in the Asian Indian population.     

Introduction to the Special Section on “Chinese Earthquake Risk Management”

Natural disasters by definition are surprises, causing a great deal of damage and inconvenience. Earthquakes are among the most terrifying and destructive natural disasters threatening humans. Emergency management has been described as the process of coordinating an emergency or its aftermath through communication and organization for deployment and the use of emergency resources. This special section of HERA provides the state of the art studies of risk and emergency management related to the Wenchuan earthquake that occurred in China in May 2008.     

SH2 Modified STAT1 Induces HLA-I Expression and Improves IFN-γ Signaling in IFN-α Resistant HCV Replicon Cells

Background

We have developed multiple stable cell lines containing subgenomic HCV RNA that are resistant to treatment with interferon alpha (IFN-α. Characterization of these IFN-α resistant replicon cells showed defects in the phosphorylation and nuclear translocation of STAT1 and STAT2 proteins due to a defective Jak-STAT pathway.

Methodology/Principal Findings

In this study, we have developed an alternative strategy to overcome interferon resistance in a cell culture model by improving intracellular STAT1 signaling. An engineered STAT1-CC molecule with double cysteine substitutions in the Src-homology 2 (SH2) domains of STAT1 (at Ala-656 and Asn-658) efficiently phosphorylates and translocates to the nucleus of IFN-resistant cells in an IFN-γ dependent manner. Transfection of a plasmid clone containing STAT1-CC significantly activated the GAS promoter compared to wild type STAT1 and STAT3. The activity of the engineered STAT1-CC is dependent upon the phosphorylation of tyrosine residue 701, since the construct with a substituted phenylalanine residue at position 701 (STAT1-CC-Y701F) failed to activate GAS promoter in the replicon cells. Intracellular expression of STAT1-CC protein showed phosphorylation and nuclear translocation in the resistant cell line after IFN-γ treatment. Transient transfection of STAT1-CC plasmid clone into an interferon resistant cell line resulted in inhibition of viral replication and viral clearance in an IFN-γ dependent manner. Furthermore, the resistant replicon cells transfected with STAT1-CC constructs significantly up regulated surface HLA-1 expression when compared to the wild type and Y to F mutant controls.

Conclusions

These results suggest that modification of the SH2 domain of the STAT1 molecule allows for improved IFN-γ signaling through increased STAT1 phosphorylation, nuclear translocation, HLA-1 surface expression, and prolonged interferon antiviral gene activation.     

Differential Modulation of Angiogenesis by Erythropoiesis-Stimulating Agents in a Mouse Model of Ischaemic Retinopathy

Background

Erythropoiesis stimulating agents (ESAs) are widely used to treat anaemia but concerns exist about their potential to promote pathological angiogenesis in some clinical scenarios. In the current study we have assessed the angiogenic potential of three ESAs; epoetin delta, darbepoetin alfa and epoetin beta using in vitro and in vivo models.

Methodology/Principal Findings

The epoetins induced angiogenesis in human microvascular endothelial cells at high doses, although darbepoetin alfa was pro-angiogenic at low-doses (1–20 IU/ml). ESA-induced angiogenesis was VEGF-mediated. In a mouse model of ischaemia-induced retinopathy, all ESAs induced generation of reticulocytes but only epoetin beta exacerbated pathological (pre-retinal) neovascularisation in comparison to controls (p<0.05). Only epoetin delta induced a significant revascularisation response which enhanced normality of the vasculature (p<0.05). This was associated with mobilisation of haematopoietic stem cells and their localisation to the retinal vasculature. Darbepoetin alfa also increased the number of active microglia in the ischaemic retina relative to other ESAs (p<0.05). Darbepoetin alfa induced retinal TNFα and VEGF mRNA expression which were up to 4 fold higher than with epoetin delta (p<0.001).

Conclusions

This study has implications for treatment of patients as there are clear differences in the angiogenic potential of the different ESAs.     

A mucoadhesive in situ gel delivery system for paclitaxel

MUC1 gene encodes a transmembrane mucin glycoprotein that is overexpressed in human breast cancer and colon cancer. The objective of this study was to develop an in situ gel delivery system containing paclitaxel (PTX) and mucoadhesives for sustained and targeted delivery of anticancer drugs. The delivery system consisted of chitosan and glyceryl monooleate (GMO) in 0.33M citric acid containing PTX. The in vitro release of PTX from the gel was performed in presence and absence of Tween 80 at drug loads of 0.18%, 0.30%, and 0.54% (wt/wt), in Sorensen’s phosphate buffer (pH 7.4) at 37°C. Different mucin-producing cell lines (Calu-3>Caco-2) were selected for PTX transport studies. Transport of PTX from solution and gel delivery system was performed in side by side diffusion chambers from apical to basal (A-B) and basal to apical (B-A) directions. In vitro release studies revealed that within 4 hours, only 7.61%±0.19%, 12.0%±0.98%, 31.7%±0.40% of PTX were released from 0.18%, 0.30%, and 0.54% drugloaded gel formulation, respectively, in absence of Tween 80. However, in presence of surfactant (0.05% wt/vol) in the dissolution medium, percentages of PTX released were 28.1%±4.35%, 44.2%±6.35%, and 97.1%±1.22%, respectively. Paclitaxel has shown a polarized transport in all the cell monolayers with B-A transport 2 to 4 times higher than in the A-B direction. The highest mucin-producing cell line (Calu-3) has shown the lowest percentage of PTX transport from gels as compared with Caco-2 cells. Transport of PTX from mucoadhesive gels was shown to be influenced by the mucin-producing capability of cell.     

Hemoglobinopathies, G6PD deficiency, and hereditary elliptocytosis in Bahrain

The native population of Bahrain has a high prevalence of hemoglobinopathies and G6PD deficiency, probably as a result of past malarial endemism. We used the Biorad-Variant hemoglobin testing system for primary screening of hemoglobinopathies in 20,000 individuals. Hemoglobin abnormalities were detected in 7,206 (36.3%) cases.     

Nitric oxide protects human extravillous trophoblast cells from apoptosis by a cyclic GMP-dependent mechanism and independently of caspase 3 nitrosylation

Apoptosis is thought to play an important regulatory role in placental development and inappropriate trophoblast apoptosis has been implicated in complications of pregnancy such as pre-eclampsia. Here we show that apoptosis of a human extravillous trophoblast-derived cell line (SGHPL-4) can be regulated by nitric oxide (NO). Nitric oxide produced exogenously by the addition of NO donors was able to delay or inhibit apoptosis induced by a combination of tumour necrosis factor alpha and actinomycin D and to suppress the activity of caspase 3. Treatment with hepatocyte growth factor (HGF) stimulated expression of the inducible isoform of NO synthase and was also able to protect SGHPL-4 cells from caspase 3 activation and apoptosis. The inhibition of basal NO production with NO synthase inhibitors was shown to sensitise cells to apoptotic stimuli and to reduce the level of endogenous caspase 3 nitrosylation. The anti-apoptotic effects of NO in these extravillous trophoblast cells appear to be mediated through the production of cyclic GMP as inhibitors of soluble guanylate cyclase inhibited the protective effect of both HGF and NO donors.     

Expression of cytochromes P450 4F4 and 4F5 in infection and injury models of inflammation

Lipopolysaccharide (LPS) treatment of rats suppresses CYP 4F4 and 4F5 expression by 50 and 40%, respectively, in a direct fashion occurring in the liver. This contention is borne out by essentially parallel dose-dependent changes observed upon treatment of rat hepatocyte cultures with LPS. An alternate avenue of triggering the inflammatory cascade is traumatic brain injury by controlled cortical impact. Such injury brings about a dramatic change in the expression of CYP 4F4 and 4F5 mRNA which reaches its greatest effect 24 h after impact compared with sham-operated but uninjured controls. At time points after 24 h the expression of both isoforms increases dramatically reaching highest levels at 2 weeks post-injury. These changes in mRNA expression are mirrored by changes in protein expression. The results are consistent with the notion that immediately after injury concentrations of leukotriene and prostaglandin mediators are elevated by decreased CYP 4F concentrations. As time after injury increases those conditions reverse. Increased CYP 4F expression leads to diminished concentrations of leukotriene and prostaglandin mediators and then to recovery and repair.