Pitfalls in screening streptococci for retrieving superior streptokinase (SK) genes: no activity correlation for streptococcal culture supernatant and recombinant SK

Streptokinase (SK), the heterogeneous protein family secreted by some groups of β-hemolytic streptococci (βHS), is a plasminogen activator and well-known drug for thrombolytic therapy. Differences in plasminogen activation property of streptococcal culture supernatants (SCS) have been traditionally used to identify superior producer strains and SK genes (skc) for recombinant SK (rSK) production. However, the role of SK heterogeneity and whether SK activities in SCS correlate with that of their corresponding rSK is a matter of debate. To address these concerns, SCS of nine group C streptococci (GCS) screened among 252 βHS clinical isolates were compared for plasminogen activation using S-2251 chromogenic assay. The GCS (Streptococcus equisimilis) showing the highest (GCS-S87) and lowest (GCS-S131) activities were selected for PCR-based isolation of skc, cloning and rSK production in Escherichia coli. The 6×His-tagged rSK proteins were purified by NI–NTA chromatography, analyzed by SDS-PAGE and Western blotting and their activities were determined. While SCS of GCS-S87 and GCS-S131 showed different plasminogen activations (95 and 35 %, respectively) compared to that of the reference strain (GCS-9542), but interestingly rSK of all three strains showed close specific activities (1.33, 1.70, and 1.55 × 10⁴ IU mg^?1). Accordingly, SKS87 and SKS131 had more than 90 % sequence identity at the amino acids level compared to SK9542. Therefore, SK heterogeneity by itself may not contribute to the differences in plasminogen activation properties of SCS and evaluation of this activity in SCS might not be a proper assay for screening superior skc.     

Intraguild predation by the generalist predator Orius majusculus on the parasitoid Encarsia formosa

Intraguild predation of Orius majusculus (Reuter) (Heteroptera: Anthocoridae) on Encarsia formosa (Gahan) (Hymenoptera: Aphelinidae), both natural enemies of Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae), was studied under laboratory conditions. The experiments quantified prey consumption by 5th instar nymphs and adults of O. majusculus offered unparasitised 3rd, early 4th or 4th instar B. tabaci nymphs or parasitised nymphs containing 2nd or 3rd larval instar or pupal parasitoids. In addition, prey preference of the two stages of O. majusculus for parasitised or unparasitised whitefly nymphs was studied using nine different prey combinations. Both predator stages readily preyed upon on both unparasitised and parasitised B. tabaci. In no-choice experiments, predation on 3rd instar E. formosa by adult predators was the highest, while predator nymphs preyed most on unparasitised 3rd instar B. tabaci and 2nd instar parasitoids. Predation of predator stages was lowest on 4th instar B. tabaci and E. formosa pupae. In all prey combinations, both stages of O. majusculus showed a significant preference for parasitised over unparasitised whitefly nymphs except for the combination of 5th instars of O. majusculus with early 4th instar whiteflies and E. formosa pupae. The results indicate that intraguild interactions between O. majusculus and E. formosa may have negative effects on biological control of B. tabaci.     

Modeling and analysis of soybean (Glycine max. L) Cu/Zn,Mn and Fe superoxide dismutases

Superoxide dismutase (SOD, EC 1.15.1.1) is an important metal-containing antioxidant enzyme that provides the first line of defense against toxic superoxide radicals by catalyzing their dismutation to oxygen and hydrogen peroxide. SOD is classified into four metalloprotein isoforms, namely, Cu/Zn SOD, Mn SOD, Ni SOD and Fe SOD. The structural models of soybean SOD isoforms have not yet been solved. In this study, we describe structural models for soybean Cu/Zn SOD, Mn SOD and Fe SOD and provide insights into the molecular function of this metal-binding enzyme in improving tolerance to oxidative stress in plants.     

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.     

Aspergillus fumigatus Invasion Increases with Progressive Airway Ischemia

Despite the prevalence of Aspergillus-related disease in immune suppressed lung transplant patients, little is known of the host-pathogen interaction. Because of the mould’s angiotropic nature and because of its capacity to thrive in hypoxic conditions, we hypothesized that the degree of Aspergillus invasion would increase with progressive rejection-mediated ischemia of the allograft. To study this relationship, we utilized a novel orthotopic tracheal transplant model of Aspergillus infection, in which it was possible to assess the effects of tissue hypoxia and ischemia on airway infectivity. Laser Doppler flowmetry and FITC-lectin were used to determine blood perfusion, and a fiber optic microsensor was used to measure airway tissue oxygen tension. Fungal burden and depth of invasion were graded using histopathology. We demonstrated a high efficacy (80%) for producing a localized fungal tracheal infection with the majority of infection occurring at the donor-recipient anastomosis; Aspergillus was more invasive in allogeneic compared to syngeneic groups. During the study period, the overall kinetics of both non-infected and infected allografts was similar, demonstrating a progressive loss of perfusion and oxygenation, which reached a nadir by days 10-12 post-transplantation. The extent of Aspergillus invasion directly correlated with the degree of graft hypoxia and ischemia. Compared to the midtrachea, the donor-recipient anastomotic site exhibited lower perfusion and more invasive disease; a finding consistent with clinical experience. For the first time, we identify ischemia as a putative risk factor for Aspergillus invasion. Therapeutic approaches focused on preserving vascular health may play an important role in limiting Aspergillus infections.     

Relationship between Vitreous Levels of Matrix Metalloproteinases and Vascular Endothelial Growth Factor in Proliferative Diabetic Retinopathy

To investigate which matrix metalloproteinases (MMPs) are more likely to be involved in the angiogenic process in proliferative diabetic retinopathy (PDR), we measured the levels of MMPs in the vitreous fluid from patients with PDR and controls and correlated these levels with the levels of vascular endothelial growth factor (VEGF). Vitreous samples from 32 PDR and 24 nondiabetic patients were studied by mosaic multiplex MMPs enzyme-linked immunosorbent assay (ELISA), single ELISA, Western blot and zymography analysis. Epiretinal membranes from 11 patients with PDR were studied by immunohistochemistry. MMP-8 and MMP-13 were not detected. ELISA, Western blot and gelatin ymography assays revealed significant increases in the expression levels of MMP-1, MMP-7, MMP-9 and VEGF in vitreous samples from PDR patients compared to nondiabetic controls, whereas MMP-2 and MMP-3 were not upregulated in vitreous samples from PDR patients. Significant correlations existed between ELISA and zymography assays for the quantitation of MMP-2 (r=0.407; p=0.039) and MMP-9 (r=0.711; p<0.001). Significant correlations were observed between levels of VEGF and levels of MMP-1 (r=0.845; P<0.001) and MMP-9 (r=0.775; p<0.001), and between levels of MMP-1 and MMP-9 (r=0.857; p<0.001). In epiretinal membranes, cytoplasmic immunoreactivity for MMP-9 was present in vascular endothelial cells and stromal monocytes/macrophages and neutrophils. Our findings suggest that among the MMPs measured, MMP-1 and MMP-9 may contribute to the angiogenic switch in PDR.     

Tobacco Smoking and Its Association with Illicit Drug Use among Young Men Aged 15-24 Years Living in Urban Slums of Bangladesh

Background

Tobacco smoking (TS) and illicit drug use (IDU) are of public health concerns especially in developing countries, including Bangladesh. This paper aims to (i) identify the determinants of TS and IDU, and (ii) examine the association of TS with IDU among young slum dwellers in Bangladesh.

Methodology/Principal Findings

Data on a total of 1,576 young slum dwellers aged 15–24 years were extracted for analysis from the 2006 Urban Health Survey (UHS), which covered a nationally representative sample of 13,819 adult men aged 15–59 years from slums, non-slums and district municipalities of six administrative regions in Bangladesh. Methods used include frequency run, Chi-square test of association and multivariable logistic regression. The overall prevalence of TS in the target group was 42.3%, of which 41.4% smoked cigarettes and 3.1% smoked bidis. The regression model for TS showed that age, marital status, education, duration of living in slums, and those with sexually transmitted infections were significantly (p<0.001 to p<0.05) associated with TS. The overall prevalence of IDU was 9.1%, dominated by those who had drug injections (3.2%), and smoked ganja (2.8%) and tari (1.6%). In the regression model for IDU, the significant (p<0.01 to p<0.10) predictors were education, duration of living in slums, and whether infected by sexually transmitted diseases. The multivariable logistic regression (controlling for other variables) revealed significantly (p<0.001) higher likelihood of IDU (OR = 9.59, 95% CI = 5.81–15.82) among users of any form of TS. The likelihood of IDU increased significantly (p<0.001) with increased use of cigarettes.

Conclusions/Significance

Certain groups of youth are more vulnerable to TS and IDU. Therefore, tobacco and drug control efforts should target these groups to reduce the consequences of risky lifestyles through information, education and communication (IEC) programs.     

Delphinidin Reduces Cell Proliferation and Induces Apoptosis of Non-Small-Cell Lung Cancer Cells by Targeting EGFR/VEGFR2 Signaling Pathways

Epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor 2 (VEGFR2) have emerged as two effective clinical targets for non-small-cell lung cancer (NSCLC). In the present study, we found that delphinidin, an anthocyanidin, present in pigmented fruits and vegetables, is a potent inhibitor of both EGFR and VEGFR2 in NSCLC cells that overexpress EGFR/VEGFR2. Using these cells, we next determined the effects of delphinidin on cell growth and apoptosis in vitro and on tumor growth and angiogenesis in vivo. Delphinidin (5-60 µM) treatment of NSCLC cells inhibited the activation of PI3K, and phosphorylation of AKT and MAPKs. Additionally, treatment of NSCLC cells with delphinidin resulted in inhibition of cell growth without having significant toxic effects on normal human bronchial epithelial cells. Specifically, treatment of NCI-H441 and SK-MES-1 cells with delphindin (5-60 µM) resulted in (i) cleavage of PARP protein, (ii) activation of caspase-3 and -9, (iii) downregulation of anti-apoptotic proteins (Bcl2, Bcl-xL and Mcl-1), (iv) upregulation of pro-apoptotic proteins (Bax and Bak), and (v) decreased expression of PCNA and cyclin D1. Furthermore, in athymic nude mice subcutaneously implanted with human NSCLC cells, delphinidin treatment caused a (i) significant inhibition of tumor growth, (ii) decrease in the expression of markers for cell proliferation (Ki67 and PCNA) and angiogenesis (CD31 and VEGF), and (iii) induction of apoptosis, when compared with control mice. Based on these observations, we suggest that delphinidin, alone or as an adjuvant to current therapies, could be used for the management of NSCLC, especially those that overexpress EGFR and VEGFR2.