A novel insecticidal GroEL protein from Xenorhabdus nematophila confers insect resistance in tobacco

Xenorhabdus nematophila is an entomopathogenic bacteria. It secretes a GroEL homolog, XnGroEL protein, toxic to its larval prey. GroEL belongs to the family of molecular chaperones and is required for proper folding of cellular proteins. Oral ingestion of insecticidal XnGroEL protein is toxic to Helicoverpa armigera, leading to cessation of growth and development of the larvae. In the present study, the insecticidal efficacy of XnGroEL against H. armigera has been evaluated in transgenic tobacco plant expressing the protein. A 1.7-kb gene encoding the 58-kDa XnGroEL protein was incorporated into the tobacco genome via Agrobacterium-mediated transformation. The stable integration of the transgene was confirmed by Southern blot analysis and its expression by RT-PCR and western blot analyses in transgenic plants. The transgenic lines showed healthy growth and were phenotypically normal. Insect bioassays revealed significant reduction of 100 % in the survival of larvae (p < 0.001) and 55–77 % reduction in plant damage (p < 0.05 and p < 0.001) compared to the untransformed and vector control plants. The results demonstrate that XnGroEL is a novel potential candidate for imparting insect resistance against H. armigera in plants.     

The Effects of School Closures on Influenza Outbreaks and Pandemics: Systematic Review of Simulation Studies

Background

School closure is a potential intervention during an influenza pandemic and has been investigated in many modelling studies.

Objectives

To systematically review the effects of school closure on influenza outbreaks as predicted by simulation studies.

Methods

We searched Medline and Embase for relevant modelling studies published by the end of October 2012, and handsearched key journals. We summarised the predicted effects of school closure on the peak and cumulative attack rates and the duration of the epidemic. We investigated how these predictions depended on the basic reproduction number, the timing and duration of closure and the assumed effects of school closures on contact patterns.

Results

School closures were usually predicted to be most effective if they caused large reductions in contact, if transmissibility was low (e.g. a basic reproduction number <2), and if attack rates were higher in children than in adults. The cumulative attack rate was expected to change less than the peak, but quantitative predictions varied (e.g. reductions in the peak were frequently 20–60% but some studies predicted >90% reductions or even increases under certain assumptions). This partly reflected differences in model assumptions, such as those regarding population contact patterns.

Conclusions

Simulation studies suggest that school closure can be a useful control measure during an influenza pandemic, particularly for reducing peak demand on health services. However, it is difficult to accurately quantify the likely benefits. Further studies of the effects of reactive school closures on contact patterns are needed to improve the accuracy of model predictions.     

Stem Cell Selection In Vivo Using Foamy Vectors Cures Canine Pyruvate Kinase Deficiency

Background

Hematopoietic stem cell (HSC) gene therapy has cured immunodeficiencies including X-linked severe combined immunodeficiency (SCID-X1) and adenine deaminase deficiency (ADA). For these immunodeficiencies corrected cells have a selective advantage in vivo, and low numbers of gene-modified cells are sufficient to provide therapeutic benefit. Strategies to efficiently transduce and/or expand long-term repopulating cells in vivo are needed for treatment of diseases that require higher levels of corrected cells, such as hemoglobinopathies. Here we expanded corrected stem cells in vivo in a canine model of a severe erythroid disease, pyruvate kinase deficiency.

Methodology/Principal Findings

We used a foamy virus (FV) vector expressing the P140K mutant of methylguanine methyltransferase (MGMTP140K) for in vivo expansion of corrected hematopoietic repopulating cells. FV vectors are attractive gene transfer vectors for hematopoietic stem cell gene therapy since they efficiently transduce repopulating cells and may be safer than more commonly used gammaretroviral vectors. Following transplantation with HSCs transduced ex vivo using a tri-cistronic FV vector that expressed EGFP, R-type pyruvate kinase, and MGMTP140K, we were able to increase marking from approximately 3.5% to 33% in myeloid long-term repopulating cells resulting in a functional cure.

Conclusions/Significance

Here we describe in one affected dog a functional cure for a severe erythroid disease using stem cell selection in vivo. In addition to providing a potential cure for patients with pyruvate kinase deficiency, in vivo selection using foamy vectors with MGMTP140K has broad potential for several hematopoietic diseases including hemoglobinopathies.     

Quantitative restoration of immune defense in old animals determined by naive antigen‐specific CD8 T‐cell numbers

Older humans and animals often exhibit reduced immune responses to infection and vaccination, and this often directly correlates to the numbers and frequency of naive T (Tn) cells. We found such a correlation between reduced numbers of blood CD8+ Tn cells and severe clinical outcomes of West Nile virus (WNV) in both humans naturally exposed to, and mice experimentally infected with, WNV. To examine possible causality, we sought to increase the number of CD8 Tn cells by treating C57BL/6 mice with IL‐7 complexes (IL‐7C, anti‐IL‐7 mAb bound to IL‐7), shown previously to efficiently increase peripheral T‐cell numbers by homeostatic proliferation. T cells underwent robust expansion following IL‐7C administration to old mice increasing the number of total T cells (>fourfold) and NS4b:H‐2D^b‐restricted antigen‐specific CD8 T cells (twofold). This improved the numbers of NS4b‐specific CD8 T cells detected at the peak of the response against WNV, but not survival of WNV challenge. IL‐7C‐treated old animals also showed no improvement in WNV‐specific effector immunity (neutralizing antibody and in vivo T‐cell cytotoxicity). To test quantitative limits to which CD8 Tn cell restoration could improve protective immunity, we transferred graded doses of Ag‐specific precursors into old mice and showed that injection of 5400 (but not of 1800 or 600) adult naive WNV‐specific CD8 T cells significantly increased survival after WNV. These results set quantitative limits to the level of Tn reconstitution necessary to improve immune defense in older organisms and are discussed in light of targets of immune reconstitution.     

Vulnerability to winter mortality in elderly people in Britain: population based study

Objective To examine the determinants of vulnerability to winter mortality in elderly British people.Design Population based cohort study (119 389 person years of follow up).Setting 106 general practices from the Medical Research Council trial of assessment and management of older people in Britain.Participants People aged ≥ 75 years.Main outcome measures Mortality (10 123 deaths) determined by follow up through the Office for National Statistics.Results Month to month variation accounted for 17% of annual all cause mortality, but only 7.8% after adjustment for temperature. The overall winter:non-winter rate ratio was 1.31 (95% confidence interval 1.26 to 1.36). There was little evidence that this ratio varied by geographical region, age, or any of the personal, socioeconomic, or clinical factors examined, with two exceptions: after adjustment for all major covariates the winter:non-winter ratio in women compared with men was 1.11 (1.00 to 1.23), and those with a self reported history of respiratory illness had a winter:non-winter ratio of 1.20 (1.08 to 1.34) times that of people without a history of respiratory illness. There was no evidence that socioeconomic deprivation or self reported financial worries were predictive of winter death.Conclusion Except for female sex and pre-existing respiratory illness, there was little evidence for vulnerability to winter death associated with factors thought to lead to vulnerability. The lack of socioeconomic gradient suggests that policies aimed at relief of fuel poverty may need to be supplemented by additional measures to tackle the burden of excess winter deaths in elderly people.     

Effect of influenza vaccination on excess deaths occurring during periods of high circulation of influenza: cohort study in elderly people

Objective To estimate the protection against death provided by vaccination against influenza.Design Prospective cohort follow up supplemented by weekly national counts of influenza confirmed in the community.Setting Primary care.Participants 24 535 patients aged over 75 years from 73 general practices in Great Britain.Main outcome measure Death.Results In unvaccinated members of the cohort daily all cause mortality was strongly associated with an index of influenza circulating in the population (mortality ratio 1.16, 95% confidence interval 1.04 to 1.29 at 90th centile of circulating influenza). The association was strongest for respiratory deaths but was also present for cardiovascular deaths. In contrast, in vaccinated people mortality from any cause was not associated with circulating influenza. The difference in patterns between vaccinated and unvaccinated people could not easily be due to chance (P = 0.02, all causes).Conclusions This study, using a novel and robust approach to control for confounding, provides robust evidence of a protective effect on mortality of vaccination against influenza.     

Chaperone-assisted refolding of Escherichia coli maltodextrin glucosidase

In vitro refolding of maltodextrin glucosidase, a 69 kDa monomeric Escherichia coli protein, was studied in the presence of glycerol, dimethylsulfoxide, trimethylamine-N-oxide, ethylene glycol, trehalose, proline and chaperonins GroEL and GroES. Different osmolytes, namely proline, glycerol, trimethylamine-N-oxide and dimethylsulfoxide, also known as chemical chaperones, assist in protein folding through effective inhibition of the aggregation process. In the present study, it was observed that a few chemical chaperones effectively reduced the aggregation process of maltodextrin glucosidase and hence the in vitro refolding was substantially enhanced, with ethylene glycol being the exception. Although, the highest recovery of active maltodextrin glucosidase was achieved through the ATP-mediated GroEL/GroES-assisted refolding of denatured protein, the yield of correctly folded protein from glycerol- or proline-assisted spontaneous refolding process was closer to the chaperonin-assisted refolding. It was also observed that the combined application of chemical chaperones and molecular chaperone was more productive than their individual contribution towards the in vitro refolding of maltodextrin glucosidase. The chemical chaperones, except ethylene glycol, were found to provide different degrees of protection to maltodextrin glucosidase from thermal denaturation, whereas proline caused the highest protection. The observations from the present studies conclusively demonstrate that chemical or molecular chaperones, or the combination of both chaperones, could be used in the efficient refolding of recombinant E. coli maltodextrin glucosidase, which enhances the possibility of identifying or designing suitable small molecules that can act as chemical chaperones in the efficient refolding of various aggregate-prone proteins of commercial and medical importance.     

The Influence of Mineral Matrices on the Thermal Behavior of Glycine

On the Hadean–Early Archean Earth, the first islands must have provided hot and dry environments for abiotically formed organic molecules. The heat sources, mainly volcanism and meteorite impacts, were also available on Mars during the Noachian period. In recent work simulating this scenario, we have shown that neat glycine forms a black, sparingly water-soluble polymer (“thermomelanoid”) when dry-heated at 200 °C under pure nitrogen. The present study explores whether relevant minerals and mineral mixtures can change this thermal behavior. Most experiments were conducted at 200 or 250 °C for 2 or 7 days. The mineral matrices used were phyllosilicates (Ca-montmorillonites SAz-1 and STx-1, Na-montmorillonite SAz-1-Na, nontronite NAu-1, kaolinite KGa-1), salts (NaCl, NaCl-KCl, CaCl₂, artificial sea salt, gypsum, magnesite), picritic basalt, and three Martian regolith simulants (P-MRS, S-MRS, JSC Mars-1A). The main analytical method employed was high-performance liquid chromatography (HPLC). Glycine intercalated in SAz-1 and SAz-1-Na was well protected against thermomelanoid formation and sublimation at 200 °C: after 2 days, 95 and 79 %, respectively, had either survived unaltered or been transformed into the cyclic dipeptide (DKP) and linear peptides up to Gly₆. The glycine survival rate followed the order SAz-1?>?SAz-1-Na?>?STx-1?≈?NAu-1?>?KGa-1. Very good protection was also provided by artificial sea salt (84 % unaltered glycine after 200 °C for 7 days). P-MRS promoted the condensation up to Gly₆, consistent with its high phyllosilicate content. The remaining matrices were less effective in preserving glycine as such or as peptides.     

Antiangiogenic properties of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol/one analogs and their derivatives

In the past half century research efforts have defined a critical role for angiogenesis in tumor growth and metastasis. We previously reported that inhibition of a novel target, ENOX1, by a (Z)-2-benzylindol-3-ylmethylene) quinuclidin-3-ol, suppressed tumor angiogenesis. The present study was undertaken in order to establish structure-activity relationships for quinuclidine analogs. The angiogenesis inhibiting activity of a series of substituted (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ols (1a-1k), (Z)-2-benzylindol-3-ylmethylene)quinuclidin-3-ones (2a-2h), (Z)-(±)-2-(1H/N-methyl-indol-3-ylmethylene)quinuclidin-3-ols (3a-3b), and substituted (Z)-(±)-2-(N-benzenesulfonylindol-3-yl-methylene)quinuclidin-3-ols and their derivatives (4a-4d) that incorporate a variety of substituents in both the indole and N-benzyl moieties was evaluated using Human Umbilical Vein Endothelial Cells (HUVECs) subjected to in vitro cell migration scratch assays, tubule formation in Matrigel, cell viability and proliferation assays. In total, 25 different analogs were evaluated. Based on in vitro cell migration scratch assays, eight analogs were identified as potent angiogenesis inhibitors at 10 μM, a concentration that was determined to be nontoxic by colony formation assay. In addition, this approach identified a potent antiangiogenic ENOX1 inhibitor, analog 4b.     

Diagnostic value of fine needle aspiration cytology in gouty tophi: a report of 7 cases

BACKGROUND The diagnosis of gout can be problematic when the presentation is atypical and serum uric acid is borderline elevated. Demonstration of monosodium urate (MSU) crystals in fine needle aspiration (FNA) smears from nodular masses clinically suspected to be tophi establishes the diagnosis unequivocally. CASES: Of the 7 cases in this study, 4 were suspected clinically to have gouty tophi. Giant cell tumor of tendon sheath, giant cell tumor of bone and metastatic tumor with multicentric involvement of bone were the clinical diagnoses in 1 case each. Serum uric acid levels high enough to be in the diagnostic range for gout were reported in 3 cases, within normal limits in 3 cases and low in 1 chronic alcoholic patient. Bright field microscopy of FNA smears revealed singly scattered or stacks of MSU crystals with variable number of inflammatory cells, with or without foreign body giant cells in 6 cases. In 1 patient, FNA showed stacks of MSU crystals only. Characteristic birefringence of MSU crystals was observed on polarizing microscopy. CONCLUSION: FNA demonstration of MSU crystals on polarizing microscopy can easily establish the nature of the nodules in and around the joints and in soft tissue as gouty tophi and is thus an investigation differentiating this lesion from other masses clinically simulating it.