Autophagy Limits Endotoxemic Acute Kidney Injury and Alters Renal Tubular Epithelial Cell Cytokine Expression

Sepsis related acute kidney injury (AKI) is a common in-hospital complication with a dismal prognosis. Our incomplete understanding of disease pathogenesis has prevented the identification of hypothesis-driven preventive or therapeutic interventions. Increasing evidence in ischemia-reperfusion and nephrotoxic mouse models of AKI support the theory that autophagy protects renal tubular epithelial cells (RTEC) from injury. However, the role of RTEC autophagy in septic AKI remains unclear. We observed that lipopolysaccharide (LPS), a mediator of gram-negative bacterial sepsis, induces RTEC autophagy in vivo and in vitro through TLR4-initiated signaling. We modeled septic AKI through intraperitoneal LPS injection in mice in which autophagy-related protein 7 was specifically knocked out in the renal proximal tubules (ATG7KO). Compared to control littermates, ATG7KO mice developed more severe renal dysfunction (24hr BUN 100.1mg/dl +/- 14.8 vs 54.6mg/dl +/- 11.3) and parenchymal injury. After injection with LPS, analysis of kidney lysates identified higher IL-6 expression and increased STAT3 activation in kidney lysates from ATG7KO mice compared to controls. In vitro experiments confirmed an altered response to LPS in RTEC with genetic or pharmacological impairment of autophagy. In conclusion, RTEC autophagy protects against endotoxin induced injury and regulates downstream effects of RTEC TLR4 signaling.     

Coupling historical prospection data and a remotely-sensed vegetation index for the preventative control of Desert locusts

Locusts are grasshopper species that exhibit phase polyphenism resulting in the expression of gregarious behaviors that favor the development of large devastating bands and swarms. Desert locust preventative management aims to prevent crop damage by controlling populations before they can reach high densities and form mass migrating swarms. The areas of potential gregarization for Desert locust are large and need to be physically assessed by survey teams for efficient preventative management. An ongoing challenge is to be able to guide where prospection surveys should occur depending on local meteorological and vegetation conditions. In this study, we analyzed the relationship between historical prospection data of Desert locust observations from 2005 to 2009 and spatio-temporal statistics of a vegetation index gathered by remote-sensing with the help of multiple models of logistic regression. The vegetation index was a composite Normalized Difference Vegetation Index (NDVI) given every 16 days and at 250 m spatial resolution (MOD13Q1 from MODIS satellite). The statistics extracted from this index were: (1) spatial means at different scales around the prospection point, (2) relative differences of NDVI variation through time before the prospection, and (3) large-scale summary of vegetation quantity. The multi-model framework showed that vegetation development a month and a half before the survey was amongst the best predictors of locust presence. Also, the local vegetation quantity was not enough to predict locust presence. Vegetation quantity on a scale of a few kilometers was a better predictor but varied non-linearly, reflecting specific biotope types that support Desert locust development. Using one of the best logistic regression models and NDVI data, we were able to derive a predictive model of probability of finding locusts in specific areas. This methodology should help in more efficiently focusing survey efforts on specific parts of the gregarization areas based on the predicted probability of locusts being present.     

Separation of lipid classes by solid phase extraction

A rapid and reliable method for the separation of lipid classes is described using aminopropyl disposable columns. This method is a modification to an existing procedure that allows the separation of both neutral and acidic phospholipid fractions and a high recovery of the latter. Acidic phospholipids were eluted with a mixture of hexane-2-propanol-ethanol-0.1 M ammonium acetate-formic acid 420:350:100:50:0.5 containing 5% phosphoric acid after neutral phospholipids had been eluted with methanol. It was verified that extremely high recoveries of cholesterol (CH), triglycerides (TG), free fatty acids (FFA), phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyelin (SM), and cerebrosides were obtained with this method. In addition, there appeared to be no preferential losses or degradation of any particular molecular species as the fatty acid distribution of bovine brain PS and the molecular species profile of plant PI were unaltered by the procedure. Depending on the tissue, this method may yield fractions containing pure lipid classes and/or simple mixtures of lipid classes of similar polarity. These fractions may then be more easily separated by thin-layer chromatography or high performance liquid chromatography for a complete lipid class analysis.     

Markers of atopic dermatitis,allergic rhinitis and bronchial asthma in pediatric patients: correlation with filaggrin,eosinophil major basic protein and immunoglobulin E

Background

Allergic reactions have been implicated as contributions in a number of atopic disorders, including atopic dermatitis (AD), allergic rhinitis (AR) and bronchial asthma (BA). However, the potential for filaggrin protein, eosinophil major basic protein (MBP) and immunoglobulin E (IgE) to elicit allergic response or to contribute to atopic disorders remains largely unexplored in pediatric patients. This study was undertaken to investigate the status and contribution of filaggrin protein, eosinophil MBP and total IgE in pediatric patients with AD, AR and BA.

Methods

Sera from 395 pediatric patients of AD, AR or BA with varying levels of disease activity according to the disease activity index and 410 age-matched non-atopic healthy controls were evaluated for serum levels of atopic markers, including filaggrin, eosinophil MBP and IgE.

Results

Serum analysis showed that filaggrin levels were remarkably high in pediatric patients with AD, followed by BA and AR, whereas its levels were low in non-atopic pediatric controls. Eosinophil MBP levels in sera of atopic patients were significantly high as compared with their respective controls, but its levels were highest in AR patients, followed by AD and BA. Total IgE in sera of AD patients was markedly high, followed by AR and BA patients, whereas its levels were low in non-atopic pediatric controls. Interestingly, not only was an increased number of subjects positive for filaggrin protein, eosinophil MBP or total IgE, but also their levels were statistically significantly higher among those atopic patients whose disease activity scores were higher as compared with atopic patients with lower disease activity scores.

Conclusions

These findings strongly support a role of filaggrin protein, eosinophil MBP and IgE in the onset of allergic reactions in pediatric patients with AD, AR and BA. The data suggest that filaggrin, eosinophil MBP or IgE might be useful in evaluating the progression of AD, AR or BA and in elucidating the mechanisms involved in the pathogenesis of these pediatric disorders.

     

Accumulation of essential oils by Agrobacterium tumefaciens-transformed shoot cultures of Pimpinella anisum

Axenic transformed shoot cultures of Pimpinella anisum (anise) were established following inoculation of plant stems with the nopaline strain T37 of Agrobacterium tumefaciens. The stable incorporation of T-DNA in the transformed tissues was demonstrated by polymerase chain reaction. Total essential oil accumulated by transformed shoot cultures grown under continuous light was found to be 18% lower (per unit fresh weight of tissue) than that produced by untransformed shoot cultures incubated under similar conditions, but more than 89% lower than the yield of oil from the intact plant. The relative amounts of the principal components of the essential oil of the transformed shoot cultures, namely geraniol, -bisabolene, trans-pseudoisoeugenol-2-methylbutyrate and transanethole, were similar to those present in the parent plant, but significantly different from those of the untransformed shoot cultures.Abbreviations T-DNA transfer-DNA - MS Murashige and Skoog medium - 2,4-D 2,4-dichlorophenoxyacetic acid - TY tryptone and yeast extract medium - t_D doubling time - GC-MS gas chromatography coupled with mass spectrometry - FID flame ionisation detector - PCR polymerase chain reaction - TE Tris-HCl, EDTA buffer - TBE Tris, borate, EDTA buffer     

Step and ramp induction of myocardial ischemia: comparison of in vivo and in silico results.

This study tested the robustness of our computational model of myocardial metabolism by comparing responses to two different inputs with experimental data obtained in pigs under similar conditions. Accordingly, an abrupt and a gradual reduction in coronary flow of similar magnitude were implemented and used as model input. After flow reductions reached 60% from control values, ischemia was kept constant for 60 min in both groups. Our hypotheses were that: (1) these two flow-reduction profiles would result in different transients (concentrations and flux rates) while having similar steady-state values and (2) our model-simulated responses would predict the experimental results in an anesthetized swine model of myocardial ischemia. The two different ischemia-induction patterns resulted in the same decrease in steady-state MVO2 and in similar steady-state values for metabolite concentrations and flux rates at 60 min of ischemia. While both the simulated and experimental results showed decreased glycogen concentration, accumulation of lactate, and net lactate release with ischemia, the onset of glycogen depletion and the switch to lactate efflux were more rapid in the experiments than in the simulations. This study demonstrates the utility of computer models for predicting experimental outcomes in studies of metabolic regulation under physiological and pathological conditions.     

Autologous transplantation of CD34+ bone marrow derived mononuclear cells in management of non-reconstructable critical lower limb ischemia

Patients with a decrease in limb perfusion with a potential threat to limb viability manifested by ischemic rest pain, ischemic ulcers, and/or gangrene are considered to have critical limb ischemia (CLI). Because of this generally poor outcome, there is a strong need for attempting any procedure to save the affected limb. The aim of this work is to evaluate the possibility to use stem cell therapy as a treatment option for patients with chronic critical lower limb ischemia with no distal run off. This study includes 20 patients with chronic critical lower limb ischemia with no distal run off who are unsuitable for vascular or endovascular option. These patients underwent stem cell therapy (SCT) by autologous transplantation of bone marrow derived mononuclear cells. 55 % of patients treated with SCT showed improvement of the rest pain after the first month, 60 % continued improvement of the rest pain after 6 months, 75 % after 1 year and 80 % after 2 years and continued without any deterioration till the third year. Limb salvage rate after STC was 80 % after the first year till the end of the second and third years. SCT can result in angiogenesis in patients with no-option CLI, providing a foundation for the application of this therapy to leg ischemia.