Design and Application of Membrane Nanodiscs for Biophysical Studies of Influenza A Proteins

Membrane proteins play a range of important roles in biological systems, yet they are underrepresented in the data base of high-resolution structures of all proteins. There is intense interest in developing new methodologies for studying membrane proteins. An essential step to membrane protein method development is devising reliable membrane mimics in which to embed membrane proteins. The goal of this thesis was to develop and apply nanodisc membrane mimics to the study of an influenza A membrane protein called M2. Nanodiscs provide a lipid bilayer environment with access to both sides of the bilayer and are smaller than commonly used liposome model membranes whose size provides challenges for some biophysical methods. This thesis shows how the sample composition of M2 containing nanodiscs was optimized. Dynamic light scattering and size exclusion chromatography was used to characterize M2-nanodiscs. Electrophysiological and budding assays showed that M2 in liposomes were in a functionally relevant conformation. Extensive previous work has been done on studying M2 protein in spherical liposome using site-directed spin label electron paramagnetic resonance (SDSL-EPR). We carried out SDSL-EPR studies of M2-nanodiscs and compared them to published work on M2 in liposomes. Our EPR data is consistent with M2 protein in nanodiscs having a similar conformation, mobility and membrane topology as that seen in previously published M2-liposome work. Furthermore, we probed the ability of nanodiscs to allow for conformational exchange by comparing the impact of drug binding on M2-nanodiscs with M2-liposomes

Efficacy and effectiveness of dihydroartemisinin-piperaquine versus artesunate-mefloquine in falciparum malaria: an open-label randomised comparison.

BACKGROUND: Artemisinin-based combinations are judged the best treatments for multidrug-resistant Plasmodium falciparum malaria. Artesunate-mefloquine is widely recommended in southeast Asia, but its high cost and tolerability profile remain obstacles to widespread deployment. To assess whether dihydroartemisinin-piperaquine is a suitable alternative to artesunate-mefloquine, we compared the safety, tolerability, efficacy, and effectiveness of the two regimens for the treatment of uncomplicated falciparum in western Myanmar (Burma). METHODS: We did an open randomised comparison of 3-day regimens of artesunate-mefloquine (12/25 mg/kg) versus dihydroartemisinin-piperaquine (6.3/50 mg/kg) for the treatment of children aged 1 year or older and in adults with uncomplicated falciparum malaria in Rakhine State, western Myanmar. Within each group, patients were randomly assigned supervised or non-supervised treatment. The primary endpoint was the PCR-confirmed parasitological failure rate by day 42. Failure rates at day 42 were estimated by Kaplan-Meier survival analysis. This study is registered as an International Standard Randomised Controlled Trial, number ISRCTN27914471. FINDINGS: Of 652 patients enrolled, 327 were assigned dihydroartemisinin-piperaquine (156 supervised and 171 not supervised), and 325 artesunate-mefloquine (162 and 163, respectively). 16 patients were lost to follow-up, and one patient died 22 days after receiving dihydroartemisinin-piperaquine. Recrudescent parasitaemias were confirmed in only two patients; the day 42 failure rate was 0.6% (95% CI 0.2-2.5) for dihydroartemisinin-piperaquine and 0 (0-1.2) for artesunate-mefloquine. Whole-blood piperaquine concentrations at day 7 were similar for patients with observed and non-observed dihydroartemisinin-piperaquine treatment. Gametocytaemia developed more frequently in patients who had received dihydroartemisinin-piperaquine than in those on artesunate-mefloquine: day 7, 18 (10%) of 188 versus five (2%) of 218; relative risk 4.2 (1.6-11.0) p=0.011. INTERPRETATION: Dihydroartemisinin-piperaquine is a highly efficacious and inexpensive treatment of multidrug-resistant falciparum malaria and is well tolerated by all age groups. The effectiveness of the unsupervised treatment, as in the usual context of use, equalled its supervised efficacy, indicating good adherence without supervision. Dihydroartemisinin-piperaquine is a good alternative to artesunate-mefloquine

A Rapid Cellular FRET Assay of Polyglutamine Aggregation Identifies a Novel Inhibitor

AbstractMany neurodegenerative diseases, including tauopathies, Parkinson's disease, amyotrophic lateral sclerosis, and the polyglutamine diseases, are characterized by intracellular aggregation of pathogenic proteins. It is difficult to study modifiers of this process in intact cells in a high-throughput and quantitative manner, although this could facilitate molecular insights into disease pathogenesis. Here we introduce a high-throughput assay to measure intracellular polyglutamine protein aggregation using fluorescence resonance energy transfer (FRET). We screened over 2800 biologically active small molecules for inhibitory activity and have characterized one lead compound in detail. Y-27632, an inhibitor of the Rho-associated kinase p160ROCK, diminished polyglutamine protein aggregation (EC50 ≅ 5 μM) and reduced neurodegeneration in a Drosophila model of polyglutamine disease. This establishes a novel high-throughput approach to study protein misfolding and aggregation associated with neurodegenerative diseases and implicates a signaling pathway of previously unrecognized importance in polyglutamine protein processing

Susceptibility of the Asian Corn Borer, Ostrinia Furnacalis, to Bacillus Thuringiensis Toxin CRY1AC

The larval susceptibility of the Asian corn borer, Ostrinia furnacalis (Guenee) (Lepidoptera: Crambidae), to a Bacillus thuringiensis protein (Cry1Ac) was evaluated using insect feeding bioassays. The founding population of O. furnacalis was originally collected from the experimental station of UGM at Kalitirto and had been reared in the laboratory for three generations using an artificial diet “InsectaLf”. The tested instars were exposed on diets treated with a series of concentrations of Cry1Ac for one week. The LC50 values on the seventh day after treatment for 1st, 2nd, 3rd and 4th instars were 7.79, 21.12, 113.66, and 123.17 ppm, respectively, showing that the higher the instars the lesser the susceptibility to Cry1Ac. When the neonates were exposed to sublethal concentrations of Cry1Ac (0.0583, 0.116, and 0.5830 ppm), growth and development of the surviving larvae were inhibited. The fecundity and viability of females produced from treated larvae decreased with increasing the concentrations. These findings indicate that Cry1Ac is toxic to larva of O. furnacalis and has chronic effects to larvae surviving from Cry1Ac ingestion. Kepekaan larva penggerek batang jagung Asia, Ostrinia furnacalis (Guenee) (Lepidoptera: Crambidae), terhadap protein Bacillus thuringiensis Cry1Ac diuji dengan metode celup pakan. Larva berasal dari pertanaman jagung di KP-4, UGM di Kalitirto dan telah dikembangbiakkan di laboratorium menggunakan pakan buatan (InsectaLF) selama tiga generasi sebelum digunakan untuk pengujian. Larva O. furnacalis yang diuji dipaparkan pada pakan buatan yang telah dicelupkan pada seri konsentrasi Cry1Ac. Nilai LC50 pada hari ketujuh setelah perlakukan untuk instar 1, 2, 3, dan 4 berturut-turut adalah 0,79; 21,12; 113,66; dan 123,17 ppm. Hal ini menunjukkan bahwa instar yang semakin tinggi tingkat kepekaannya terhadap Cry1Ac semakin menurun. Larva yang baru menetas dan diberi pakan yang telah dicelupkan pada konsentrasi sublethal Cry1Ac sebagian akan mati, sedangkan larva yang hidup pertumbuhan dan perkembangannya terhambat. Fekunditas dan vialibitas serangga betina hasil dari larva yang diperlakukan dengan Cry1Ac menurun. Penelitian ini menunjukkan bahwa Cry1Ac toksik terhadap larva O. furnacalis dan juga mempunyai efek kronik terhadap larva yang tetap hidup setelah memakan toksin

Impairment in biochemical indices in sprayers exposed to commonly used organophosphorus and pyrethroids: A case study

This study was carried out to evaluate the effect of spraying organophosphate, carbamate and synthetic pyrethroid pesticides on biochemical parameters viz., red blood cell (RBC) acetylcholine esterase (AchE) and plasma butyrylcholinesterase (BChE) activities, oxidative stress parameters viz., plasma T-BARs, catalase activity and antioxidant levels such as reduced glutathione, and α-tocopherol and pesticide residues (analysed using the GC/LC-MS/MS) in sprayers cultivating cotton, chilly and paddy crops in Guntur district for a period of >10 years. The study population included sprayers (120) with a mean age of 37.8 ± 10.8 years and controls (60) with a mean age of 37.3 ± 12.2 years. A significant difference was observed for AchE, BChE activities, thiobarbituric acid reactive substances (TBARs), catalase activity, reduced glutathione and the concentration of pesticide residues in the sprayers cultivating different crops for a period of >10 years as compared to the controls. However, there is no such significant difference observed for α-tocopherol.Key words: Pesticide residues, chronic exposure, biochemical indices,  sprayers running head, biochemical indices, pesticide residues in sprayer

Differences in decision-making behavior between elite and amateur team-handball players in a near-game test situation

Athletic features distinguishing experts from non-experts in team sports are relevant for performance analyses, talent identification and successful training. In this respect, perceptual-cognitive factors like decision making have been proposed to be important predictor of talent but, however, assessing decision making in team sports remains a challenging endeavor. In particular, it is now known that decisions expressed by verbal reports or micro-movements in the laboratory differ from those actually made in on-field situations in play. To address this point, our study compared elite and amateur players' decision-making behavior in a near-game test environment including sport-specific sensorimotor responses. Team-handball players (N = 44) were asked to respond as quickly as possible to representative, temporally occluded attack sequences in a team-handball specific defense environment on a contact plate system. Specifically, participants had to choose and perform the most appropriate out of four prespecified, defense response actions. The frequency of responses and decision time were used as dependent variables representing decision-making behavior. We found that elite players responded significantly more often with offensive responses (p < 0.05, odds ratios: 2.76-3.00) in left-handed attack sequences. Decision time decreased with increasing visual information, but no expertise effect was found. We suppose that expertise-related knowledge and processing of kinematic information led to distinct decision-making behavior between elite and amateur players, evoked in a domain-specific and near-game test setting. Results also indicate that the quality of a decision might be of higher relevance than the required time to decide. Findings illustrate application opportunities in the context of performance analyses and talent identification processes

Planet Four: A Neural Network’s search for polar spring-time fans on Mars

Dark deposits visible from orbit appear in the Martian south polar region during the springtime. These are thought to form from explosive jets of carbon dioxide gas breaking through the thawing seasonal ice cap, carrying dust and dirt which is then deposited onto the ice as dark ‘blotches’, or blown by the surface winds into streaks or ‘fans’. We investigate machine learning (ML) methods for automatically identifying these seasonal features in High Resolution Imaging Science Experiment (HiRISE) satellite imagery. We designed deep Convolutional Neural Networks (CNNs) that were trained and tested using the catalog generated by Planet Four, an online citizen science project mapping the south polar seasonal deposits. We validated the CNNs by comparing their results with those of ISODATA (Iterative Self-Organizing Data Analysis Technique) clustering and as expected, the CNNs were significantly better at predicting the results found by Planet Four, in both the area of predicted seasonal deposits and in delineating their boundaries. We found neither the CNNs or ISODATA were suited to predicting the source point and directions of seasonal fans, which is a strength of the citizen science approach. The CNNs showed good agreement with Planet Four in cross-validation metrics and detected some seasonal deposits in the HiRISE images missed in the Planet Four catalog; the total area of seasonal deposits predicted by the CNNs was 27% larger than that of the Planet Four catalog, but this aspect varied considerably on a per-image basis

Health and human rights in eastern Myanmar prior to political transition: a population-based assessment using multistaged household cluster sampling

Background: Myanmar/Burma has received increased development and humanitarian assistance since the election in November 2010. Monitoring the impact of foreign assistance and economic development on health and human rights requires knowledge of pre-election conditions. Methods: From October 2008-January 2009, community-based organizations conducted household surveys using three-stage cluster sampling in Shan, Kayin, Bago, Kayah, Mon and Tanintharyi areas of Myanmar. Data was collected from 5,592 heads of household on household demographics, reproductive health, diarrhea, births, deaths, malaria, and acute malnutrition of children 6–59 months and women aged 15–49 years. A human rights focused survey module evaluated human rights violations (HRVs) experienced by household members during the previous year. Results: Estimated infant and under-five rates were 77 (95% CI 56 to 98) and 139 (95% CI 107 to 171) deaths per 1,000 live births; and the crude mortality rate was 13 (95% CI 11 to 15) deaths per thousand persons. The leading respondent-reported cause of death was malaria, followed by acute respiratory infection and diarrhea, causing 21.2% (95% CI 16.5 to 25.8), 16.6% (95% CI 11.8 to 21.4), and 12.3% (95% CI 8.7 to 15.8), respectively. Over a third of households suffered at least one human rights violation in the preceding year (36.2%; 30.7 to 41.7). Household exposure to forced labor increased risk of death among infants (rate ratio (RR) = 2.2; 95% CI 1.1 to 4.4) and children under five (RR = 2.1; 95% CI 1.3 to 3.6). The proportion of children suffering from moderate to severe acute malnutrition was higher among households that were displaced (prevalence ratio (PR) = 3.3; 95% CI 1.9 to 5.6). Conclusions: Prior to the 2010 election, populations of eastern Myanmar experienced high rates of disease and death and high rates of HRVs. These population-based data provide a baseline that can be used to monitor national and international efforts to improve the health and human rights situation in the region

Linear Lattice and Trajectory Reconstruction and Correction at FAST Linear Accelerator

The low energy part of the FAST linear accelerator based on 1.3 GHz superconducting RF cavities was successfully commissioned. During commissioning, beam based model dependent methods were used to correct linear lattice and trajectory. Lattice correction algorithm is based on analysis of beam shape from profile monitors and trajectory responses to dipole correctors. Trajectory responses to field gradient variations in quadrupoles and phase variations in superconducting RF cavities were used to correct bunch offsets in quadrupoles and accelerating cavities relative to their magnetic axes. Details of used methods and experimental results are presented.Comment: 3 p

Sensor-based precision nutrient and irrigation management enhances the physiological performance, water productivity, and yield of soybean under system of crop intensification

Sensor-based decision tools provide a quick assessment of nutritional and physiological health status of crop, thereby enhancing the crop productivity. Therefore, a 2-year field study was undertaken with precision nutrient and irrigation management under system of crop intensification (SCI) to understand the applicability of sensor-based decision tools in improving the physiological performance, water productivity, and seed yield of soybean crop. The experiment consisted of three irrigation regimes [I1: standard flood irrigation at 50% depletion of available soil moisture (DASM) (FI), I2: sprinkler irrigation at 80% ETC (crop evapo-transpiration) (Spr 80% ETC), and I3: sprinkler irrigation at 60% ETC (Spr 60% ETC)] assigned in main plots, with five precision nutrient management (PNM) practices{PNM1-[SCI protocol], PNM2-[RDF, recommended dose of fertilizer: basal dose incorporated (50% N, full dose of P and K)], PNM3-[RDF: basal dose point placement (BDP) (50% N, full dose of P and K)], PNM4-[75% RDF: BDP (50% N, full dose of P and K)] and PNM5-[50% RDF: BDP (50% N, full P and K)]} assigned in sub-plots using a split-plot design with three replications. The remaining 50% N was top-dressed through SPAD assistance for all the PNM practices. Results showed that the adoption of Spr 80% ETC resulted in an increment of 25.6%, 17.6%, 35.4%, and 17.5% in net-photosynthetic rate (Pn), transpiration rate (Tr), stomatal conductance (Gs), and intercellular CO2 concentration (Ci), respectively, over FI. Among PNM plots, adoption of PNM3 resulted in a significant (p=0.05) improvement in photosynthetic characters like Pn (15.69 µ mol CO2 m−2 s−1), Tr (7.03 m mol H2O m−2 s−1), Gs (0.175 µmol CO2 mol−1 year−1), and Ci (271.7 mol H2O m2 s−1). Enhancement in SPAD (27% and 30%) and normalized difference vegetation index (NDVI) (42% and 52%) values were observed with nitrogen (N) top dressing through SPAD-guided nutrient management, helped enhance crop growth indices, coupled with better dry matter partitioning and interception of sunlight. Canopy temperature depression (CTD) in soybean reduced by 3.09–4.66°C due to adoption of sprinkler irrigation. Likewise, Spr 60% ETc recorded highest irrigation water productivity (1.08 kg ha−1 m−3). However, economic water productivity (27.5 INR ha−1 m−3) and water-use efficiency (7.6 kg ha−1 mm−1 day−1) of soybean got enhanced under Spr 80% ETc over conventional cultivation. Multiple correlation and PCA showed a positive correlation between physiological, growth, and yield parameters of soybean. Concurrently, the adoption of Spr 80% ETC with PNM3 recorded significantly higher grain yield (2.63 t ha−1) and biological yield (8.37 t ha−1) over other combinations. Thus, the performance of SCI protocols under sprinkler irrigation was found to be superior over conventional practices. Hence, integrating SCI with sensor-based precision nutrient and irrigation management could be a viable option for enhancing the crop productivity and enhance the resource-use efficiency in soybean under similar agro-ecological regions