Advances in genome-wide RNAi cellular screens: a case study using the Drosophila JAK/STAT pathway

BACKGROUND: Genome-scale RNA-interference (RNAi) screens are becoming ever more common gene discovery tools. However, whilst every screen identifies interacting genes, less attention has been given to how factors such as library design and post-screening bioinformatics may be effecting the data generated. RESULTS: Here we present a new genome-wide RNAi screen of the Drosophila JAK/STAT signalling pathway undertaken in the Sheffield RNAi Screening Facility (SRSF). This screen was carried out using a second-generation, computationally optimised dsRNA library and analysed using current methods and bioinformatic tools. To examine advances in RNAi screening technology, we compare this screen to a biologically very similar screen undertaken in 2005 with a first-generation library. Both screens used the same cell line, reporters and experimental design, with the SRSF screen identifying 42 putative regulators of JAK/STAT signalling, 22 of which verified in a secondary screen and 16 verified with an independent probe design. Following reanalysis of the original screen data, comparisons of the two gene lists allows us to make estimates of false discovery rates in the SRSF data and to conduct an assessment of off-target effects (OTEs) associated with both libraries. We discuss the differences and similarities between the resulting data sets and examine the relative improvements in gene discovery protocols. CONCLUSIONS: Our work represents one of the first direct comparisons between first- and second-generation libraries and shows that modern library designs together with methodological advances have had a significant influence on genome-scale RNAi screens

Deciphering the unique cellulose degradation mechanism of the ruminal bacterium Fibrobacter succinogenes S85

Fibrobacter succinogenes S85, isolated from the rumen of herbivores, is capable of robust lignocellulose degradation. However, the mechanism by which it achieves this is not fully elucidated. In this study, we have undertaken the most comprehensive quantitative proteomic analysis, to date, of the changes in the cell envelope protein profile of F. succinogenes S85 in response to growth on cellulose. Our results indicate that the cell envelope proteome undergoes extensive rearrangements to accommodate the cellulolytic degradation machinery, as well as associated proteins involved in adhesion to cellulose and transport and metabolism of cellulolytic products. Molecular features of the lignocellulolytic enzymes suggest that the Type IX secretion system is involved in the translocation of these enzymes to the cell envelope. Finally, we demonstrate, for the first time, that cyclic-di-GMP may play a role in mediating catabolite repression, thereby facilitating the expression of proteins involved in the adhesion to lignocellulose and subsequent lignocellulose degradation and utilisation. Understanding the fundamental aspects of lignocellulose degradation in F. succinogenes will aid the development of advanced lignocellulosic biofuels

In situ high pressure powder diffraction study of proton conductors based on metal phosphonates

Soft Porous Metal Organic frameworks (MOFs) are referred to as a class of coordination polymers that exhibit structural flexibility in response to guest interactions or physical stimuli [1]. By combining softness and regularity, the responsive crystalline frameworks show, for instance, unique mechanisms of separation and storage of gases. Here we report the effects of high pressures of CO2 on the frameworks of two types of coordination polymers based on multifunctional metal phosphonates, which exhibit proton conductivity at high relative humidity in addition to porous properties. The first one, Ni2(H2O)2(O3PCH2N(C4H8)NCH2PO3)⋅8H2O (Ni-STA-12) is a well-known MOF material structural featured by 1D channels build from MO5N octahedra linked by the piperazinyl moieties [2]. The second solid, Mg[(HO3PCH2)2NHCH2C6H4CH2NH-(CH2PO3H)2]·2H2O, (MgHDTMP·2H2O), is a pillared layer metal phosphonate containing flexible alkyldiaminetetraphosphonate as linker of the inorganic layers. For both solids, in situ synchrotron powder diffraction data were collected on BL04-MSPD under different pressures of CO2 (up to ~10 bar) and temperatures at ALBA (Barcelona, Spain). The resulting structural changes observed on their frameworks as well as their proton conductivities will be discussed.Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. Junta de Andalucía,Proyecto de Excelencia P12-FQM-1656 MINECO: MAT2013-41836-R

Rates and Factors Associated with Major Modifications to First-Line Combination Antiretroviral Therapy: Results from the Asia-Pacific Region

Background: In the Asia-Pacific region many countries have adopted the WHO's public health approach to HIV care and treatment. We performed exploratory analyses of the factors associated with first major modification to first-line combination antiretroviral therapy (ART) in resource-rich and resource-limited countries in the region. Methods: We selected treatment naive HIV-positive adults from the Australian HIV Observational Database (AHOD) and the TREAT Asia HIV Observational Database (TAHOD). We dichotomised each country's per capita income into high/upper-middle (T-H) and lower-middle/low (T-L). Survival methods stratified by income were used to explore time to first major modification of first-line ART and associated factors. We defined a treatment modification as either initiation of a new class of antiretroviral (ARV) or a substitution of two or more ARV agents from within the same ARV class. Results: A total of 4250 patients had 961 major modifications to first-line ART in the first five years of therapy. The cumulative incidence (95% CI) of treatment modification was 0.48 (0.44-0.52), 0.33 (0.30-0.36) and 0.21 (0.18-0.23) for AHOD, T-H and T-L respectively. We found no strong associations between typical patient characteristic factors and rates of treatment modification. In AHOD, relative to sites that monitor twice-yearly (both CD4 and HIV RNA-VL), quarterly monitoring corresponded with a doubling of the rate of treatment modifications. In T-H, relative to sites that monitor once-yearly (both CD4 and HIV RNA-VL), monitoring twice-yearly corresponded to a 1.8 factor increase in treatment modifications. In T-L, no sites on average monitored both CD4 & HIV RNA-VL concurrently once-yearly. We found no differences in rates of modifications for once- or twice-yearly CD4 count monitoring. Conclusions: Low-income countries tended to have lower rates of major modifications made to first-line ART compared to higher-income countries. In higher-income countries, an increased rate of RNA-VL monitoring was associated with increased modifications to first-line ART. © 2013 Wright et al

Kernel density classification and boosting: an L2 sub analysis

Kernel density estimation is a commonly used approach to classification. However, most of the theoretical results for kernel methods apply to estimation per se and not necessarily to classification. In this paper we show that when estimating the difference between two densities, the optimal smoothing parameters are increasing functions of the sample size of the complementary group, and we provide a small simluation study which examines the relative performance of kernel density methods when the final goal is classification. A relative newcomer to the classification portfolio is “boosting”, and this paper proposes an algorithm for boosting kernel density classifiers. We note that boosting is closely linked to a previously proposed method of bias reduction in kernel density estimation and indicate how it will enjoy similar properties for classification. We show that boosting kernel classifiers reduces the bias whilst only slightly increasing the variance, with an overall reduction in error. Numerical examples and simulations are used to illustrate the findings, and we also suggest further areas of research

Practices in scholarly publishing : making sense of rejection

Abstract: In South Africa, criticisms of peer review often hinge on allegations of racism, anti-African attitudes, and viewpoint discrimination. This article discusses the issue of peer-review, and examines these allegations in terms of claims of Western conceptual gatekeeping. Cautions are offered on allegations of exceptionalism, as are some strategies on dealing with the process of peer review

Agr quorum sensing influences the Wood-Ljungdahl pathway in Clostridium autoethanogenum

Acetogenic bacteria are capable of fermenting CO2 and carbon monoxide containing waste-gases into a range of platform chemicals and fuels. Despite major advances in genetic engineering and improving these biocatalysts, several important physiological functions remain elusive. Among these is quorum sensing, a bacterial communication mechanism known to coordinate gene expression in response to cell population density. Two putative agr systems have been identified in the genome of Clostridium autoethanogenum suggesting bacterial communication via autoinducing signal molecules. Signal molecule-encoding agrD1 and agrD2 genes were targeted for in-frame deletion. During heterotrophic growth on fructose as a carbon and energy source, single deletions of either gene did not produce an observable phenotype. However, when both genes were simultaneously inactivated, final product concentrations in the double mutant shifted to a 1.5:1 ratio of ethanol:acetate, compared to a 0.2:1 ratio observed in the wild type control, making ethanol the dominant fermentation product. Moreover, CO2 re-assimilation was also notably reduced in both hetero- and autotrophic growth conditions. These findings were supported through comparative proteomics, which showed lower expression of carbon monoxide dehydrogenase, formate dehydrogenase A and hydrogenases in the ∆agrD1∆agrD2 double mutant, but higher levels of putative alcohol and aldehyde dehydrogenases and bacterial micro-compartment proteins. These findings suggest that Agr quorum sensing, and by inference, cell density play a role in carbon resource management and use of the Wood-Ljungdahl pathway as an electron sink

Tisa: A Language Design and Modular Verification Technique for Temporal Policies in Web Services

Web services are distributed software components, that are decoupled from each other using interfaces with specified functional behaviors. However, such behavioral specifications are insufficient to demonstrate compliance with certain temporal non-functional policies. An example is demonstrating that a patient’s health-related query sent to a health care service is answered only by a doctor (and not by a secretary). Demonstrating compliance with such policies is important for satisfying governmental privacy regulations. It is often necessary to expose the internals of the web service implementation for demonstrating such compliance, which may compromise modularity. In this work, we provide a language design that enables such demonstrations, while hiding majority of the service’s source code. The key idea is to use greybox specifications to allow service providers to selectively hide and expose parts of their implementation. The overall problem of showing compliance is then reduced to two subproblems: whether the desired properties are satisfied by the service’s greybox specification, and whether this greybox specification is satisfied by the service’s implementation. We specify policies using LTL and solve the first problem by model checking. We solve the second problem by refinement techniques

Search for Pairs of Isolated Radio Pulsars - Components in Disrupted Binary Systems

We have developed a method for analyzing the kinematic association of isolated relativistic objects - possible remnants of disrupted close binary systems. We investigate pairs of fairly young radio pulsars with known proper motions and estimated distances (dispersion measures) that are spaced no more than 2-3 kpc apart. Using a specified radial velocity distribution for these objects, we have constructed 100-300 thousand trajectories of their possible motion in the Galactic gravitational field on a time scale of several million years. The probabilities of their close encounters at epochs consistent with the age of the younger pulsar in the pair are analyzed. When these probabilities exceed considerably their reference values obtained by assuming a purely random encounter between the pulsars under consideration, we conclude that the objects may have been gravitationally bound in the past. As a result, we have detected six pulsar pairs (J0543+2329/J0528+2200, J1453-6413/J1430-6623, J2354+6155/J2321+6024, J1915+1009/J1909+1102, J1832-0827/J1836-1008, and J1917+1353/J1926+1648) that are companions in disrupted binary systems with a high probability. Estimates of their kinematic ages and velocities at binary disruption and at the present epoch are provided

The molecular basis of glucose galactose malabsorption in a large Swedish pedigree

Glucose-galactose malabsorption (GGM) is due to mutations in the gene coding for the intestinal sodium glucose cotransporter SGLT1 (SLC5A1). Here we identify the rare variant Gln457Arg (Q457R) in a large pedigree of patients in the Vasterbotten County in Northern Sweden with the clinical phenotype of GGM. The functional effect of the Q457R mutation was determined in protein expressed in Xenopus laevis oocytes using biophysical and biochemical methods. The mutant failed to transport the specific SGLT1 sugar analog alpha-methyl-D-glucopyranoside (alphaMDG). Q457R SGLT1 was synthesized in amounts comparable to the wild-type (WT) transporter. SGLT1 charge measurements and freeze-fracture electron microscopy demonstrated that the mutant protein was inserted into the plasma membrane. Electrophysiological experiments, both steady-state and presteady-state, demonstrated that the mutant bound sugar with an affinity lower than the WT transporter. Together with our previous studies on Q457C and Q457E mutants, we established that the positive charge on Q457R prevented the translocation of sugar from the outward-facing to inward-facing conformation. This is contrary to other GGM cases where missense mutations caused defects in trafficking SGLT1 to the plasma membrane. Thirteen GGM patients are now added to the pedigree traced back to the late 17th century. The frequency of the Q457R variant in Vasterbotten County genomes, 0.0067, is higher than in the general Swedish population, 0.0015..