Systematic Screening of Drosophila Deficiency Mutations for Embryonic Phenotypes and Orphan Receptor Ligands

This paper defines a collection of Drosophila deletion mutations (deficiencies) that can be systematically screened for embryonic phenotypes, orphan receptor ligands, and genes affecting protein localization. It reports the results of deficiency screens we have conducted that have revealed new axon guidance phenotypes in the central nervous system and neuromuscular system and permitted a quantitative assessment of the number of potential genes involved in regulating guidance of specific motor axon branches. Deficiency “kits” that cover the genome with a minimum number of lines have been established to facilitate gene mapping. These kits cannot be systematically analyzed for phenotypes, however, since embryos homozygous for many deficiencies in these kits fail to develop due to the loss of key gene products encoded within the deficiency. To create new kits that can be screened for phenotype, we have examined the development of the nervous system in embryos homozygous for more than 700 distinct deficiency mutations. A kit of ∼400 deficiency lines for which homozygotes have a recognizable nervous system and intact body walls encompasses >80% of the genome. Here we show examples of screens of this kit for orphan receptor ligands and neuronal antigen expression. It can also be used to find genes involved in expression, patterning, and subcellular localization of any protein that can be visualized by antibody staining. A subset kit of 233 deficiency lines, for which homozygotes develop relatively normally to late stage 16, covers ∼50% of the genome. We have screened it for axon guidance phenotypes, and we present examples of new phenotypes we have identified. The subset kit can be used to screen for phenotypes affecting all embryonic organs. In the future, these deficiency kits will allow Drosophila researchers to rapidly and efficiently execute genome-wide anatomical screens that require examination of individual embryos at high magnification

Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation

Background: Recent research supports that aggregation of islet amyloid polypeptide (IAPP) leads to cell death and this makes islet amyloid a plausible cause for the reduction of beta cell mass, demonstrated in patients with type 2 diabetes. IAPP is produced by the beta cells as a prohormone, and proIAPP is processed into IAPP by the prohormone convertases PC1/3 and PC2 in the secretory granules. Little is known about the pathogenesis for islet amyloid and which intracellular mechanisms are involved in amyloidogenesis and induction of cell death. Methodology/Principal Findings: We have established expression of human proIAPP (hproIAPP), human IAPP (hIAPP) and the non-amyloidogenic mouse IAPP (mIAPP) in Drosophila melanogaster, and compared survival of flies with the expression driven to different cell populations. Only flies expressing hproIAPP in neurons driven by the Gal4 driver elavC(155,Gal4) showed a reduction in lifespan whereas neither expression of hIAPP or mIAPP influenced survival. Both hIAPP and hproIAPP expression caused formation of aggregates in CNS and fat body region, and these aggregates were both stained by the dyes Congo red and pFTAA, both known to detect amyloid. Also, the morphology of the highly organized protein granules that developed in the fat body of the head in hIAPP and hproIAPP expressing flies was characterized, and determined to consist of 15.8 nm thick pentagonal rod-like structures. Conclusions/Significance: These findings point to a potential for Drosophila melanogaster to serve as a model system for studies of hproIAPP and hIAPP expression with subsequent aggregation and developed pathology.Original Publication: Sebastian Schultz, Peter Nilsson and Gunilla Torstensdotter Westermark, Drosophila Melanogaster as a Model System for Studies of Islet Amyloid Polypeptide Aggregation, 2011, PLoS ONE, (6), 6. http://dx.doi.org/10.1371/journal.pone.0020221 Copyright: Public Library of Science (PLoS) http://www.plos.org/</p

Enhanced tuberculosis case detection among substitution treatment patients: a randomized controlled trial

<p>Abstract</p> <p>Background</p> <p>Historically, HIV, TB (tuberculosis) and illegal drug treatment services in Estonia have been developed as vertical structures. Related health care services are often provided by different health care institutions and in different locations. This may present obstacles for vulnerable groups, such as injecting drug users (IDU), to access the needed services. We conducted a small scale randomized controlled trial to evaluate a case management intervention aimed at increasing TB screening and treatment entry among IDUs referred from a methadone drug treatment program in Jõhvi, North-Eastern Estonia.</p> <p>Findings</p> <p>Of the 189 potential subjects, 112 (59%) participated. HIV prevalence was 86% (n = 96) and 7.4% (n = 8) of participants were interferon gamma release assay (IGRA) positive (6.5% were both HIV and IGRA-positive, n = 7). Overall, 44% of participants (n = 49) attended TB clinic, 17 (30%) from control group and 32 (57%) from case management group (p = 0.004). None of the participants were diagnosed with TB. In a multivariate model, those randomized to case management group were more likely to access TB screening services.</p> <p>Conclusions</p> <p>These findings demonstrate the urgent need for scaling up TB screening among IDUs and the value of more active approach in referring substitution treatment patients to TB services.</p> <p>Trial registration</p> <p>ClinicalTrials.gov: <a href="http://www.clinicaltrials.gov/ct2/show/NCT01290081">NCT01290081</a></p

The role of Probiotics in allergic diseases

Allergic disorders are very common in the pediatric age group. While the exact etiology is unclear, evidence is mounting to incriminate environmental factors and an aberrant gut microbiota with a shift of the Th1/Th2 balance towards a Th2 response. Probiotics have been shown to modulate the immune system back to a Th1 response. Several in vitro studies suggest a role for probiotics in treating allergic disorders. Human trials demonstrate a limited benefit for the use of probiotics in atopic dermatitis in a preventive as well as a therapeutic capacity. Data supporting their use in allergic rhinitis are less robust. Currently, there is no role for probiotic therapy in the treatment of bronchial asthma. Future studies will be critical in determining the exact role of probiotics in allergic disorders

Identification and Characterization of Alternative Promoters, Transcripts and Protein Isoforms of Zebrafish R2 Gene

Ribonucleotide reductase (RNR) is the rate-limiting enzyme in the de novo synthesis of deoxyribonucleoside triphosphates. Expression of RNR subunits is closely associated with DNA replication and repair. Mammalian RNR M2 subunit (R2) functions exclusively in DNA replication of normal cells due to its S phase-specific expression and late mitotic degradation. Herein, we demonstrate the control of R2 expression through alternative promoters, splicing and polyadenylation sites in zebrafish. Three functional R2 promoters were identified to generate six transcript variants with distinct 5′ termini. The proximal promoter contains a conserved E2F binding site and two CCAAT boxes, which are crucial for the transcription of R2 gene during cell cycle. Activity of the distal promoter can be induced by DNA damage to generate four transcript variants through alternative splicing. In addition, two novel splice variants were found to encode distinct N-truncated R2 isoforms containing residues for enzymatic activity but no KEN box essential for its proteolysis. These two N-truncated R2 isoforms remained in the cytoplasm and were able to interact with RNR M1 subunit (R1). Thus, our results suggest that multilayered mechanisms control the differential expression and function of zebrafish R2 gene during cell cycle and under genotoxic stress

High-Content Chemical and RNAi Screens for Suppressors of Neurotoxicity in a Huntington's Disease Model

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.National Institutes of Health (U.S.) (grant R01 EB007042)National Institutes of Health (U.S.

A Serum Factor Induces Insulin-Independent Translocation of GLUT4 to the Cell Surface which Is Maintained in Insulin Resistance

In response to insulin, glucose transporter GLUT4 translocates from intracellular compartments towards the plasma membrane where it enhances cellular glucose uptake. Here, we show that sera from various species contain a factor that dose-dependently induces GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes, human adipocytes, myoblasts and myotubes. Notably, the effect of this factor on GLUT4 is fully maintained in insulin-resistant cells. Our studies demonstrate that the serum-induced increase in cell surface GLUT4 levels is not due to inhibition of its internalization and is not mediated by insulin, PDGF, IGF-1, or HGF. Similarly to insulin, serum also augments cell surface levels of GLUT1 and TfR. Remarkably, the acute effect of serum on GLUT4 is largely additive to that of insulin, while it also sensitizes the cells to insulin. In accordance with these findings, serum does not appear to activate the same repertoire of downstream signaling molecules that are implicated in insulin-induced GLUT4 translocation. We conclude that in addition to insulin, at least one other biological proteinaceous factor exists that contributes to GLUT4 regulation and still functions in insulin resistance. The challenge now is to identify this factor

Measurement of polarization amplitudes and CP asymmetries in B-0 -> phi K*(892)(0)

An angular analysis of the decay B (0) -> phi K (*)(892)(0) is reported based on a pp collision data sample, corresponding to an integrated luminosity of 1.0 fb(-1), collected at a centre-of-mass energy of root S = 7 TeV with the LHCb detector. The P-wave amplitudes and phases are measured with a greater precision than by previous experiments, and confirm about equal amounts of longitudinal and transverse polarization. The S-wave K+ pi(-) and K+ K- contributions are taken into account and found to be significant. A comparison of the B (0) -> phi K (*)(892)(0) and results shows no evidence for direct CP violation in the rate asymmetry, in the triple-product asymmetries or in the polarization amplitudes and phases

Search for the rare decay K0S→μ+μ−

A search for the decay K0S→μ+μ− is performed, based on a data sample of 1.0 fb−1 of pp collisions at &#8730;&lt;span style="text-decoration:overline"&gt;s&lt;/span&gt;=7 TeV collected by the LHCb experiment at the Large Hadron Collider. The observed number of candidates is consistent with the background-only hypothesis, yielding an upper limit of B(K0S→μ+μ−) &#60; 11(9) × 10−9 at 95 (90)% confidence level. This limit is a factor of thirty below the previous measurement