A deficiency screen of the 3rd chromosome for dominant modifiers of the Drosophila ER integral membrane protein, Jagunal.

The mechanism surrounding chromosome inheritance during cell division has been well documented, however, organelle inheritance during mitosis is less understood. Recently, the endoplasmic reticulum (ER) has been shown to reorganize during mitosis, dividing asymmetrically in proneuronal cells prior to cell fate selection, indicating a programmed mechanism of inheritance. ER asymmetric partitioning in proneural cells relies on the highly conserved ER integral membrane protein, Jagunal (Jagn). Knockdown of Jagn in the compound Drosophila eye displays a pleotropic rough eye phenotype in 48% of the progeny. To identify genes involved in Jagn dependent ER partitioning pathway, we performed a dominant modifier screen of the 3rd chromosome for enhancers and suppressors of this Jagn-RNAi-induced rough eye phenotype. We screened through 181 deficiency lines covering the 3L and 3R chromosomes and identified 12 suppressors and 10 enhancers of the Jagn-RNAi phenotype. Based on the functions of the genes covered by the deficiencies, we identified genes that displayed a suppression or enhancement of the Jagn-RNAi phenotype. These include Division Abnormally Delayed (Dally), a heparan sulfate proteoglycan, the γ-secretase subunit Presenilin, and the ER resident protein Sec63. Based on our understanding of the function of these targets, there is a connection between Jagn and the Notch signaling pathway. Further studies will elucidate the role of Jagn and identified interactors within the mechanisms of ER partitioning during mitosis.

Sleep-related eating disorder associated with zolpidem: cases compiled from a literature review.

OBJECTIVE: Zolpidem is associated with sleep-related eating disorder (SRED). We compiled case reports and performed a descriptive study to identify etiology and aggravating factors. METHODS: A literature search on PubMed's MeSH search feature, CINAHL, and SciFinder was performed using search terms "Zolpidem," "Feeding and Eating Disorders/chemically induced," "Dyssomnias," "sleep eating disorder," and "sleep-related eating disorder." Three reviewers examined all English and Spanish citations and extracted pertinent information. A narrative synthesis of the evidence was prepared. RESULTS: We identified 40 case reports of which 65% were female, and the mean age was 53 years. SRED onset was most commonly seen with daily zolpidem doses of 10 mg or higher (95% of patients). Prior medical history included obstructive sleep apnea (OSA) (35%), depression (32.5%), and restless leg syndrome (RLS) (25%). Even with controlled RLS and OSA, SRED developed in some patients. All patients had either partial or full amnesia with compulsive eating. Onset of SRED occurred as early as the first dose to after 9 years of use. SRED symptoms occurred nightly in 57.5% of patients. Discontinuation of zolpidem resolved SRED in all patients (n = 36). CONCLUSION: SRED associated with zolpidem can occur with any dose, but was most common with higher doses of zolpidem. Therefore, prescribers should initiate lower doses of zolpidem. Interestingly, many patients had underlying disorders known to affect sleep (RLS, OSA, depression). Although it is recommended to control these underlying disorders prior to initiating zolpidem, SRED may still occur. Zolpidem discontinuation resolved all cases of SRED.

A systematic exploration of the interactions between bacterial effector proteins and host cell membranes.

Membrane-bound organelles serve as platforms for the assembly of multi-protein complexes that function as hubs of signal transduction in eukaryotic cells. Microbial pathogens have evolved virulence factors that reprogram these host signaling responses, but the underlying molecular mechanisms are poorly understood. Here we test the ability of ~200 type III and type IV effector proteins from six Gram-negative bacterial species to interact with the eukaryotic plasma membrane and intracellular organelles. We show that over 30% of the effectors localize to yeast and mammalian cell membranes, including a subset of previously uncharacterized Legionella effectors that appear to be able to regulate yeast vacuolar fusion. A combined genetic, cellular, and biochemical approach supports that some of the tested bacterial effectors can bind to membrane phospholipids and may regulate membrane trafficking. Finally, we show that the type III effector IpgB1 from Shigella flexneri may bind to acidic phospholipids and regulate actin filament dynamics.Microbial pathogens secrete effector proteins into host cells to affect cellular functions. Here, the authors use a yeast-based screen to study around 200 effectors from six bacterial species, showing that over 30% of them interact with the eukaryotic plasma membrane or intracellular organelles.

nanos-Driven expression of piggyBac transposase induces mobilization of a synthetic autonomous transposon in the malaria vector mosquito, Anopheles stephensi.

Transposons are a class of selfish DNA elements that can mobilize within a genome. If mobilization is accompanied by an increase in copy number (replicative transposition), the transposon may sweep through a population until it is fixed in all of its interbreeding members. This introgression has been proposed as the basis for drive systems to move genes with desirable phenotypes into target species. One such application would be to use them to move a gene conferring resistance to malaria parasites throughout a population of vector mosquitos. We assessed the feasibility of using the piggyBac transposon as a gene-drive mechanism to distribute anti-malarial transgenes in populations of the malaria vector, Anopheles stephensi. We designed synthetic gene constructs that express the piggyBac transposase in the female germline using the control DNA of the An. stephensi nanos orthologous gene linked to marker genes to monitor inheritance. Two remobilization events were observed with a frequency of one every 23 generations, a rate far below what would be useful to drive anti-pathogen transgenes into wild mosquito populations. We discuss the possibility of optimizing this system and the impetus to do so.

Cell-Based Screen Identifies Human Interferon-Stimulated Regulators of Listeria monocytogenes Infection.

The type I interferon (IFN) activated transcriptional response is a critical antiviral defense mechanism, yet its role in bacterial pathogenesis remains less well characterized. Using an intracellular pathogen Listeria monocytogenes (Lm) as a model bacterial pathogen, we sought to identify the roles of individual interferon-stimulated genes (ISGs) in context of bacterial infection. Previously, IFN has been implicated in both restricting and promoting Lm growth and immune stimulatory functions in vivo. Here we adapted a gain-of-function flow cytometry based approach to screen a library of more than 350 human ISGs for inhibitors and enhancers of Lm infection. We identify 6 genes, including UNC93B1, MYD88, AQP9, and TRIM14 that potently inhibit Lm infection. These inhibitors act through both transcription-mediated (MYD88) and non-transcriptional mechanisms (TRIM14). Further, we identify and characterize the human high affinity immunoglobulin receptor FcγRIa as an enhancer of Lm internalization. Our results reveal that FcγRIa promotes Lm uptake in the absence of known host Lm internalization receptors (E-cadherin and c-Met) as well as bacterial surface internalins (InlA and InlB). Additionally, FcγRIa-mediated uptake occurs independently of Lm opsonization or canonical FcγRIa signaling. Finally, we established the contribution of FcγRIa to Lm infection in phagocytic cells, thus potentially linking the IFN response to a novel bacterial uptake pathway. Together, these studies provide an experimental and conceptual basis for deciphering the role of IFN in bacterial defense and virulence at single-gene resolution.

How Bacteria Subvert Animal Cell Structure and Function.

Bacterial pathogens encode a wide variety of effectors and toxins that hijack host cell structure and function. Of particular importance are virulence factors that target actin cytoskeleton dynamics critical for cell shape, stability, motility, phagocytosis, and division. In addition, many bacteria target organelles of the general secretory pathway (e.g., the endoplasmic reticulum and the Golgi complex) and recycling pathways (e.g., the endolysosomal system) to establish and maintain an intracellular replicative niche. Recent research on the biochemistry and structural biology of bacterial effector proteins and toxins has begun to shed light on the molecular underpinnings of these host-pathogen interactions. This exciting work is revealing how pathogens gain control of the complex and dynamic host cellular environments, which impacts our understanding of microbial infectious disease, immunology, and human cell biology.

Group B Streptococcus CovR regulation modulates host immune signalling pathways to promote vaginal colonization.

Streptococcus agalactiae (Group B Streptococcus, GBS) is a frequent commensal organism of the vaginal tract of healthy women. However, GBS can transition to a pathogen in susceptible hosts, but host and microbial factors that contribute to this conversion are not well understood. GBS CovR/S (CsrR/S) is a two component regulatory system that regulates key virulence elements including adherence and toxin production. We performed global transcription profiling of human vaginal epithelial cells exposed to WT, CovR deficient, and toxin deficient strains, and observed that insufficient regulation by CovR and subsequent increased toxin production results in a drastic increase in host inflammatory responses, particularly in cytokine signalling pathways promoted by IL-8 and CXCL2. Additionally, we observed that CovR regulation impacts epithelial cell attachment and intracellular invasion. In our mouse model of GBS vaginal colonization, we further demonstrated that CovR regulation promotes vaginal persistence, as infection with a CovR deficient strainresulted in a heightened host immune response as measured by cytokine production and neutrophil activation. Using CXCr2 KO mice, we determined that this immune alteration occurs, at least in part, via signalling through the CXCL2 receptor. Taken together, we conclude that CovR is an important regulator of GBS vaginal colonization and loss of this regulatory function may contribute to the inflammatory havoc seen during the course of infection.

Serine-rich repeat proteins and pili promote Streptococcus agalactiae colonization of the vaginal tract.

Streptococcus agalactiae (group B streptococcus [GBS]) is a Gram-positive bacterium found in the female rectovaginal tract and is capable of producing severe disease in susceptible hosts, including newborns and pregnant women. The vaginal tract is considered a major reservoir for GBS, and maternal vaginal colonization poses a significant risk to the newborn; however, little is known about the specific bacterial factors that promote GBS colonization and persistence in the female reproductive tract. We have developed in vitro models of GBS interaction with the human female cervicovaginal tract using human vaginal and cervical epithelial cell lines. Analysis of isogenic mutant GBS strains deficient in cell surface organelles such as pili and serine-rich repeat (Srr) proteins shows that these factors contribute to host cell attachment. As Srr proteins are heavily glycosylated, we confirmed that carbohydrate moieties contribute to the effective interaction of Srr-1 with vaginal epithelial cells. Antibody inhibition assays identified keratin 4 as a possible host receptor for Srr-1. Our findings were further substantiated in an in vivo mouse model of GBS vaginal colonization, where mice inoculated with an Srr-1-deficient mutant exhibited decreased GBS vaginal persistence compared to those inoculated with the wild-type (WT) parental strain. Furthermore, competition experiments in mice showed that WT GBS exhibited a significant survival advantage over the ΔpilA or Δsrr-1 mutant in the vaginal tract. Our results suggest that these GBS surface proteins contribute to vaginal colonization and may offer new insights into the mechanisms of vaginal niche establishment.

Nanos (nos) genes of the vector mosquitoes, Anopheles gambiae, Anopheles stephensi and Aedes aegypti.

A number of genetics-based strategies for the control of vector-borne diseases require the development of genetic drive systems for introgressing antipathogen effector genes into wild populations of insects. Modified transposons whose mobilization is controlled by the DNA elements of developmentally regulated genes offer a potential solution for introducing effector genes into mosquitoes. Such elements could exhibit sex-, stage- and species-specific transposition, thus mitigating some of the concerns associated with autonomous transposition. Hybridizations in situ show that the transcription products of the nanos orthologous genes of Anopheles gambiae (Anga nos), An. stephensi (Anst nos) and Aedes aegypti (Aeae nos) accumulate in developing oocytes in adult females and localize to the posterior pole in early embryos. These features make nos genes promising candidates for donating control sequences to modified transposons.