The RNA-binding protein Arrest (Bruno) regulates alternative splicing to enable myofibril maturation in Drosophila flight muscle.

In Drosophila, fibrillar flight muscles (IFMs) enable flight, while tubular muscles mediate other body movements. Here, we use RNA-sequencing and isoform-specific reporters to show that spalt major (salm) determines fibrillar muscle physiology by regulating transcription and alternative splicing of a large set of sarcomeric proteins. We identify the RNA-binding protein Arrest (Aret, Bruno) as downstream of salm. Aret shuttles between the cytoplasm and nuclei and is essential for myofibril maturation and sarcomere growth of IFMs. Molecularly, Aret regulates IFM-specific splicing of various salm-dependent sarcomeric targets, including Stretchin and wupA (TnI), and thus maintains muscle fiber integrity. As Aret and its sarcomeric targets are evolutionarily conserved, similar principles may regulate mammalian muscle morphogenesis

Efficient Bayesian-based Multi-View Deconvolution

Light sheet fluorescence microscopy is able to image large specimen with high resolution by imaging the sam- ples from multiple angles. Multi-view deconvolution can significantly improve the resolution and contrast of the images, but its application has been limited due to the large size of the datasets. Here we present a Bayesian- based derivation of multi-view deconvolution that drastically improves the convergence time and provide a fast implementation utilizing graphics hardware.Comment: 48 pages, 20 figures, 1 table, under review at Nature Method

A genome-wide resource for the analysis of protein localisation in Drosophila

The Drosophila genome contains >13000 protein-coding genes, the majority of which remain poorly investigated. Important reasons include the lack of antibodies or reporter constructs to visualise these proteins. Here, we present a genome-wide fosmid library of 10000 GFP-tagged clones, comprising tagged genes and most of their regulatory information. For 880 tagged proteins, we created transgenic lines, and for a total of 207 lines, we assessed protein expression and localisation in ovaries, embryos, pupae or adults by stainings and live imaging approaches. Importantly, we visualised many proteins at endogenous expression levels and found a large fraction of them localising to subcellular compartments. By applying genetic complementation tests, we estimate that about two-thirds of the tagged proteins are functional. Moreover, these tagged proteins enable interaction proteomics from developing pupae and adult flies. Taken together, this resource will boost systematic analysis of protein expression and localisation in various cellular and developmental contexts

Prevalence of head lice and hygiene practices among women over twelve years of age in Sindh, Balochistan, and North West Frontier Province: National Health Survey of Pakistan, 1990-1994

Background: Head lice infestation is an infection of the scalp and skin which causes blood loss, discomfort, and social and psychological distress with the possibility of secondary bacterial infections occurring at scratch sites. In Pakistan, although some small scale studies have been conducted to investigate prevalence of head lice in school children and the general population, no population based estimates have been reported. The National Health Survey of Pakistan (NHSP 1990 - 94) was a nationally representative health examination survey of the Pakistani population. The NHSP is the first population based household survey to collect data on the prevalence of head lice in adult women in Pakistan. In this paper we use data from the NHSP to present an epidemiological profile of personal hygiene practices and head lice infestation among women aged 12 years or older in three provinces of Pakistan, Balochistan, Sindh and North West Frontier Province (NWFP). Results: Overall about 7% women aged 12 years and older suffered from head lice infestation. Multivariable logistic regression analysis identified factors independently associated with presence of head lice. Age less than 16 years and crowding at home were associated with higher infestation-rates. The impact of household socio-economic status on infestation rates among women was different in urban and rural settings, urban women with low socio-economic status were more vulnerable than similar women in rural settings. Bathing infrequently in summer was associated with higher prevalence rates only in Sindh, possibly due to the fact that among the three provinces Sindh has a hotter and more humid summer. Conclusions: The results of our analysis of NHSP indicate high levels of head lice infestation among girls and women in the three Provinces. The epidemiological profile of hygienic practices of women indicated that NWFP and Balochistan as compared to Sindh, and rural as compared to urban areas were less developed with respect to access to water supply and soap for maintaining personal hygiene. Simple and cost-effective measures such as provision of water and soap, and improving awareness regarding maintaining personal hygiene can contribute significantly towards improving public health status of the women in Pakistan

A Widespread Distribution of Genomic CeMyoD Binding Sites Revealed and Cross Validated by ChIP-Chip and ChIP-Seq Techniques

Identifying transcription factor binding sites genome-wide using chromatin immunoprecipitation (ChIP)-based technology is becoming an increasingly important tool in addressing developmental questions. However, technical problems associated with factor abundance and suitable ChIP reagents are common obstacles to these studies in many biological systems. We have used two completely different, widely applicable methods to determine by ChIP the genome-wide binding sites of the master myogenic regulatory transcription factor HLH-1 (CeMyoD) in C. elegans embryos. The two approaches, ChIP-seq and ChIP-chip, yield strongly overlapping results revealing that HLH-1 preferentially binds to promoter regions of genes enriched for E-box sequences (CANNTG), known binding sites for this well-studied class of transcription factors. HLH-1 binding sites were enriched upstream of genes known to be expressed in muscle, consistent with its role as a direct transcriptional regulator. HLH-1 binding was also detected at numerous sites unassociated with muscle gene expression, as has been previously described for its mouse homolog MyoD. These binding sites may reflect several additional functions for HLH-1, including its interactions with one or more co-factors to activate (or repress) gene expression or a role in chromatin organization distinct from direct transcriptional regulation of target genes. Our results also provide a comparison of ChIP methodologies that can overcome limitations commonly encountered in these types of studies while highlighting the complications of assigning in vivo functions to identified target sites

Acute Spotted Fever Rickettsiosis among Febrile Patients, Cameroon

Although potential arthropod vectors are abundant in Cameroon, acute febrile illnesses are rarely evaluated for arboviral or rickettsial infections. Serum samples from 234 acutely febrile patients at clinics in Tiko and Buea, Cameroon, were examined for antibodies to Rickettsia africae and African alphaviruses and flaviviruses. These serum samples did not contain antibodies against typhoid, and blood malarial parasites were not detected. Serum samples of 32% contained immunoglobulin M antibodies reactive with R. africae by immunofluorescence assay and were reactive with outer membrane proteins A and B of R. africae by immunoblotting. These findings established a diagnosis of acute rickettsiosis, most likely African tick-bite fever. Hemagglutination inhibition testing of the serum samples also detected antibodies to Chikungunya virus (47%) and flaviviruses (47%). High prevalence of antibodies to arboviruses may represent a major, previously unrecognized public health problem in an area where endemic malaria and typhoid fever have been the principal diagnostic considerations

Whole Cell Cryo-Electron Tomography Reveals Distinct Disassembly Intermediates of Vaccinia Virus

At each round of infection, viruses fall apart to release their genome for replication, and then reassemble into stable particles within the same host cell. For most viruses, the structural details that underlie these disassembly and assembly reactions are poorly understood. Cryo-electron tomography (cryo-ET), a unique method to investigate large and asymmetric structures at the near molecular resolution, was previously used to study the complex structure of vaccinia virus (VV). Here we study the disassembly of VV by cryo-ET on intact, rapidly frozen, mammalian cells, infected for up to 60 minutes. Binding to the cell surface induced distinct structural rearrangements of the core, such as a shape change, the rearrangement of its surface spikes and de-condensation of the viral DNA. We propose that the cell surface induced changes, in particular the decondensation of the viral genome, are a prerequisite for the subsequent release of the vaccinia DNA into the cytoplasm, which is followed by its cytoplasmic replication. Generally, this is the first study that employs whole cell cryo-ET to address structural details of pathogen-host cell interaction

A Toolkit and Robust Pipeline for the Generation of Fosmid-Based Reporter Genes in C. elegans

Engineering fluorescent proteins into large genomic clones, contained within BACs or fosmid vectors, is a tool to visualize and study spatiotemporal gene expression patterns in transgenic animals. Because these reporters cover large genomic regions, they most likely capture all cis-regulatory information and can therefore be expected to recapitulate all aspects of endogenous gene expression. Inserting tags at the target gene locus contained within genomic clones by homologous recombination (“recombineering”) represents the most straightforward method to generate these reporters. In this methodology paper, we describe a simple and robust pipeline for recombineering of fosmids, which we apply to generate reporter constructs in the nematode C. elegans, whose genome is almost entirely covered in an available fosmid library. We have generated a toolkit that allows for insertion of fluorescent proteins (GFP, YFP, CFP, VENUS, mCherry) and affinity tags at specific target sites within fosmid clones in a virtually seamless manner. Our new pipeline is less complex and, in our hands, works more robustly than previously described recombineering strategies to generate reporter fusions for C. elegans expression studies. Furthermore, our toolkit provides a novel recombineering cassette which inserts a SL2-spliced intercistronic region between the gene of interest and the fluorescent protein, thus creating a reporter controlled by all 5′ and 3′ cis-acting regulatory elements of the examined gene without the direct translational fusion between the two. With this configuration, the onset of expression and tissue specificity of secreted, sub-cellular compartmentalized or short-lived gene products can be easily detected. We describe other applications of fosmid recombineering as well. The simplicity, speed and robustness of the recombineering pipeline described here should prompt the routine use of this strategy for expression studies in C. elegans

The amyloid precursor protein controls PIKfyve function

While the Amyloid Precursor Protein (APP) plays a central role in Alzheimer's disease, its cellular function still remains largely unclear. It was our goal to establish APP function which will provide insights into APP's implication in Alzheimer's disease. Using our recently developed proteo-liposome assay we established the interactome of APP's intracellular domain (known as AICD), thereby identifying novel APP interactors that provide mechanistic insights into APP function. By combining biochemical, cell biological and genetic approaches we validated the functional significance of one of these novel interactors. Here we show that APP binds the PIKfyve complex, an essential kinase for the synthesis of the endosomal phosphoinositide phosphatidylinositol-3,5-bisphosphate. This signalling lipid plays a crucial role in endosomal homeostasis and receptor sorting. Loss of PIKfyve function by mutation causes profound neurodegeneration in mammals. Using C. elegans genetics we demonstrate that APP functionally cooperates with PIKfyve in vivo. This regulation is required for maintaining endosomal and neuronal function. Our findings establish an unexpected role for APP in the regulation of endosomal phosphoinositide metabolism with dramatic consequences for endosomal biology and important implications for our understanding of Alzheimer's disease