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Millions of patients worldwide suffer from gastrointestinal (GI) motility disorders such as gastroparesis. These disorders typically include debilitating symptoms, such as chronic nausea and vomiting. As no cures are currently available, clinical care is limited to symptom management, while the underlying causes of impaired GI motility remain unaddressed. The efficient movement of contents through the GI tract is facilitated by peristalsis. These rhythmic slow waves of GI muscle contraction are mediated by several cell types, including smooth muscle cells, enteric neurons, telocytes, and specialised gut pacemaker cells called interstitial cells of Cajal (ICC). As ICC dysfunction or loss has been implicated in several GI motility disorders, ICC represent a potentially valuable therapeutic target. Due to their availability, murine ICC have been extensively studied at the molecular level using both normal and diseased GI tissue. In contrast, relatively little is known about the biology of human ICC or their involvement in GI disease pathogenesis. Here, we demonstrate human gastric tissue as a source of primary human cells with ICC phenotype. Further characterisation of these cells will provide new insights into human GI biology, with the potential for developing novel therapies to address the fundamental causes of GI dysmotility.

Toys are us (Trus) is the Drosophila melanogaster ortholog of mammalian Programmed Cell Death 2-Like (PDCD2L), a protein that has been implicated in ribosome biogenesis, cell cycle regulation, and oncogenesis. In this study, we examined the function of Trus during Drosophila development. CRISPR/Cas9 generated null mutations in trus lead to partial embryonic lethality, significant larval developmental delay, and complete pre-pupal lethality. In mutant larvae, we found decreased cell proliferation and growth defects in the brain and imaginal discs. Mapping relevant tissues for Trus function using trus RNAi and trus mutant rescue experiments revealed that imaginal disc defects are primarily responsible for the developmental delay, while the pre-pupal lethality is likely associated with faulty central nervous system (CNS) development. Examination of the molecular mechanism behind the developmental delay phenotype revealed that trus mutations induce the Xrp1-Dilp8 ribosomal stress-response in growth-impaired imaginal discs, and this signaling pathway attenuates production of the hormone ecdysone in the prothoracic gland. Additional Tap-tagging and mass spectrometry of components in Trus complexes isolated from Drosophila Kc cells identified Ribosomal protein subunit 2 (RpS2), which is coded by string of pearls (sop) in Drosophila, and Eukaryotic translation elongation factor 1 alpha 1 (eEF1α1) as interacting factors. We discuss the implication of these findings with respect to the similarity and differences in trus genetic null mutant phenotypes compared to the haplo-insufficiency phenotypes produced by heterozygosity for mutants in Minute genes and other genes involved in ribosome biogenesis.

Steroid hormones are crucial for many biological events in multicellular organisms. In insects, the principal steroid hormones are ecdysteroids, which play essential roles in regulating molting and metamorphosis. During larval and pupal development, ecdysteroids are synthesized in the prothoracic gland (PG) from dietary cholesterol via a series of hydroxylation and oxidation steps. The expression of all but one of the known ecdysteroid biosynthetic enzymes is restricted to the PG, but the transcriptional regulatory networks responsible for generating such exquisite tissue-specific regulation is only beginning to be elucidated. Here, we report identification and characterization of the C2H2-type zinc finger transcription factor Ouija board (Ouib) necessary for ecdysteroid production in the PG in the fruit fly Drosophila melanogaster. Expression of ouib is predominantly limited to the PG, and genetic null mutants of ouib result in larval developmental arrest that can be rescued by administrating an active ecdysteroid. Interestingly, ouib mutant animals exhibit a strong reduction in the expression of one ecdysteroid biosynthetic enzyme, spookier. Using a cell culture-based luciferase reporter assay, Ouib protein stimulates transcription of spok by binding to a specific ~15 bp response element in the spok PG enhancer element. Most remarkable, the developmental arrest phenotype of ouib mutants is rescued by over-expression of a functionally-equivalent paralog of spookier. These observations imply that the main biological function of Ouib is to specifically regulate spookier transcription during Drosophila development.

Cyclin D1 is an essential regulator of the G1–S cell-cycle transition and is overexpressed in many cancers. Expression of cyclin D1 is under tight cellular regulation that is controlled by many signaling pathways. Here we report that PARP14, a member of the poly(ADP-ribose) polymerase (PARP) family, is a regulator of cyclin D1 expression. Depletion of PARP14 leads to decreased cyclin D1 protein levels. In cells with a functional retinoblastoma (RB) protein pathway, this results in G1 cell-cycle arrest and reduced proliferation. Mechanistically, we found that PARP14 controls cyclin D1 mRNA levels. Using luciferase assays, we show that PARP14 specifically regulates cyclin D1 3′UTR mRNA stability. Finally, we also provide evidence that G1 arrest in PARP14-depleted cells is dependent on an intact p53–p21 pathway. Our work uncovers a new role for PARP14 in promoting cell-cycle progression through both cyclin D1 and the p53 pathway.

Speciation constrains the flow of genetic information between populations of sexually reproducing organisms. Gaining control over mechanisms of speciation would enable new strategies to manage wild populations of disease vectors, agricultural pests, and invasive species. Additionally, such control would provide safe biocontainment of transgenes and gene drives. Here, we demonstrate a general approach to create engineered genetic incompatibilities (EGIs) in the model insect Drosophila melanogaster . EGI couples a dominant lethal transgene with a recessive resistance allele. Strains homozygous for both elements are fertile and fecund when they mate with similarly engineered strains, but incompatible with wild-type strains that lack resistant alleles. EGI genotypes can also be tuned to cause hybrid lethality at different developmental life-stages. Further, we demonstrate that multiple orthogonal EGI strains of D. melanogaster can be engineered to be mutually incompatible with wild-type and with each other. EGI is a simple and robust approach in multiple sexually reproducing organisms. Natural speciation can be driven by pre-zygotic and post-zygotic barriers. Here the authors use a dominant lethal transgene coupled to a recessive resistance allele to engineer species-like barriers in Drosophila.

The climatic variability hypothesis (CVH) is a cornerstone of thermal ecology, predicting the evolution of wider organismal thermal tolerance ranges in more thermally variable environments. Thermal tolerance ranges depend on both upper and lower tolerance limits (critical thermal maxima [CTmax] and critical thermal minima [CTmin]), which may show different responses to environmental gradients. To delineate the relative effects of mean and extreme temperatures on thermal tolerances, we conducted a within-latitude comparative test of CVH predictions for army ants (Dorylinae) at multiple scales: across elevations, in seasonal versus aseasonal forests, and in subterranean versus surface microhabitats. Consistent with the CVH, thermally buffered subterranean species had narrower thermal tolerance ranges. Both CTmin and CTmax decreased with elevation for subterranean species. In contrast, aboveground species (those exposed to insolation) showed a decrease in CTmin but no change in CTmax across elevations. Furthermore, greater seasonal temperature variation in dry forests correlated with increased CTmax but not CTmin. These patterns suggest that CTmin and CTmin respond to different abiotic selective forces: habitat-specific exposure to extreme insolation corresponds to CTmin differences but not to CTmin variation. We predict that increasingly frequent heat spikes associated with climate change will have habitat-specific physiological consequences for ectothermic animals. Models predicting climate change impacts should account for species microhabitat uses and within-latitude differences in temperature seasonality.

Correct scaling of body and organ size is crucial for proper development, and the survival of all organisms. Perturbations in circulating hormones, including insulins and steroids, are largely responsible for changing body size in response to both genetic and environmental factors. Such perturbations typically produce adults whose organs and appendages scale proportionately with final size. The identity of additional factors that might contribute to scaling of organs and appendages with body size is unknown. Here, we report that loss-of-function mutations in Drosophila  Activinβ (Actβ), a member of the TGF-β superfamily, lead to the production of small larvae/pupae and undersized rare adult escapers. Morphometric measurements of escaper adult appendage size (wings and legs), as well as heads, thoraxes, and abdomens, reveal a disproportional reduction in abdominal size compared to other tissues. Similar size measurements of selected Actβ mutant larval tissues demonstrate that somatic muscle size is disproportionately smaller when compared to the fat body, salivary glands, prothoracic glands, imaginal discs, and brain. We also show that Actβ control of body size is dependent on canonical signaling through the transcription-factor dSmad2 and that it modulates the growth rate, but not feeding behavior, during the third-instar period. Tissue- and cell-specific knockdown, and overexpression studies, reveal that motoneuron-derived Actβ is essential for regulating proper body size and tissue scaling. These studies suggest that, unlike in vertebrates, where Myostatin and certain other Activin-like factors act as systemic negative regulators of muscle mass, in Drosophila, Actβ is a positive regulator of muscle mass that is directly delivered to muscles by motoneurons. We discuss the importance of these findings in coordinating proportional scaling of insect muscle mass to appendage size.

To test intravenously injected gold nanoparticles for x-ray imaging and radiotherapy enhancement of large, imminently lethal, intracerebral malignant gliomas. Gold nanoparticles approximately 11 nm in size were injected intravenously and brains imaged using microcomputed tomography. A total of 15 h after an intravenous dose of 4 g Au/kg was administered, brains were irradiated with 30 Gy 100 kVp x-rays. Gold uptake gave a 19:1 tumor to normal brain ratio with 1.5% w/w gold in tumor, calculated to increase local radiation dose by approximately 300%. Mice receiving gold and radiation (30 Gy) demonstrated 50% long term (>1 year) tumor-free survival, whereas all mice receiving radiation only died. Intravenously injected gold nanoparticles cross the blood-tumor barrier, but are largely blocked by the normal blood-brain barrier, enabling high-resolution computed tomography tumor imaging. Gold radiation enhancement significantly improved long-term survival compared with radiotherapy alone. This approach holds promise to improve therapy of human brain tumors and other cancers. Original submitted 14 February 2012; Revised submitted 16 September 2012; Published online 24 December 2012

Simulating drug binding and unbinding is a challenge, as the rugged energy landscapes that separate bound and unbound states require extensive sampling that consumes significant computational resources. Here, we describe the use of interactive molecular dynamics in virtual reality (iMD-VR) as an accurate low-cost strategy for flexible protein-ligand docking. We outline an experimental protocol which enables expert iMD-VR users to guide ligands into and out of the binding pockets of trypsin, neuraminidase, and HIV-1 protease, and recreate their respective crystallographic protein-ligand binding poses within 5-10 minutes. Following a brief training phase, our studies shown that iMD-VR novices were able to generate unbinding and rebinding pathways on similar timescales as iMD-VR experts, with the majority able to recover binding poses within 2.15 Å RMSD of the crystallographic binding pose. These results indicate that iMD-VR affords sufficient control for users to carry out the detailed atomic manipulations required to dock flexible ligands into dynamic enzyme active sites and recover crystallographic poses, offering an interesting new approach for simulating drug docking and generating binding hypotheses.