Stage-Specific Expression Profiling of Drosophila Spermatogenesis Suggests that Meiotic Sex Chromosome Inactivation Drives Genomic Relocation of Testis-Expressed Genes

In Drosophila, genes expressed in males tend to accumulate on autosomes and are underrepresented on the X chromosome. In particular, genes expressed in testis have been observed to frequently relocate from the X chromosome to the autosomes. The inactivation of X-linked genes during male meiosis (i.e., meiotic sex chromosome inactivation—MSCI) was first proposed to explain male sterility caused by X-autosomal translocation in Drosophila, and more recently it was suggested that MSCI might provide the conditions under which selection would favor the accumulation of testis-expressed genes on autosomes. In order to investigate the impact of MSCI on Drosophila testis-expressed genes, we performed a global gene expression analysis of the three major phases of D. melanogaster spermatogenesis: mitosis, meiosis, and post-meiosis. First, we found evidence supporting the existence of MSCI by comparing the expression levels of X- and autosome-linked genes, finding the former to be significantly reduced in meiosis. Second, we observed that the paucity of X-linked testis-expressed genes was restricted to those genes highly expressed in meiosis. Third, we found that autosomal genes relocated through retroposition from the X chromosome were more often highly expressed in meiosis in contrast to their X-linked parents. These results suggest MSCI as a general mechanism affecting the evolution of some testis-expressed genes

Multivesicular exocytosis in rat pancreatic beta cells

AIMS/HYPOTHESIS: To establish the occurrence, modulation and functional significance of compound exocytosis in insulin-secreting beta cells. METHODS: Exocytosis was monitored in rat beta cells by electrophysiological, biochemical and optical methods. The functional assays were complemented by three-dimensional reconstruction of confocal imaging, transmission and block face scanning electron microscopy to obtain ultrastructural evidence of compound exocytosis. RESULTS: Compound exocytosis contributed marginally (<5% of events) to exocytosis elicited by glucose/membrane depolarisation alone. However, in beta cells stimulated by a combination of glucose and the muscarinic agonist carbachol, 15-20% of the release events were due to multivesicular exocytosis, but the frequency of exocytosis was not affected. The optical measurements suggest that carbachol should stimulate insulin secretion by ∼40%, similar to the observed enhancement of glucose-induced insulin secretion. The effects of carbachol were mimicked by elevating [Ca(2+)](i) from 0.2 to 2 μmol/l Ca(2+). Two-photon sulforhodamine imaging revealed exocytotic events about fivefold larger than single vesicles and that these structures, once formed, could persist for tens of seconds. Cells exposed to carbachol for 30 s contained long (1-2 μm) serpentine-like membrane structures adjacent to the plasma membrane. Three-dimensional electron microscopy confirmed the existence of fused multigranular aggregates within the beta cell, the frequency of which increased about fourfold in response to stimulation with carbachol. CONCLUSIONS/INTERPRETATION: Although contributing marginally to glucose-induced insulin secretion, compound exocytosis becomes quantitatively significant under conditions associated with global elevation of cytoplasmic calcium. These findings suggest that compound exocytosis is a major contributor to the augmentation of glucose-induced insulin secretion by muscarinic receptor activation

Analysis of Interactions of Salmonella Type Three Secretion Mutants with 3-D Intestinal Epithelial Cells

The prevailing paradigm of Salmonella enteropathogenesis based on monolayers asserts that Salmonella pathogenicity island-1 Type Three Secretion System (SPI-1 T3SS) is required for bacterial invasion into intestinal epithelium. However, little is known about the role of SPI-1 in mediating gastrointestinal disease in humans. Recently, SPI-1 deficient nontyphoidal Salmonella strains were isolated from infected humans and animals, indicating that SPI-1 is not required to cause enteropathogenesis and demonstrating the need for more in vivo-like models. Here, we utilized a previously characterized 3-D organotypic model of human intestinal epithelium to elucidate the role of all characterized Salmonella enterica T3SSs. Similar to in vivo reports, the Salmonella SPI-1 T3SS was not required to invade 3-D intestinal cells. Additionally, Salmonella strains carrying single (SPI-1 or SPI-2), double (SPI-1/2) and complete T3SS knockout (SPI-1/SPI-2: flhDC) also invaded 3-D intestinal cells to wildtype levels. Invasion of wildtype and TTSS mutants was a Salmonella active process, whereas non-invasive bacterial strains, bacterial size beads, and heat-killed Salmonella did not invade 3-D cells. Wildtype and T3SS mutants did not preferentially target different cell types identified within the 3-D intestinal aggregates, including M-cells/M-like cells, enterocytes, or Paneth cells. Moreover, each T3SS was necessary for substantial intracellular bacterial replication within 3-D cells. Collectively, these results indicate that T3SSs are dispensable for Salmonella invasion into highly differentiated 3-D models of human intestinal epithelial cells, but are required for intracellular bacterial growth, paralleling in vivo infection observations and demonstrating the utility of these models in predicting in vivo-like pathogenic mechanisms

A Scalable System for Production of Functional Pancreatic Progenitors from Human Embryonic Stem Cells

Development of a human embryonic stem cell (hESC)-based therapy for type 1 diabetes will require the translation of proof-of-principle concepts into a scalable, controlled, and regulated cell manufacturing process. We have previously demonstrated that hESC can be directed to differentiate into pancreatic progenitors that mature into functional glucose-responsive, insulin-secreting cells in vivo. In this study we describe hESC expansion and banking methods and a suspension-based differentiation system, which together underpin an integrated scalable manufacturing process for producing pancreatic progenitors. This system has been optimized for the CyT49 cell line. Accordingly, qualified large-scale single-cell master and working cGMP cell banks of CyT49 have been generated to provide a virtually unlimited starting resource for manufacturing. Upon thaw from these banks, we expanded CyT49 for two weeks in an adherent culture format that achieves 50–100 fold expansion per week. Undifferentiated CyT49 were then aggregated into clusters in dynamic rotational suspension culture, followed by differentiation en masse for two weeks with a four-stage protocol. Numerous scaled differentiation runs generated reproducible and defined population compositions highly enriched for pancreatic cell lineages, as shown by examining mRNA expression at each stage of differentiation and flow cytometry of the final population. Islet-like tissue containing glucose-responsive, insulin-secreting cells was generated upon implantation into mice. By four- to five-months post-engraftment, mature neo-pancreatic tissue was sufficient to protect against streptozotocin (STZ)-induced hyperglycemia. In summary, we have developed a tractable manufacturing process for the generation of functional pancreatic progenitors from hESC on a scale amenable to clinical entry

Applications of CRISPR–Cas systems in neuroscience

Genome-editing tools, and in particular those based on CRISPR-Cas (clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal models and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research.National Institute of Mental Health (U.S.) (Grant 5DP1-MH100706)National Institute of Mental Health (U.S.) (Grant 1R01-MH110049)National Institute of Diabetes and Digestive and Kidney Diseases (U.S.) (Grant 5R01DK097768-03

Comparative Linkage Meta-Analysis Reveals Regionally-Distinct, Disparate Genetic Architectures: Application to Bipolar Disorder and Schizophrenia

New high-throughput, population-based methods and next-generation sequencing capabilities hold great promise in the quest for common and rare variant discovery and in the search for ”missing heritability.” However, the optimal analytic strategies for approaching such data are still actively debated, representing the latest rate-limiting step in genetic progress. Since it is likely a majority of common variants of modest effect have been identified through the application of tagSNP-based microarray platforms (i.e., GWAS), alternative approaches robust to detection of low-frequency (1–5% MAF) and rare (<1%) variants are of great importance. Of direct relevance, we have available an accumulated wealth of linkage data collected through traditional genetic methods over several decades, the full value of which has not been exhausted. To that end, we compare results from two different linkage meta-analysis methods—GSMA and MSP—applied to the same set of 13 bipolar disorder and 16 schizophrenia GWLS datasets. Interestingly, we find that the two methods implicate distinct, largely non-overlapping, genomic regions. Furthermore, based on the statistical methods themselves and our contextualization of these results within the larger genetic literatures, our findings suggest, for each disorder, distinct genetic architectures may reside within disparate genomic regions. Thus, comparative linkage meta-analysis (CLMA) may be used to optimize low-frequency and rare variant discovery in the modern genomic era

The glial growth factors deficiency and synaptic destabilization hypothesis of schizophrenia

BACKGROUND: A systems approach to understanding the etiology of schizophrenia requires a theory which is able to integrate genetic as well as neurodevelopmental factors. PRESENTATION OF THE HYPOTHESIS: Based on a co-localization of loci approach and a large amount of circumstantial evidence, we here propose that a functional deficiency of glial growth factors and of growth factors produced by glial cells are among the distal causes in the genotype-to-phenotype chain leading to the development of schizophrenia. These factors include neuregulin, insulin-like growth factor I, insulin, epidermal growth factor, neurotrophic growth factors, erbB receptors, phosphatidylinositol-3 kinase, growth arrest specific genes, neuritin, tumor necrosis factor alpha, glutamate, NMDA and cholinergic receptors. A genetically and epigenetically determined low baseline of glial growth factor signaling and synaptic strength is expected to increase the vulnerability for additional reductions (e.g., by viruses such as HHV-6 and JC virus infecting glial cells). This should lead to a weakening of the positive feedback loop between the presynaptic neuron and its targets, and below a certain threshold to synaptic destabilization and schizophrenia. TESTING THE HYPOTHESIS: Supported by informed conjectures and empirical facts, the hypothesis makes an attractive case for a large number of further investigations. IMPLICATIONS OF THE HYPOTHESIS: The hypothesis suggests glial cells as the locus of the genes-environment interactions in schizophrenia, with glial asthenia as an important factor for the genetic liability to the disorder, and an increase of prolactin and/or insulin as possible working mechanisms of traditional and atypical neuroleptic treatments

A narrative review on the similarities and dissimilarities between myalgic encephalomyelitis/chronic fatigue syndrome (me/cfs) and sickness behavior

It is of importance whether myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a variant of sickness behavior. The latter is induced by acute infections/injury being principally mediated through proinflammatory cytokines. Sickness is a beneficial behavioral response that serves to enhance recovery, conserves energy and plays a role in the resolution of inflammation. There are behavioral/symptomatic similarities (for example, fatigue, malaise, hyperalgesia) and dissimilarities (gastrointestinal symptoms, anorexia and weight loss) between sickness and ME/CFS. While sickness is an adaptive response induced by proinflammatory cytokines, ME/CFS is a chronic, disabling disorder, where the pathophysiology is related to activation of immunoinflammatory and oxidative pathways and autoimmune responses. While sickness behavior is a state of energy conservation, which plays a role in combating pathogens, ME/CFS is a chronic disease underpinned by a state of energy depletion. While sickness is an acute response to infection/injury, the trigger factors in ME/CFS are less well defined and encompass acute and chronic infections, as well as inflammatory or autoimmune diseases. It is concluded that sickness behavior and ME/CFS are two different conditions