Cell proliferation and differentiation kinetics during spermatogenesis in Hydra carnea

Spermatogenesis inHydra carnea was investigated. The cell proliferation and differentiation kinetics of intermediates in the spermatogenesis pathway were determined, using quantitative determinations of cell abundance, pulse and continuous labelling with3H-thymidine and nuclear DNA measurements. Testes develop in the ectoderm of male hydra as a result of interstitial cell proliferation. Gonial stem cells and proliferating spermatogonia have cell cycles of 28 h and 22 h, respectively. Stem cells undergo four, five or six cell divisions prior to meiosis which includes a premeiotic S+G2 phase of 20 h followed by a long meiotic prophase (22 h). Spermatid differentiation requires 12–29 h. When they first appear, testes contain only proliferating spermatogonia; meiotic and postmeiotic cells appear after 2 and 3 days, respectively and release of mature sperm begins after 4 days. Mature testes produce about 27,000 sperm per day over a period of 4–6 days: about 220 gonial stem cells per testis are required to support this level of sperm differentiation. Further results indicate that somatic (e.g. nematocyte) differentiation does not occur in testes although it continues normally in ectodermal tissue outside testes. Our results support the hypothesis that spermatogenesis is controlled locally in regions of the ectoderm where testes develop

The transcription factor Spores Absent A is a PKA dependent inducer of Dictyostelium sporulation

Abstract Sporulation in Dictyostelium fruiting bodies evolved from amoebozoan encystation with both being induced by cAMP acting on PKA, but with downstream components still being unknown. Using tagged mutagenesis to find missing pathway components, we identified a sporeless mutant defective in a nuclear protein, SpaA. Expression of prespore genes was strongly reduced in spaA- cells, while expression of many spore stage genes was absent. Chromatin immunoprecipitation (ChIP) of a SpaA-YFP gene fusion showed that (pre)spore gene promoters bind directly to SpaA, identifying SpaA as a transcriptional regulator. SpaA dependent spore gene expression required PKA in vivo and was stimulated in vitro by the membrane-permeant PKA agonist 8Br-cAMP. The PKA agonist also promoted SpaA binding to (pre)spore promoters, placing SpaA downstream of PKA. Sequencing of SpaA-YFP ChIPed DNA fragments revealed that SpaA binds at least 117 (pre)spore promoters, including those of other transcription factors that activate some spore genes. These factors are not in turn required for spaA expression, identifying SpaA as the major trancriptional inducer of sporulation

Roadless and Low-Traffic Areas as Conservation Targets in Europe

With increasing road encroachment, habitat fragmentation by transport infrastructures has been a serious threat for European biodiversity. Areas with no roads or little traffic (“roadless and low-traffic areas”) represent relatively undisturbed natural habitats and functioning ecosystems. They provide many benefits for biodiversity and human societies (e.g., landscape connectivity, barrier against pests and invasions, ecosystem services). Roadless and low-traffic areas, with a lower level of anthropogenic disturbances, are of special relevance in Europe because of their rarity and, in the context of climate change, because of their contribution to higher resilience and buffering capacity within landscape ecosystems. An analysis of European legal instruments illustrates that, although most laws aimed at protecting targets which are inherent to fragmentation, like connectivity, ecosystem processes or integrity, roadless areas are widely neglected as a legal target. A case study in Germany underlines this finding. Although the Natura 2000 network covers a significant proportion of the country (16%), Natura 2000 sites are highly fragmented and most low-traffic areas (75%) lie unprotected outside this network. This proportion is even higher for the old Federal States (western Germany), where only 20% of the low-traffic areas are protected. We propose that the few remaining roadless and low-traffic areas in Europe should be an important focus of conservation efforts; they should be urgently inventoried, included more explicitly in the law and accounted for in transport and urban planning. Considering them as complementary conservation targets would represent a concrete step towards the strengthening and adaptation of the Natura 2000 network to climate change

ACAP-A/B Are ArfGAP Homologs in Dictyostelium Involved in Sporulation but Not in Chemotaxis

Arfs and Arf GTPase-activating proteins (ArfGAPs) are regulators of membrane trafficking and actin dynamics in mammalian cells. In this study, we identified a primordial Arf, ArfA, and two ArfGAPs (ACAP-A/B) containing BAR, PH, ArfGAP and Ankyrin repeat domains in the eukaryote Dictyostelium discoideum. In vitro, ArfA has similar nucleotide binding properties as mammalian Arfs and, with GTP bound, is a substrate for ACAP-A and B. We also investigated the physiological functions of ACAP-A/B by characterizing cells lacking both ACAP-A and B. Although ACAP-A/B knockout cells showed no defects in cell growth, migration or chemotaxis, they exhibited abnormal actin protrusions and ∼50% reduction in spore yield. We conclude that while ACAP-A/B have a conserved biochemical mechanism and effect on actin organization, their role in migration is not conserved. The absence of an effect on Dictyostelium migration may be due to a specific requirement for ACAPs in mesenchymal migration, which is observed in epithelial cancer cells where most studies of mammalian ArfGAPs were performed

The Toronto prehospital hypertonic resuscitation-head injury and multi organ dysfunction trial (TOPHR HIT) - Methods and data collection tools

<p>Abstract</p> <p>Background</p> <p>Clinical trials evaluating the use of hypertonic saline in the treatment of hypovolemia and head trauma suggest no survival superiority over normal saline; however subgroup analyses suggest there may be a reduction in the inflammatory response and multiorgan failure which may lead to better survival and enhanced neurocognitive function. We describe a feasibility study of randomizing head injured patients to hypertonic saline and dextran vs. normal saline administration in the out of hospital setting.</p> <p>Methods/Design</p> <p>This feasibility study employs a randomized, placebo-controlled design evaluating normal saline compared with a single dose of 250 ml of 7.5% hypertonic saline in 6% dextran 70 in the management of traumatic brain injuries. The primary feasibility endpoints of the trial were: 1) baseline survival rates for the treatment and control group to aid in the design of a definitive multicentre trial, 2) randomization compliance rate, 3) ease of protocol implementation in the out-of-hospital setting, and 4) adverse event rate of HSD infusion.</p> <p>The secondary objectives include measuring the effect of HSD in modulating the immuno-inflammatory response to severe head injury and its effect on modulating the release of neuro-biomarkers into serum; evaluating the role of serum neuro-biomarkers in predicting patient outcome and clinical response to HSD intervention; evaluating effects of HSD on brain atrophy post-injury and neurocognitive and neuropsychological outcomes.</p> <p>Discussion</p> <p>We anticipate three aspects of the trial will present challenges to trial success; ethical demands associated with a waiver of consent trial, challenging follow up and comprehensive accurate timely data collection of patient identifiers and clinical or laboratory values. In addition all the data collection tools had to be derived de novo as none existed in the literature.</p> <p>Trial registration number</p> <p>NCT00878631</p

The multicellularity genes of dictyostelid social amoebas

The evolution of multicellularity enabled specialization of cells, but required novel signalling mechanisms for regulating cell differentiation. Early multicellular organisms are mostly extinct and the origins of these mechanisms are unknown. Here using comparative genome and transcriptome analysis across eight uni- and multicellular amoebozoan genomes, we find that 80% of proteins essential for the development of multicellular Dictyostelia are already present in their unicellular relatives. This set is enriched in cytosolic and nuclear proteins, and protein kinases. The remaining 20%, unique to Dictyostelia, mostly consists of extracellularly exposed and secreted proteins, with roles in sensing and recognition, while several genes for synthesis of signals that induce cell-type specialization were acquired by lateral gene transfer. Across Dictyostelia, changes in gene expression correspond more strongly with phenotypic innovation than changes in protein functional domains. We conclude that the transition to multicellularity required novel signals and sensors rather than novel signal processing mechanisms

Deficiency of Huntingtin Has Pleiotropic Effects in the Social Amoeba Dictyostelium discoideum

Huntingtin is a large HEAT repeat protein first identified in humans, where a polyglutamine tract expansion near the amino terminus causes a gain-of-function mechanism that leads to selective neuronal loss in Huntington's disease (HD). Genetic evidence in humans and knock-in mouse models suggests that this gain-of-function involves an increase or deregulation of some aspect of huntingtin's normal function(s), which remains poorly understood. As huntingtin shows evolutionary conservation, a powerful approach to discovering its normal biochemical role(s) is to study the effects caused by its deficiency in a model organism with a short life-cycle that comprises both cellular and multicellular developmental stages. To facilitate studies aimed at detailed knowledge of huntingtin's normal function(s), we generated a null mutant of hd, the HD ortholog in Dictyostelium discoideum. Dictyostelium cells lacking endogenous huntingtin were viable but during development did not exhibit the typical polarized morphology of Dictyostelium cells, streamed poorly to form aggregates by accretion rather than chemotaxis, showed disorganized F-actin staining, exhibited extreme sensitivity to hypoosmotic stress, and failed to form EDTA-resistant cell–cell contacts. Surprisingly, chemotactic streaming could be rescued in the presence of the bivalent cations Ca2+ or Mg2+ but not pulses of cAMP. Although hd− cells completed development, it was delayed and proceeded asynchronously, producing small fruiting bodies with round, defective spores that germinated spontaneously within a glassy sorus. When developed as chimeras with wild-type cells, hd− cells failed to populate the pre-spore region of the slug. In Dictyostelium, huntingtin deficiency is compatible with survival of the organism but renders cells sensitive to low osmolarity, which produces pleiotropic cell autonomous defects that affect cAMP signaling and as a consequence development. Thus, Dictyostelium provides a novel haploid organism model for genetic, cell biological, and biochemical studies to delineate the functions of the HD protein

Identification of gene targets against dormant phase Mycobacterium tuberculosis infections

<p>Abstract</p> <p>Background</p> <p><it>Mycobacterium tuberculosis</it>, the causative agent of tuberculosis (TB), infects approximately 2 billion people worldwide and is the leading cause of mortality due to infectious disease. Current TB therapy involves a regimen of four antibiotics taken over a six month period. Patient compliance, cost of drugs and increasing incidence of drug resistant <it>M. tuberculosis </it>strains have added urgency to the development of novel TB therapies. Eradication of TB is affected by the ability of the bacterium to survive up to decades in a dormant state primarily in hypoxic granulomas in the lung and to cause recurrent infections.</p> <p>Methods</p> <p>The availability of <it>M. tuberculosis </it>genome-wide DNA microarrays has lead to the publication of several gene expression studies under simulated dormancy conditions. However, no single model best replicates the conditions of human pathogenicity. In order to identify novel TB drug targets, we performed a meta-analysis of multiple published datasets from gene expression DNA microarray experiments that modeled infection leading to and including the dormant state, along with data from genome-wide insertional mutagenesis that examined gene essentiality.</p> <p>Results</p> <p>Based on the analysis of these data sets following normalization, several genome wide trends were identified and used to guide the selection of targets for therapeutic development. The trends included the significant up-regulation of genes controlled by <it>devR</it>, down-regulation of protein and ATP synthesis, and the adaptation of two-carbon metabolism to the hypoxic and nutrient limited environment of the granuloma. Promising targets for drug discovery were several regulatory elements (<it>devR/devS</it>, <it>relA</it>, <it>mprAB</it>), enzymes involved in redox balance and respiration, sulfur transport and fixation, pantothenate, isoprene, and NAD biosynthesis. The advantages and liabilities of each target are discussed in the context of enzymology, bacterial pathways, target tractability, and drug development.</p> <p>Conclusion</p> <p>Based on our bioinformatics analysis and additional discussion of in-depth biological rationale, several novel anti-TB targets have been proposed as potential opportunities to improve present therapeutic treatments for this disease.</p

Adaptations to Climate-Mediated Selective Pressures in Humans

Humans inhabit a remarkably diverse range of environments, and adaptation through natural selection has likely played a central role in the capacity to survive and thrive in extreme climates. Unlike numerous studies that used only population genetic data to search for evidence of selection, here we scan the human genome for selection signals by identifying the SNPs with the strongest correlations between allele frequencies and climate across 61 worldwide populations. We find a striking enrichment of genic and nonsynonymous SNPs relative to non-genic SNPs among those that are strongly correlated with these climate variables. Among the most extreme signals, several overlap with those from GWAS, including SNPs associated with pigmentation and autoimmune diseases. Further, we find an enrichment of strong signals in gene sets related to UV radiation, infection and immunity, and cancer. Our results imply that adaptations to climate shaped the spatial distribution of variation in humans

Association of melanocortin 1 receptor gene (MC1R) polymorphisms with skin reflectance and freckles in Japanese.

Most studies on the genetic basis of human skin pigmentation have focused on people of European ancestry and only a few studies have focused on Asian populations. We investigated the association of skin reflectance and freckling with genetic variants of melanocortin 1 receptor (MC1R) gene in Japanese. DNA samples were obtained from a total of 653 Japanese individuals (ages 19-40 years) residing in Okinawa; skin reflectance was measured using a spectrophotometer and freckling status was determined for each individual. Lightness index (L*) and freckling status were not correlated with age, body mass index or ancestry (Ryukyuan or Main Islanders of Japan). Among the 10 nonsynonymous variants that were identified by direct sequencing of the coding region of MC1R, two variants--R163Q and V92M--with the derived allele frequencies of 78.6 and 5.5%, respectively, were most common. Multiple regression analysis showed that the 163Q allele and the presence of nonsynonymous rare variants (allele frequencies <5%) were significantly associated with an increase in sex-standardized skin lightness (L* of CIELAB (CIE 1976 (L*a*b*) color space)) of the inner upper arm. Relative to the 92V allele, the 92M allele was significantly associated with increased odds of freckling. This is the first study to show an association between the 163Q allele and skin reflectance values; this association indicated that light-toned skin may have been subjected to positive selection in East Asian people