Development of a somatic cell hybrid mapping panel and molecular probes for human chromosome 3

A somatic cell hybrid mapping panel and molecular probes have been developed for human chromosome 3. This panel defines 11 regions for the short and long arms of the chromosome. Four hundred thirty-two probes have been mapped using these hybrids. One hundred thirty-one of these probes were derived from EcoRI and HindIII flow-sorted libraries. The remaining 301 probes were isolated from NotI boundary and random (partial MboI) libraries constructed from a hybrid that provided a relative enrichment in 3p DNA sequences. For some regions of the chromosome, significant differences in the distribution of probes were noted. This was observed for both the unique sequence flow-sorted and NotI probes. These differences are in agreement with previous suggestions that Giemsa light bands are GC-rich, and therefore gene-rich (especially housekeeping genes), and that the Giemsa dark bands may contain DNA that is more highly condensed. The isolation of probes from different types of libraries, or by different screening strategies, appears to reduce deficiencies that might arise from the use of probes derived with a more limited approach. These hybrids and probes should facilitate the construction of physical and genetic linkage maps to identify various disease loci involving chromosome 3.     

Anion exchanger is present in both luminal and basolateral renal membranes

Binding of the anion-exchange inhibitor 3H2-labeled 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) to highly purified luminal and basolateral beef kidney tubular membranes was characterized. Specific binding of [3H2]DIDS is present in both luminal and basolateral membranes. Scatchard analysis revealed a Kd for [3H2]DIDS of 5.5 microM and 19.3 microM and a maximal number of binding sites of 10.9 nmol and 31.7 nmol DIDS/mg protein in basolateral and luminal membranes, respectively. To assess the role of this putative anion exchanger on transport we measured 35SO4 uptake by luminal and basolateral membranes. In both luminal and basolateral membranes sulfate uptake was significantly greater in the presence of an outward-directed Cl gradient, OH gradient or HCO3 gradient than in the absence of these gradients. There was an early anion-dependent sulfate uptake of five to ten times the equilibrium uptake at 60 min. The sulfate taken in could be released by lysis of the vesicles indicating true uptake and not binding of sulfate. No significant difference in SO4 uptake was found in the presence and in the absence of valinomycin, indicating that the anion exchanger is electroneutral. The anion-dependent sulfate uptake was completely inhibited by either DIDS or furosemide in both luminal and basolateral membranes. Dixon analysis of HCO3-dependent SO4 uptake by luminal membranes in the presence of different concentrations of DIDS revealed a Ki for DIDS of 20 microM. The similar values of the Kd for [3H2]DIDS binding and the Ki for DIDS inhibition of SO4 uptake might suggest an association between DIDS binding and the inhibition of SO4 transport. In addition, an inward-directed Na gradient stimulated sulfate uptake in luminal but not in basolateral membranes. The Na-dependent sulfate uptake in luminal membranes was also inhibited by DIDS. We conclude that, in addition to the well-known Na-dependent sulfate uptake in luminal membranes, there exists an anion exchanger in both basolateral and luminal membranes capable of sulfate transport.     

Characterization and Molecular Genetic Complementation of Mutants Affecting Dimorphism in the FungusUstilago maydis

Ustilago maydis,the causal agent of corn smut disease, displays dimorphic growth in which it alternates between a unicellular, nonpathogenic yeast-like form and a dikaryotic, pathogenic filamentous form. Previously, a constitutively filamentous haploid mutant was obtained. Complementation of this mutant led to the isolation of the gene encoding adenylate cyclase,uac1.Secondary mutagenesis of auac1disruption strain allowed the isolation of a large number of suppressor mutants, termedubc,forUstilagobypass of cyclase, lacking the filamentous phenotype. Analysis of one of these suppressor mutants previously led to the identification of theubc1gene, encoding the regulatory subunit of cAMP-dependent protein kinase. In this report we describe the isolation of cosmids containing three newubcgenes, termedubc2, ubc3,andubc4.We also describe the morphology of theubc2, ubc3,andubc4mutants in auac1⁻background as well as in a background with a functionaluac1gene. In addition, we describe several mutant strains not complemented with any of the genes currently in hand and that are thus presumed to possess mutations in additionalubcgenes.     

USEtox human exposure and toxicity factors for comparative assessment of toxic emissions in life cycle analysis: sensitivity to key chemical properties

Purpose

The aim of this paper is to provide science-based consensus and guidance for health effects modelling in comparative assessments based on human exposure and toxicity. This aim is achieved by (a) describing the USEtox? exposure and toxicity models representing consensus and recommended modelling practice, (b) identifying key mechanisms influencing human exposure and toxicity effects of chemical emissions, (c) extending substance coverage.

Methods

The methods section of this paper contains a detailed documentation of both the human exposure and toxic effects models of USEtox?, to determine impacts on human health per kilogram substance emitted in different compartments. These are considered as scientific consensus and therefore recommended practice for comparative toxic impact assessment. The framework of the exposure model is described in details including the modelling of each exposure pathway considered (i.e. inhalation through air, ingestion through (a) drinking water, (b) agricultural produce, (c) meat and milk, and (d) fish). The calculation of human health effect factors for cancer and non-cancer effects via ingestion and inhalation exposure respectively is described. This section also includes discussions regarding parameterisation and estimation of input data needed, including route-to-route and acute-to-chronic extrapolations.

Results and discussion

For most chemicals in USEtox?, inhalation, above-ground agricultural produce, and fish are the important exposure pathways with key driving factors being the compartment and place of emission, partitioning, degradation, bioaccumulation and bioconcentration, and dietary habits of the population. For inhalation, the population density is the key factor driving the intake, thus the importance to differentiate emissions in urban areas, except for very persistent and mobile chemicals that are taken in by the global population independently from their place of emission. The analysis of carcinogenic potency (TD₅₀) when volatile chemicals are administrated to rats and mice by both inhalation and an oral route suggests that results by one route can reasonably be used to represent another route. However, we first identify and mark as interim chemicals for which observed tumours are directly related to a given exposure route (e.g. for nasal or lung, or gastrointestinal cancers) or for which absorbed fraction by inhalation and by oral route differ greatly.

Conclusions

A documentation of the human exposure and toxicity models of USEtox? is provided, and key factors driving the human health characterisation factor are identified. Approaches are proposed to derive human toxic effect factors and expand the number of chemicals in USEtox?, primarily by extrapolating from an oral route to exposure in air (and optionally acute-to-chronic). Some exposure pathways (e.g. indoor inhalation, pesticide residues, dermal exposure) will be included in a later stage. USEtox? is applicable in various comparative toxicity impact assessments and not limited to LCA.     

Vertebral Pneumaticity in the Ornithomimosaur Archaeornithomimus (Dinosauria: Theropoda) Revealed by Computed Tomography Imaging and Reappraisal of Axial Pneumaticity in Ornithomimosauria

Among extant vertebrates, pneumatization of postcranial bones is unique to birds, with few known exceptions in other groups. Through reduction in bone mass, this feature is thought to benefit flight capacity in modern birds, but its prevalence in non-avian dinosaurs of variable sizes has generated competing hypotheses on the initial adaptive significance of postcranial pneumaticity. To better understand the evolutionary history of postcranial pneumaticity, studies have surveyed its distribution among non-avian dinosaurs. Nevertheless, the degree of pneumaticity in the basal coelurosaurian group Ornithomimosauria remains poorly known, despite their potential to greatly enhance our understanding of the early evolution of pneumatic bones along the lineage leading to birds. Historically, the identification of postcranial pneumaticity in non-avian dinosaurs has been based on examination of external morphology, and few studies thus far have focused on the internal architecture of pneumatic structures inside the bones. Here, we describe the vertebral pneumaticity of the ornithomimosaur Archaeornithomimus with the aid of X-ray computed tomography (CT) imaging. Complementary examination of external and internal osteology reveals (1) highly pneumatized cervical vertebrae with an elaborate configuration of interconnected chambers within the neural arch and the centrum; (2) anterior dorsal vertebrae with pneumatic chambers inside the neural arch; (3) apneumatic sacral vertebrae; and (4) a subset of proximal caudal vertebrae with limited pneumatic invasion into the neural arch. Comparisons with other theropod dinosaurs suggest that ornithomimosaurs primitively exhibited a plesiomorphic theropod condition for axial pneumaticity that was extended among later taxa, such as Archaeornithomimus and large bodied Deinocheirus. This finding corroborates the notion that evolutionary increases in vertebral pneumaticity occurred in parallel among independent lineages of bird-line archosaurs. Beyond providing a comprehensive view of vertebral pneumaticity in a non-avian coelurosaur, this study demonstrates the utility and need of CT imaging for further clarifying the early evolutionary history of postcranial pneumaticity.     

Cortical and Subcortical Grey and White Matter Atrophy in Myotonic Dystrophies Type 1 and 2 Is Associated with Cognitive Impairment,Depression and Daytime Sleepiness

Objectives

Central nervous system involvement is one important clinical aspect of myotonic dystrophy type 1 and 2 (DM1 and DM2). We assessed CNS involvement DM1 and DM2 by 3T MRI and correlated clinical and neuocognitive symptoms with brain volumetry and voxel-based morphometry (VBM).

Methods

12 patients with juvenile or classical DM1 and 16 adult DM2 patients underwent 3T MRI, a thorough neurological and neuropsychological examination and scoring of depression and daytime sleepiness. Volumes of brain, ventricles, cerebellum, brainstem, cervical cord, lesion load and VBM results of the patient groups were compared to 33 matched healthy subjects.

Results

Clinical symptoms were depression (more pronounced in DM2), excessive daytime sleepiness (more pronounced in DM1), reduced attention and flexibility of thinking, and deficits of short-term memory and visuo-spatial abilities in both patient groups. Both groups showed ventricular enlargement and supratentorial GM and WM atrophy, with prevalence for more GM atrophy and involvement of the motor system in DM1 and more WM reduction and affection of limbic structures in DM2. White matter was reduced in DM1 in the splenium of the corpus callosum and in left-hemispheric WM adjacent to the pre- and post-central gyrus. In DM2, the bilateral cingulate gyrus and subgyral medio-frontal and primary somato-sensory WM was affected.Significant structural-functional correlations of morphological MRI findings (global volumetry and VBM) with clinical findings were found for reduced flexibility of thinking and atrophy of the left secondary visual cortex in DM1 and of distinct subcortical brain structures in DM2. In DM2, depression was associated with brainstem atrophy, Daytime sleepiness correlated with volume decrease in the middle cerebellar peduncles, pons/midbrain and the right medio-frontal cortex.

Conclusion

GM and WM atrophy was significant in DM1 and DM2. Specific functional-structural associations related morphological changes to cognitive impairment, depression and daytime sleepiness, partly indicating involvement of complex neuronal networks.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Light modulated cnidocyte discharge predates the origins of eyes in Cnidaria

Complex biological traits often originate by integrating previously separate parts, but the organismal functions of these precursors are challenging to infer. If we can understand the ancestral functions of these precursors, it could help explain how they persisted and how they facilitated the origins of complex traits. Animal eyes are some of the best studied complex traits, and they include many parts, such as opsin‐based photoreceptor cells, pigment cells, and lens cells. Eye evolution is understood through conceptual models that argue these parts gradually came together to support increasingly sophisticated visual functions. Despite the well‐accepted logic of these conceptual models, explicit comparative studies to identify organismal functions of eye precursors are lacking. Here, we investigate how precursors functioned before they became part of eyes in Cnidaria, a group formed by sea anemones, corals, and jellyfish. Specifically, we test whether ancestral photoreceptor cells regulated the discharge of cnidocytes, the expensive single‐use cells with various functions including prey capture, locomotion, and protection. Similar to a previous study of Hydra, we show an additional four distantly related cnidarian groups discharge significantly more cnidocytes when exposed to dim blue light compared with bright blue light. Our comparative analyses support the hypothesis that the cnidarian ancestor was capable of modulating cnidocyte discharge with light, which we speculate uses an opsin‐based phototransduction pathway homologous to that previously described in Hydra. Although eye precursors might have had other functions like regulating timing of spawning, our findings are consistent with the hypothesis that photoreceptor cells which mediate cnidocyte discharge predated eyes, perhaps facilitating the prolific origination of eyes in Cnidaria.     

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.