Membrane entrapped Saccharomyces cerevisiae in a biosensor-like device as a generic rapid method to study cellular metabolism

We describe a new, faster and convenient method to study some metabolic characteristics - by the successful application of immobilized yeast cells (S. cerevisiae) in a microbial biosensor-like device. Microbial biosensors consist of microorganisms immobilized on the surface of a membrane or in a gel, in close contact with a transducer. Almost all works published to date have used biosensors for analyses in which a concentration-related property of the external medium is measured. A different approach is presented here; we have successfully used S. cerevisiae and a carbon dioxide electrode as the main components of a biosensor-like device, used as a proof of concept, for a system useful to characterize metabolic parameters of the microbial cells immobilized on a carbon dioxide electrode. The biosensor-like device we are presenting allows us to calculate Michaelis-Menten parameters related to the kinetics of transport and degradation of several carbohydrates (i.e., glucose, fructose, galactose, sucrose and xylose, with K(m(app)) of 6.0, 5.8, 0.9, 2.0, and 147 mM, respectively), and the study of the kinetics of expression of non-constitutive proteins related to the transport and degradation of galactose.     

Evidence for epistasis between <Emphasis Type="Italic">SLC6A4</Emphasis> and <Emphasis Type="Italic">ITGB3</Emphasis> in autism etiology and in the determination of platelet serotonin levels

Autism is a neurodevelopmental disorder of unclear etiology. The consistent finding of platelet hyperserotonemia in a proportion of patients and its heritability within affected families suggest that genes involved in the serotonin system play a role in this disorder. The role in autism etiology of seven candidate genes in the serotonin metabolic and neurotransmission pathways and mapping to autism linkage regions (SLC6A4, HTR1A, HTR1D, HTR2A, HTR5A, TPH1 and ITGB3) was analyzed in a sample of 186 nuclear families. The impact of interactions among these genes in autism was assessed using the multifactor-dimensionality reduction (MDR) method in 186 patients and 181 controls. We further evaluated whether the effect of specific gene variants or gene interactions associated with autism etiology might be mediated by their influence on serotonin levels, using the quantitative transmission disequilibrium test (QTDT) and the restricted partition method (RPM), in a sample of 109 autistic children. We report a significant main effect of the HTR5A gene in autism (P = 0.0088), and a significant three-locus model comprising a synergistic interaction between the ITGB3 and SLC6A4 genes with an additive effect of HTR5A (P < 0.0010). In addition to the previously reported contribution of SLC6A4, we found significant associations of ITGB3 haplotypes with serotonin level distribution (P = 0.0163). The most significant models contributing to serotonin distribution were found for interactions between TPH1 rs4537731 and SLC6A4 haplotypes (P = 0.002) and between HTR1D rs6300 and SLC6A4 haplotypes (P = 0.013). In addition to the significant independent effects, evidence for interaction between SLC6A4 and ITGB3 markers was also found. The overall results implicate SLC6A4 and ITGB3 gene interactions in autism etiology and in serotonin level determination, providing evidence for a common underlying genetic mechanism and a molecular explanation for the association of platelet hyperserotonemia with autism. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Vibrio navarrensis biotype pommerensis: a new biotype of V. navarrensis isolated in the German Baltic Sea

The determination of phenotypic and genotypic traits of a group of closely related Vibrio strains from the Baltic Sea did not allow species designation. DNA-DNA hybridization and fatty acid analysis revealed them as Vibrio navarrensis. Therefore we suggest these Vibrios to represent a new biotype, named V. navarrensis biotype pommerensis.     

Bypassing Iron Storage in Endodermal Vacuoles Rescues the Iron Mobilization Defect in the natural resistance associated-macrophage protein3natural resistance associated-macrophage protein4 Double Mutant

To improve seed iron (Fe) content and bioavailability, it is crucial to decipher the mechanisms that control Fe storage during seed development. In Arabidopsis (Arabidopsis thaliana) seeds, most Fe is concentrated in insoluble precipitates, with phytate in the vacuoles of cells surrounding the vasculature of the embryo. NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in Fe retrieval from vacuoles during germination. When germinated under Fe-deficient conditions, development of the nramp3nramp4 double mutant is arrested as a consequence of impaired Fe mobilization. To identify novel genes involved in seed Fe homeostasis, we screened an ethyl methanesulfonate-mutagenized population of nramp3nramp4 seedlings for mutations suppressing their phenotypes on low Fe. Here, we report that, among the suppressors, two independent mutations in the VACUOLAR IRON TRANSPORTER1 (AtVIT1) gene caused the suppressor phenotype. The AtVIT1 transporter is involved in Fe influx into vacuoles of endodermal and bundle sheath cells. This result establishes a functional link between Fe loading in vacuoles by AtVIT1 and its remobilization by AtNRAMP3 and AtNRAMP4. Moreover, analysis of subcellular Fe localization indicates that simultaneous disruption of AtVIT1, AtNRAMP3, and AtNRAMP4 limits Fe accumulation in vacuolar globoids.Iron (Fe) is an essential micronutrient. In cells, this metal may change between two oxidation states: ferrous (Fe²⁺) and ferric (Fe³⁺). This property makes Fe an important metal cofactor for electron transfer in many biochemical reactions. However, for the same reason, free Fe generates harmful reactive oxygen species via the Fenton reaction (Haber and Weiss, 1932; Halliwell, 1978). Cells thus need to tightly control Fe homeostasis through chelation and compartmentalization. For example, in yeast (Saccharomyces cerevisiae), the membrane transporter yeast Calcium-sensitive Cross-Complementer1 (ScCCC1) is required to move excess Fe into the vacuole (Li et al., 2001). The Δccc1 mutant is sensitive to extracellular Fe. In mammalian cells, excess cytosolic Fe is complexed by ferritins; the assembly of 24 ferritin monomers forms a hollow complex able to safely store up to 4,500 Fe(III) atoms (Finazzi and Arosio, 2014).Fe deficiency is an important public health issue: 2 billion people, corresponding to over 25% of the world population, are anemic (World Health Organization, 2015). To fight Fe deficiency, it has been proposed to develop crops with more available Fe according to a strategy called biofortification (Bouis, 2003). In most crops, seeds are used as food or feed. Fe stores in seeds are also important for the germination of seedlings. In seeds, Fe may be associated with ferritin in plastids, with phytate in vacuoles, or with nicotianamine. Although Fe complexed with ferritin or nicotianamine is considered highly bioavailable, Fe phytate is insoluble and poorly bioavailable (Clemens, 2014).In Arabidopsis (Arabidopsis thaliana) seeds, no more than 5% of the total seed Fe is associated with ferritin (Ravet et al., 2009). About 50% of seed Fe is concentrated in the vacuoles of endodermal and bundle sheath cells surrounding the vasculature in the radicle and cotyledons of the embryo, respectively (Lanquar et al., 2005; Kim et al., 2006; Roschzttardtz et al., 2009; Schnell Ramos et al., 2013). As endodermal and bundle sheath cells belong to the same lineage, they will be collectively referred to as endodermal cells in this report. VACUOLAR IRON TRANSPORTER1 (AtVIT1) is responsible for the loading of Fe in endodermal vacuoles during seed development (Kim et al., 2006). VIT1 is homologous to CCC1, which mediates Fe sequestration in the vacuole in yeast (Li et al., 2001). In addition to VIT1, which is mostly expressed during seed development, the Arabidopsis genome encodes five VIT1-like proteins (VTLs). VTL gene expression is down-regulated under Fe deficiency (Gollhofer et al., 2011). At least one of the VTLs also functions in Fe sequestration in the vacuole (Gollhofer et al., 2014). VIT1 homologs have been identified in most plant species for which genome sequencing data are available. In rice (Oryza sativa), OsVIT1 and OsVIT2 sequester Fe and zinc in the vacuole of the flag leaf (Zhang et al., 2012). Loss of AtVIT1 function perturbs the cell type-specific localization of Fe in mature embryos. Accordingly, in the vit1-1 knockout mutant, Fe is no longer concentrated in cells surrounding the vasculature of the hypocotyl, radicle, and cotyledons but instead is accumulated in cortical cells in the hypocotyl and radicle and in the subepidermal cells of the abaxial side of cotyledons (Kim et al., 2006). Loss of AtVIT1 function also has an impact on germination on alkaline (pH 7.9) soil: vit1-1 mutant seedlings grow poorly compared with the wild type (Kim et al., 2006).NATURAL RESISTANCE ASSOCIATED-MACROPHAGE PROTEIN3 (AtNRAMP3) and AtNRAMP4 function redundantly in the mobilization of seed Fe from vacuoles during germination (Lanquar et al., 2005). They belong to a ubiquitous family of divalent cation transporters represented in all kingdoms of life. NRAMP proteins have well-documented roles in manganese (Mn) uptake in bacteria, yeast, and Arabidopsis (Nevo and Nelson, 2006; Cailliatte et al., 2010). In mammals, NRAMP2/Divalent Cation Transporter1/Divalent Metal Transporter1 is the major uptake system involved in dietary Fe absorption (Andrews, 2004). Recently, some members of this family have been shown to transport other substrates (Xia et al., 2010; Ishikawa et al., 2012; Shin et al., 2014). The Arabidopsis genome encodes six NRAMP homologs. AtNRAMP3 and AtNRAMP4 are targeted to the vacuole and play a role in essential metal remobilization from this organelle, not only during seed germination but also in adult plants (Lanquar et al., 2005, 2010). Even though Fe content and localization are unaffected in mature seeds of the nramp3nramp4 (nr3nr4) double knockout mutant (Schnell Ramos et al., 2013), seedlings of this mutant display strong defects when grown on an Fe-deficient medium: they are chlorotic and their development is arrested (Lanquar et al., 2005).When associated with phytate in vacuolar globoids, Fe is insoluble and notoriously poorly available for animal nutrition (Clemens, 2014). To identify mutations that limit Fe storage in this compartment, we generated an ethyl methanesulfonate (EMS)-mutagenized population of the nr3nr4 mutant and looked for mutations that restore growth on Fe-deficient medium. We called these mutants isv (for bypass iron storage in vacuoles).Here, we report the characterization of two isv mutants displaying Fe distribution patterns similar to the vit1-1 mutant. These mutants carry mutations in the AtVIT1 gene. By genetic analysis, we demonstrate that the mutations in AtVIT1 are loss-of-function alleles that are responsible for the suppression phenotype. The vit1-2 allele in isv1 carries an amino acid change in the AtVIT1 protein leading to a nonfunctional protein, and the vit1-3 allele in isv2 modifies the first intron-splicing consensus sequence leading to nonfunctional RNAs or proteins. Whereas NRAMP3 and NRAMP4 are necessary for retrieving Fe from vacuoles in the wild-type background, they are not necessary for using Fe in a VIT1 loss-of-function background. Actually, combining nramp3, nramp4, and vit1 mutations modifies Fe localization at the tissue and subcellular levels. These changes likely account for the ability of vit1 mutations to rescue Fe mobilization and growth in the nr3nr4 background.     

Territory Quality and Plumage Morph Predict Offspring Sex Ratio Variation in a Raptor

Parents may adapt their offspring sex ratio in response to their own phenotype and environmental conditions. The most significant causes for adaptive sex-ratio variation might express themselves as different distributions of fitness components between sexes along a given variable. Several causes for differential sex allocation in raptors with reversed sexual size dimorphism have been suggested. We search for correlates of fledgling sex in an extensive dataset on common buzzards Buteo buteo, a long-lived bird of prey. Larger female offspring could be more resource-demanding and starvation-prone and thus the costly sex. Prominent factors such as brood size and laying date did not predict nestling sex. Nonetheless, lifetime sex ratio (LSR, potentially indicative of individual sex allocation constraints) and overall nestling sex were explained by territory quality with more females being produced in better territories. Additionally, parental plumage morphs and the interaction of morph and prey abundance tended to explain LSR and nestling sex, indicating local adaptation of sex allocation However, in a limited census of nestling mortality, not females but males tended to die more frequently in prey-rich years. Also, although females could have potentially longer reproductive careers, a subset of our data encompassing full individual life histories showed that longevity and lifetime reproductive success were similarly distributed between the sexes. Thus, a basis for adaptive sex allocation in this population remains elusive. Overall, in common buzzards most major determinants of reproductive success appeared to have no effect on sex ratio but sex allocation may be adapted to local conditions in morph-specific patterns.     

Asthma,Type 1 and Type 2 Diabetes Mellitus,and Inflammatory Bowel Disease amongst South Asian Immigrants to Canada and Their Children: A Population-Based Cohort Study

BACKGROUND

There is a high and rising rate of immune-mediated diseases in the Western world. Immigrants from South Asia have been reported to be at higher risk upon arrival to the West. We determined the risk of immune-mediated diseases in South Asian and other immigrants to Ontario, Canada, and their Ontario-born children.

METHODS

Population-based cohorts of patients with asthma, type 1 diabetes (T1DM), type 2 diabetes (T2DM), and inflammatory bowel disease (IBD) were derived from health administrative data. We determined the standardized incidence, and the adjusted risk of these diseases in immigrants from South Asia, immigrants from other regions, compared with non-immigrant residents of Ontario. The risk of these diseases in the Ontario-born children of immigrants were compared to the children of non-immigrants.

RESULTS

Compared to non-immigrants, adults from South Asia had higher risk of asthma (IRR 1.56, 95%CI 1.51-1.61) and T2DM (IRR 2.59, 95%CI 2.53-2.65). Adults from South Asia had lower incidence of IBD than non-immigrants (IRR 0.32, 95%CI 0.22-0.49), as did immigrants from other regions (IRR 0.29, 95%CI 0.20-0.42). Compared to non-immigrant children, the incidence of asthma (IRR 0.66, 95%CI 0.62-0.71) and IBD (IRR 0.47, 95%CI 0.33-0.67) was low amongst immigrant children from South Asia. However, the risk in Ontario-born children of South Asian immigrants relative to the children of non-immigrants was higher for asthma (IRR 1.75, 95%CI 1.69-1.81) and less attenuated for IBD (IRR 0.90, 95%CI 0.65-1.22).

CONCLUSION

Early-life environmental exposures may trigger a genetic predisposition to the development of asthma and IBD in South Asian immigrants and their Canada-born children.     

3D Algebraic Iterative Reconstruction for Cone-Beam X-Ray Differential Phase-Contrast Computed Tomography

Due to the potential of compact imaging systems with magnified spatial resolution and contrast, cone-beam x-ray differential phase-contrast computed tomography (DPC-CT) has attracted significant interest. The current proposed FDK reconstruction algorithm with the Hilbert imaginary filter will induce severe cone-beam artifacts when the cone-beam angle becomes large. In this paper, we propose an algebraic iterative reconstruction (AIR) method for cone-beam DPC-CT and report its experiment results. This approach considers the reconstruction process as the optimization of a discrete representation of the object function to satisfy a system of equations that describes the cone-beam DPC-CT imaging modality. Unlike the conventional iterative algorithms for absorption-based CT, it involves the derivative operation to the forward projections of the reconstructed intermediate image to take into account the differential nature of the DPC projections. This method is based on the algebraic reconstruction technique, reconstructs the image ray by ray, and is expected to provide better derivative estimates in iterations. This work comprises a numerical study of the algorithm and its experimental verification using a dataset measured with a three-grating interferometer and a mini-focus x-ray tube source. It is shown that the proposed method can reduce the cone-beam artifacts and performs better than FDK under large cone-beam angles. This algorithm is of interest for future cone-beam DPC-CT applications.     

Spastin Binds to Lipid Droplets and Affects Lipid Metabolism

Mutations in SPAST, encoding spastin, are the most common cause of autosomal dominant hereditary spastic paraplegia (HSP). HSP is characterized by weakness and spasticity of the lower limbs, owing to progressive retrograde degeneration of the long corticospinal axons. Spastin is a conserved microtubule (MT)-severing protein, involved in processes requiring rearrangement of the cytoskeleton in concert to membrane remodeling, such as neurite branching, axonal growth, midbody abscission, and endosome tubulation. Two isoforms of spastin are synthesized from alternative initiation codons (M1 and M87). We now show that spastin-M1 can sort from the endoplasmic reticulum (ER) to pre- and mature lipid droplets (LDs). A hydrophobic motif comprised of amino acids 57 through 86 of spastin was sufficient to direct a reporter protein to LDs, while mutation of arginine 65 to glycine abolished LD targeting. Increased levels of spastin-M1 expression reduced the number but increased the size of LDs. Expression of a mutant unable to bind and sever MTs caused clustering of LDs. Consistent with these findings, ubiquitous overexpression of Dspastin in Drosophila led to bigger and less numerous LDs in the fat bodies and increased triacylglycerol levels. In contrast, Dspastin overexpression increased LD number when expressed specifically in skeletal muscles or nerves. Downregulation of Dspastin and expression of a dominant-negative variant decreased LD number in Drosophila nerves, skeletal muscle and fat bodies, and reduced triacylglycerol levels in the larvae. Moreover, we found reduced amount of fat stores in intestinal cells of worms in which the spas-1 homologue was either depleted by RNA interference or deleted. Taken together, our data uncovers an evolutionarily conserved role of spastin as a positive regulator of LD metabolism and open up the possibility that dysfunction of LDs in axons may contribute to the pathogenesis of HSP.