Manganese efficiency in barley: identification and characterization of the metal ion transporter HvIRT1

Manganese (Mn) deficiency is an important plant nutritional disorder in many parts of the world. Barley (Hordeum vulgare) genotypes differ considerably in their ability to grow in soils with low Mn(2+) availability. Differential genotypic Mn efficiency can be attributed to differences in Mn(2+) uptake kinetics in the low nanomolar concentration range. However, the molecular basis for these differences has not yet been clarified. We present here the identification and characterization of the first barley gene encoding a plasma membrane-localized metal transport protein able to transport Mn(2+). The gene is designated HvIRT1 (for IRON-REGULATED TRANSPORTER1) because it belongs to the ZIP gene family and has a high similarity to rice (Oryza sativa) OsIRT1. A novel yeast uptake assay based on inductively coupled plasma-mass spectrometry analysis of 31 different metal and metalloid ions showed that the HvIRT1 protein, in addition to Mn(2+), also transported Fe(2+)/Fe(3+), Zn(2+), and Cd(2+). Both Mn and iron deficiency induced an up-regulation of HvIRT1 in two barley genotypes differing in Mn efficiency, but the expression levels in all cases were highest (up to 40%) in the Mn-efficient genotype. The higher expression of HvIRT1 correlated with an increased Mn(2+) uptake rate. We conclude that HvIRT1 is an important component controlling Mn(2+) uptake in barley roots and contributes to genotypic differences in Mn(2+) uptake kinetics.     

Number of Published Randomized Controlled Multi Center Trials Testing Pharmacological Interventions or Devices Is Increasing in Both Medical and Surgical Specialties

Background

In general, there is a need for testing new interventions in large randomized controlled trials. Depending on the research question it may be advantageous to establish multicenter studies as a way of organizing clinical trials in order to increase study power.

Main Objectives

The object of this study was to investigate the development in the organization of multicenter studies, the distribution of studies within different clinical specialties, across continents, and investigate the differences related to testing various interventions.

Methods and Materials

A literature search was done in MEDLINE for multicenter studies published in 1995, 2000, 2005, and 2010, respectively. Data extraction identified data related to clinical specialties, interventions, participating patients, departments, countries, and continents.

Results

The number of multicenter studies increased from 112 in 1995 to 1,273 in 2010, with a larger share of multicenter studies being performed in Europe and North America. The pharmacological interventions were primarily being tested in medical studies followed by the device tests predominantly in surgical studies. The number of included patients as well as the number of participating departments increased during the time span, though the increase in studies was most evident in Europe and North America compared with the rest of the world.     

Lack of the Lysosomal Membrane Protein,GLMP, in Mice Results in Metabolic Dysregulation in Liver

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmp^gt/gt mice (formerly known as Ncu-g1^gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmp^gt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmp^gt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmp^gt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmp^gt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmp^gt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmp^gt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmp^gt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury.     

Low-Volume High-Intensity Interval Training in a Gym Setting Improves Cardio-Metabolic and Psychological Health

Background

Within a controlled laboratory environment, high-intensity interval training (HIT) elicits similar cardiovascular and metabolic benefits as traditional moderate-intensity continuous training (MICT). It is currently unclear how HIT can be applied effectively in a real-world environment.

Purpose

To investigate the hypothesis that 10 weeks of HIT, performed in an instructor-led, group-based gym setting, elicits improvements in aerobic capacity (VO_2max), cardio-metabolic risk and psychological health which are comparable to MICT.

Methods

Ninety physically inactive volunteers (42±11 y, 27.7±4.8 kg.m^-2) were randomly assigned to HIT or MICT group exercise classes. HIT consisted of repeated sprints (15–60 seconds, >90% HR_max) interspersed with periods of recovery cycling (≤25 min.session^-1, 3 sessions.week^-1). MICT participants performed continuous cycling (~70% HR_max, 30–45 min.session^-1, 5 sessions.week^-1). VO_2max, markers of cardio-metabolic risk, and psychological health were assessed pre and post-intervention.

Results

Mean weekly training time was 55±10 (HIT) and 128±44 min (MICT) (p<0.05), with greater adherence to HIT (83±14% vs. 61±15% prescribed sessions attended, respectively; p<0.05). HIT improved VO_2max, insulin sensitivity, reduced abdominal fat mass, and induced favourable changes in blood lipids (p<0.05). HIT also induced beneficial effects on health perceptions, positive and negative affect, and subjective vitality (p<0.05). No difference between HIT and MICT was seen for any of these variables.

Conclusions

HIT performed in a real-world gym setting improves cardio-metabolic risk factors and psychological health in physically inactive adults. With a reduced time commitment and greater adherence than MICT, HIT offers a viable and effective exercise strategy to target the growing incidence of metabolic disease and psychological ill-being associated with physical inactivity.     

Axo-Glia Interaction Preceding CNS Myelination Is Regulated by Bidirectional Eph-Ephrin Signaling

In the central nervous system, myelination of axons is required to ensure fast saltatory conduction and for survival of neurons. However, not all axons are myelinated, and the molecular mechanisms involved in guiding the oligodendrocyte processes toward the axons to be myelinated are not well understood. Only a few negative or positive guidance clues that are involved in regulating axo-glia interaction prior to myelination have been identified. One example is laminin, known to be required for early axo-glia interaction, which functions through α6β1 integrin. Here, we identify the Eph-ephrin family of guidance receptors as novel regulators of the initial axo-glia interaction, preceding myelination. We demonstrate that so-called forward and reverse signaling, mediated by members of both Eph and ephrin subfamilies, has distinct and opposing effects on processes extension and myelin sheet formation. EphA forward signaling inhibits oligodendrocyte process extension and myelin sheet formation, and blocking of bidirectional signaling through this receptor enhances myelination. Similarly, EphB forward signaling also reduces myelin membrane formation, but in contrast to EphA forward signaling, this occurs in an integrin-dependent manner, which can be reversed by overexpression of a constitutive active β1-integrin. Furthermore, ephrin-B reverse signaling induced by EphA4 or EphB1 enhances myelin sheet formation. Combined, this suggests that the Eph-ephrin receptors are important mediators of bidirectional signaling between axons and oligodendrocytes. It further implies that balancing Eph-ephrin forward and reverse signaling is important in the selection process of axons to be myelinated.     

Vacuolar ATPase in Phagosome-Lysosome Fusion

The vacuolar H⁺-ATPase (v-ATPase) complex is instrumental in establishing and maintaining acidification of some cellular compartments, thereby ensuring their functionality. Recently it has been proposed that the transmembrane V₀ sector of v-ATPase and its a-subunits promote membrane fusion in the endocytic and exocytic pathways independent of their acidification functions. Here, we tested if such a proton-pumping independent role of v-ATPase also applies to phagosome-lysosome fusion. Surprisingly, endo(lyso)somes in mouse embryonic fibroblasts lacking the V₀ a3 subunit of the v-ATPase acidified normally, and endosome and lysosome marker proteins were recruited to phagosomes with similar kinetics in the presence or absence of the a3 subunit. Further experiments used macrophages with a knockdown of v-ATPase accessory protein 2 (ATP6AP2) expression, resulting in a strongly reduced level of the V₀ sector of the v-ATPase. However, acidification appeared undisturbed, and fusion between latex bead-containing phagosomes and lysosomes, as analyzed by electron microscopy, was even slightly enhanced, as was killing of non-pathogenic bacteria by V₀ mutant macrophages. Pharmacologically neutralized lysosome pH did not affect maturation of phagosomes in mouse embryonic cells or macrophages. Finally, locking the two large parts of the v-ATPase complex together by the drug saliphenylhalamide A did not inhibit in vitro and in cellulo fusion of phagosomes with lysosomes. Hence, our data do not suggest a fusion-promoting role of the v-ATPase in the formation of phagolysosomes.     

Exophiala angulospora causes systemic inflammation in atlantic cod Gadus morhua

Species of Exophiala are opportunistic fungal pathogens that may infect a broad range of warm- and cold-blooded animals, including salmonids and Atlantic cod. In the present study, we observed abnormal swimming behaviour and skin pigmentation and increased mortality in cod kept in an indoor tank. Necropsy revealed foci of different sizes with a greyish to brownish colour in internal organs of diseased fish. The foci consisted of ramifying darkly pigmented fungal hyphae surrounded by distinct layers of inflammatory cells, including macrophage-like cells. In the inner layer with many hyphae, the macrophage-like cells were dead. We observed no apparent restriction of fungal growth by the inflammatory response. A darkly pigmented fungus was repeatedly isolated in pure culture from foci of diseased fish and identified as Exophiala angulospora using morphological and molecular characters. This species has not been previously reported to cause disease in cod, but has been reported as an opportunistic pathogen of both marine and freshwater fish. Based on the morphology and sequence analysis presented here, we conclude that E. angulospora caused the observed chronic multifocal inflammation in internal organs of cod, leading to severe disease and mortality.     

Francisella noatunensis in Atlantic cod (Gadus morhua L.); waterborne transmission and immune responses

This is the first report that confirms waterborne transmission of francisellosis in Atlantic cod. To investigate the transmission of disease, particle reduced water was transferred from a tank with intraperitoneally infected cod to a tank with healthy cod. Waterborne transmission of Francisella noatunensis was confirmed in the effluent group using immunohistochemistry and real-time quantitative PCR (RT-qPCR). The bacteria were located inside the accumulated macrophage-like cells. Specific and high antibody responses against live and inactivated bacteria were observed. Oil adjuvant had no effect on the antibody responses against inactivated F.?noatunensis compared to saline formulation. The antigen epitope was a 20-25?kDa component of F.?noatunensis suggested to be lipopolysaccharide detected by Western blot, Sypro Ruby and Silver staining. Systemic immune reactions were investigated by measuring the expression of IFN-γ, IL-1β and IL-10 genes with RT-qPCR. After i.p. injection of live bacteria, a significant up-regulation of IFN-γ and IL-1β expression was observed from 15 to 60 days post infection in spleen and head kidney. In intestine, IFN-γ was significantly up-regulated after 30 days whereas rectum showed no significant differences in expression. Elevated expression of IL-10 was observed in all the organs tested but was only significantly up-regulated at 60 days post infection in intestine from i.p. infected fish. For the cohabitant group, IL-1β and IFN-γ was up-regulated in spleen whereas intestine and rectum showed a down-regulation after 60 days. IL-10 was up-regulated in intestine of cohabitant fish from day 30 to day 60. These results indicate that F.?noatunensis infection provokes both specific antibody responses and long term inflammatory responses in cod. The present study provides new knowledge about infection routes and shows that both humoral and cellular defence mechanisms are triggered by F.?noatunensis in cod.     

Strikingly different properties of uracil-DNA glycosylases UNG2 and SMUG1 may explain divergent roles in processing of genomic uracil

Genomic uracil resulting from spontaneously deaminated cytosine generates mutagenic U:G mismatches that are usually corrected by error-free base excision repair (BER). However, in B-cells, activation-induced cytosine deaminase (AID) generates U:G mismatches in hot-spot sequences at Ig loci. These are subject to mutagenic processing during somatic hypermutation (SHM) and class switch recombination (CSR). Uracil N-glycosylases UNG2 and SMUG1 (single strand-selective monofunctional uracil-DNA glycosylase 1) initiate error-free BER in most DNA contexts, but UNG2 is also involved in mutagenic processing of AID-induced uracil during the antibody diversification process, the regulation of which is not understood. AID is strictly single strand-specific. Here we show that in the presence of Mg2+ and monovalent salts, human and mouse SMUG1 are essentially double strand-specific, whereas UNG2 efficiently removes uracil from both single and double stranded DNA under all tested conditions. Furthermore, SMUG1 and UNG2 display widely different sequence preferences. Interestingly, uracil in a hot-spot sequence for AID is 200-fold more efficiently removed from single stranded DNA by UNG2 than by SMUG1. This may explain why SMUG1, which is not excluded from Ig loci, is unable to replace UNG2 in antibody diversification. We suggest a model for mutagenic processing in which replication protein A (RPA) recruits UNG2 to sites of deamination and keeps DNA in a single stranded conformation, thus avoiding error-free BER of the deaminated cytosine.     

Hyperactivity of the Ero1α Oxidase Elicits Endoplasmic Reticulum Stress but No Broad Antioxidant Response

Oxidizing equivalents for the process of oxidative protein folding in the endoplasmic reticulum (ER) of mammalian cells are mainly provided by the Ero1α oxidase. The molecular mechanisms that regulate Ero1α activity in order to harness its oxidative power are quite well understood. However, the overall cellular response to oxidative stress generated by Ero1α in the lumen of the mammalian ER is poorly characterized. Here we investigate the effects of overexpressing a hyperactive mutant (C104A/C131A) of Ero1α. We show that Ero1α hyperactivity leads to hyperoxidation of the ER oxidoreductase ERp57 and induces expression of two established unfolded protein response (UPR) targets, BiP (immunoglobulin-binding protein) and HERP (homocysteine-induced ER protein). These effects could be reverted or aggravated by N-acetylcysteine and buthionine sulfoximine, respectively. Because both agents manipulate the cellular glutathione redox buffer, we conclude that the observed effects of Ero1α-C104A/C131A overexpression are likely caused by an oxidative perturbation of the ER glutathione redox buffer. In accordance, we show that Ero1α hyperactivity affects cell viability when cellular glutathione levels are compromised. Using microarray analysis, we demonstrate that the cell reacts to the oxidative challenge caused by Ero1α hyperactivity by turning on the UPR. Moreover, this analysis allowed the identification of two new targets of the mammalian UPR, CRELD1 and c18orf45. Interestingly, a broad antioxidant response was not induced. Our findings suggest that the hyperoxidation generated by Ero1α-C104A/C131A is addressed in the ER lumen and is unlikely to exert oxidative injury throughout the cell.