Dysferlin-Peptides Reallocate Mutated Dysferlin Thereby Restoring Function

Mutations in the dysferlin gene cause the most frequent adult-onset limb girdle muscular dystrophy, LGMD2B. There is no therapy. Dysferlin is a membrane protein comprised of seven, beta-sheet enriched, C2 domains and is involved in Ca²⁺dependent sarcolemmal repair after minute wounding. On the protein level, point mutations in DYSF lead to misfolding, aggregation within the endoplasmic reticulum, and amyloidogenesis. We aimed to restore functionality by relocating mutant dysferlin. Therefore, we designed short peptides derived from dysferlin itself and labeled them to the cell penetrating peptide TAT. By tracking fluorescently labeled short peptides we show that these dysferlin-peptides localize in the endoplasmic reticulum. There, they are capable of reducing unfolded protein response stress. We demonstrate that the mutant dysferlin regains function in membrane repair in primary human myotubes derived from patients’ myoblasts by the laser wounding assay and a novel technique to investigate membrane repair: the interventional atomic force microscopy. Mutant dysferlin abuts to the sarcolemma after peptide treatment. The peptide-mediated approach has not been taken before in the field of muscular dystrophies. Our results could redirect treatment efforts for this condition.     

Bread matters: a national initiative to profile the genetic diversity of Australian wheat

The large and complex genome of wheat makes genetic and genomic analysis in this important species both expensive and resource intensive. The application of next-generation sequencing technologies is particularly resource intensive, with at least 17?Gbp of sequence data required to obtain minimal (1×) coverage of the genome. A similar volume of data would represent almost 40× coverage of the rice genome. Progress can be made through the establishment of consortia to produce shared genomic resources. Australian wheat genome researchers, working with Bioplatforms Australia, have collaborated in a national initiative to establish a genetic diversity dataset representing Australian wheat germplasm based on whole genome next-generation sequencing data. Here, we describe the establishment and validation of this resource which can provide a model for broader international initiatives for the analysis of large and complex genomes.     

Expression and Characterization of Recombinant Ecarin

The snake venom protease ecarin from Echis carinatus was expressed in stable transfected CHO-S cells grown in animal component free cell culture medium. Recombinant ecarin (r-ecarin) was secreted from the suspension adapted Chinese Hamster Ovary (CHO-S) host cells as a pro-protein and activation to the mature form of r-ecarin occurred spontaneously during continued incubation of the cell culture at 37?°C after death of the host cells. Maximal ecarin activity was reached 7?days or more after cell culture viability had dropped to zero. The best producing CHO-S clone obtained produced up to 7,000 EU ecarin/litre in lab scale shaker cultures. The conversion of different concentrations of both prothrombin and prethrombin-2 as substrates for native and r-ecarin were examined with a chromogenic thrombin substrate. At low concentrations both these proteins were converted into thrombin by the two ecarin preparations with comparable rates. However, with prothrombin concentrations above 250?nM r-ecarin apparently had a two times higher turnover than native ecarin, consistent with the observed rapid complete conversion of prothrombin into thrombin by r-ecarin. With r-ecarin a K _m value of 0.4???M prethrombin-2 was determined but only a rough estimate could be made of the K _m for prothrombin of 0.9???M. In conclusion, r-ecarin was identified as a promising candidate for replacement of native ecarin in assays utilizing conversion of prothrombin to thrombin.     

Amino acid screening based on structural modeling identifies critical residues for the function, ion selectivity and structure of Arabidopsis MTP1

Arabidopsis thaliana MTP1 is a vacuolar membrane Zn(2+)/H(+) antiporter of the cation diffusion facilitator family. Here we present a structure-function analysis of AtMTP1-mediated transport and its remarkable Zn(2+) selectivity by functional complementation tests of more than 50 mutant variants in metal-sensitive yeast strains. This was combined with homology modeling of AtMTP1 based on the crystal structure of the Escherichia coli broad-specificity divalent cation transporter YiiP. The Zn(2+)-binding sites of EcYiiP in the cytoplasmic C-terminus, and the pore formed by transmembrane helices TM2 and TM5, are conserved in AtMTP1. Although absent in EcYiiP, Cys31 and Cys36 in the extended N-terminal cytosolic domain of AtMTP1 are necessary for complementation of a Zn-sensitive yeast strain. On the cytosolic side of the active Zn(2+)-binding site inside the transmembrane pore, Ala substitution of either Asn258 in TM5 or Ser101 in TM2 non-selectively enhanced the metal tolerance conferred by AtMTP1. Modeling predicts that these residues obstruct the movement of cytosolic Zn(2+) into the intra-membrane Zn(2+)-binding site of AtMTP1. A conformational change in the immediately preceding His-rich cytosolic loop may displace Asn258 and permit Zn(2+) entry into the pore. This would allow dynamic coupling of Zn(2+) transport to the His-rich loop, thus acting as selectivity filter or sensor of cytoplasmic Zn(2+) levels. Individual mutations at diverse sites within AtMTP1 conferred Co and Cd tolerance in yeast, and included deletions in N-terminal and His-rich intra-molecular cytosolic domains, and mutations of single residues flanking the transmembrane pore or participating in intra- or inter-molecular domain interactions, all of which are not conserved in the non-selective EcYiiP.     

Tolerability and efficacy of the intestinal phosphate binder Lantharenol<Superscript>®</Superscript> in cats

Background

Tolerability and efficacy of the intestinal phosphate binder Lantharenol^® (lanthanum carbonate octahydrate) were tested in two prospective, randomized and negative controlled laboratory studies with healthy adult cats fed commercial maintenance diets non-restricted in phosphorus. In the first study, the maximal tolerated dose was determined. Starting from a dose of 0.125 g/kg body weight mixed with the daily feed ration, the dose of Lantharenol^® was doubled every other week until signs of intolerability were observed (N = 10 cats compared to 5 untreated controls). In the second study, the effects of feed supplementation for two weeks with approximately 2, 6, and 20% of the maximal tolerated dose on phosphorus excretion patterns and balance were assessed (N = 8 cats per group).

Results

Lantharenol^® was found to be safe and well tolerated up to the dose of 1 g/kg bodyweight, corresponding to a concentration of 84 g Lantharenol^®/kg complete feed, defined as dry matter with a standard moisture content of 12%. Feed supplementation for two weeks with approximately 2-20% of this dosage (i.e., 1.6, 4.8, and 16 g/kg complete feed) resulted in a shift from urinary to faecal phosphorus excretion. Apparent phosphorus digestibility was dose-dependently reduced compared to the control group fed with diet only (N = 8).

Conclusions

The feed additive was well accepted and tolerated by all cats. Therefore, Lantharenol^® presents a well tolerated and efficacious option to individually tailor restriction of dietary phosphorus as indicated, for instance, in feline chronic kidney disease.

     

Assessing the complex sponge microbiota: core,variable and species-specific bacterial communities in marine sponges

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the world''s oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.     

Glatiramer Acetate Increases Phagocytic Activity of Human Monocytes In Vitro and in Multiple Sclerosis Patients

Beside its effects on T cells, a direct influence on cells of the myelo-monocytic lineage by GA becomes evident. Recently, we demonstrated that GA drives microglia to adopt properties of type II antigen presenting cells (APC) and increases their phagocytic activity. In the present work, we focused on human blood monocytes in order to examine whether GA may increase phagocytic activity in vivo and to evaluate the molecular mechanisms explaining this new discovered mode of action. Peripheral blood mononuclear cells (PBMC) were obtained using a Biocoll-Isopaque gradient and monocytes were subsequently isolated by using CD14 MicroBeads. Phagocytic activity was determined by flow cytometric measurement of the ingestion of fluorescent beads. Flow cytometry was also used to assess monocytic differentiation and expression of phagocytic receptors. Monocytes of GA treated MS patients exhibited a significantly higher phagocytic activity than those of healthy controls or non-treated MS patients. In vitro, a significant phagocytic response was already detectable after 1 h of GA treatment at the concentrations of 62.5 and 125 µg/ml. A significant increase at all concentrations of GA was observed after 3 h and 24 h, respectively. Only monocytes co-expressing CD16, particularly CD14⁺⁺CD16⁺ cells, were observed to phagocytose. Treatment of monocytes with IL-10 and supernatants from GA-treated monocytes did not alter phagocytosis. We observed a decrease in CD11c expression by GA while no changes were found in the expression of CD11b, CD36, CD51/61, CD91, TIM-3, and CD206. In our blocking assays, treatment with anti-CD14, anti-CD16, anti-TIM3, anti-CD210, and particularly anti-CD36 antibodies led to a decrease in phagocytosis. Our results demonstrate a new mechanism of action of GA treatment that augments phagocytic activity of human monocytes in vivo and in vitro. This activity seems to arise from the CD14⁺⁺CD16⁺ monocyte subset.     

Influence of a non-NK complex region of chromosome 6 on CD4+ invariant NK T cell homeostasis

The number and function of immunoregulatory invariant NKT (iNKT) cells are genetically controlled. A defect of iNKT cell ontogeny and function has been implicated as one causal factor of NOD mouse susceptibility to type 1 diabetes. Other factors of diabetes susceptibility, such as a decrease of regulatory T cell function or an increase in TLR1 expression, are corrected in diabetes-resistant Idd6 NOD.C3H 6.VIII congenic mice. Thus, we surmised that the iNKT cell defects found in NOD mice may also be rescued in congenic mice. Unexpectedly, we found, in both the thymus and the periphery, a 50% reduction in iNKT cell number in NOD.C3H 6.VIII mice as compared with NOD mice. This reduction only affected CD4(+) iNKT cells, and left the double negative iNKT cells unchanged. In parallel, the production of IL-4 and IFN-gamma following alpha-GalCer stimulation was proportionally reduced. Using three subcongenic strains, we have narrowed down the region controlling iNKT development within Idd6 (5.8 Mb) to Idd6.2 region (2.5 Mb). Idd6 region had no effect on NK cell number and in vivo cytotoxic activity. These results indicate that the role of iNKT cells in diabetes development is equivocal and more complex than initially considered. In addition, they bring strong evidence that the regulation of CD4(+) iNKT cell production is independent from that of DN iNKT cells, and involves genes of the Idd6 locus.     

Exceptional diversity of mouse lemurs (Microcebus spp.) in the Makira region with the description of one new species

Although the number of described lemur species has increased considerably over the last 20 years, detailed biogeographic data are still lacking from many geographic regions, in particular in the eastern part of Madagascar. This study investigated mouse lemur species diversity in a previously unstudied Inter-River-System in the eastern Makira region. Three sites were visited and 26 individuals were sampled and characterized with 13 external morphometric measurements. Standard phylogenetic analyses were performed on the basis of sequences of three mitochondrial loci by including representatives of all other published mouse lemur species for comparison. The analyses revealed the presence of three mouse lemur species in one study site, two of which were previously undescribed. The two new species are genetically distinct and belong to the larger-bodied mouse lemur species on the island, whereas the third species, Microcebus mittermeieri, belongs to the smaller-bodied mouse lemur species. The study fully describes one of the new species. This study and other lemur inventories suggest that the Makira region is particularly rich in lemur species and the lack of any protected zone in this area should now attract the urgent attention of conservation stakeholders.