The novel IMAGE001 genotyping array as a valuable alternative for genetic diversity screening in chicken: a demonstration in a local chicken breed in Belgium.

Screening for genetic diversity in livestock species breeds is of utmost importance, especially for local, small populations that are at the risk of extinction. Luckily, recent developments in technology increase access to genotyping, also for numerically small breeds. One of these new technologies is the IMAGE001 single nucleotide polymorphism genotyping array that includes markers for 6 different species (cow, pig, sheep, chicken, horse and goat). For our current study, we studied the Turkey-headed Malines chicken, a local chicken breed in Belgium, for the first time. A total of 110 animals were genotyped, together with 29 samples from 4 supposedly related breeds. The genotypes were used to assess the genetic diversity of this local breed. Our analysis revealed an average inbreeding coefficient of 0.20 through runs of homozygosity analysis, and effective population size estimation based on linkage disequilibrium indicated a low genetic diversity (Ne = 34). Moreover, a principal component analysis and a genetic differentiation study (FST) were performed using these marker data to position the Turkey-headed Malines relative to the 4 other indigenous Belgian chicken breeds. Finally, we discussed the practical implications of the overlap between the IMAGE001 array and other existing chicken genotyping arrays. This study is the first use of the novel IMAGE001 array to evaluate a local chicken breed, and demonstrates it as a viable option for genomic characterization a breed. Moreover, with this research, we are able to provide a good basis for further evaluation of the Belgian chicken heritage.

Persistent blood glucose reduction upon repeated transcranial electric stimulation in men.

BACKGROUND: Transcranial direct current stimulation (tDCS) of the human brain increases systemic glucose tolerance. OBJECTIVE/HYPOTHESIS: To investigate whether this effect persists after one week of repeated stimulation. Because systemic glucose uptake relates to brain energy homeostasis, we concomitantly measured cerebral high-energy phosphate metabolites. METHODS: In a sham-controlled crossover design, 14 healthy men were tested under daily anodal tDCS vs. sham for 8 days. Systemic glucose metabolism was examined by concentrations of circulating glucose and insulin. Cerebral energy metabolism - i.e. adenosine triphosphate (ATP) and phosphocreatine (PCr) levels - was assessed by 31phosphorous magnetic resonance spectroscopy. RESULTS: Blood glucose concentrations were distinctly lower upon tDCS compared with sham stimulation on day 1. This effect persisted on day 8, while serum insulin levels remained persistently unchanged. Transcranial stimulation increased mean levels of ATP and PCr compared with sham on day 1 only. Blood glucose concentrations negatively correlated with PCr content after repeated daily stimulation. CONCLUSIONS: Our data confirm that tDCS reduces blood glucose through an insulin-independent mechanism. This effect persists after 8 days of repeated stimulation and relates to brain energy metabolism. Therefore, transcranial electric stimulation may be a promising non-pharmacological adjuvant option to treat systemic disorders such as glucose intolerance or type 2 diabetes mellitus with a low side-effect profile.

Lipidomic analysis of molecular cardiolipin species in livers exposed to ischemia/reperfusion.

Transient hepatic ischemia can cause significant liver injury. A central and early event in ischemia/reperfusion (I/R) injury is the impairment of mitochondria. The phospholipid cardiolipin (CL) is required for efficient mitochondrial function. The aim of this study was to analyze composition, content, and oxidation of CL in dependence of I/R stress. Therefore, we exposed rat livers to 20 min ischemia by interrupting the perfusion with Krebs-Ringer solution in situ. Tissue histology as well as increased activities of LDH, GLDH, and ASAT analysed in the efflux after 50 min reperfusion indicated impairment of the liver. For the analysis of local CL distribution the liver homogenate was separated according to density into 11 fractions. The fractions displayed different contents of CL and citrate synthase peaking at density of about 1.07 g/cm(3). Among the fractions, the distribution of molecular CL species significantly differed. I/R caused loss of about 30 % CL and 17 % citrate synthase activity. Further, I/R shifted the CL and citrate synthase activity profile toward lower densities. Oxidized CL was exclusively found in fractions with high CL and citrate synthase content after I/R stress. I/R treatment caused significant changes in the distribution of molecular CL species. Our data demonstrate that I/R causes significant decrease in CL content and increase of oxidized CL that may be of impact for impairment of mitochondrial function by I/R. These results lead to the suggestion that strategies supporting anti-oxidative defence and CL synthesis may be beneficial to reduce I/R injury of the liver.

Oxidation of cardiolipin is involved in functional impairment and disintegration of liver mitochondria by hypoxia/reoxygenation in the presence of increased Ca²âº concentrations.

The aim of this study was to investigate the interrelationship between the mitochondrial phospholipid cardiolipin (CL), mitochondrial respiration and morphology in dependence on hypoxia/reoxygenation and Ca(2+). Therefore, we subjected rat liver mitochondria to hypoxia/reoxygenation at different extramitochondrial Ca(2+) concentrations and analysed mitochondrial respiration, morphology, CL content, the composition of molecular CL species, oxidation of CL and two mono-lyso-CL species. Hypoxia/reoxygenation in the presence of elevated extramitochondrial Ca(2+) concentration caused dramatic impairment of mitochondrial respiration and morphology. Concomitantly, increased amounts of oxidised CL were detected in the incubation medium after the treatment. Hypoxia/reoxygenation alone caused degradation of CL. The treatments had no effect on the composition of molecular CL species. Our data support the hypothesis that CL oxidation and CL degradation are involved in mitochondrial injury caused by hypoxia/reoxygenation and Ca(2+). Our results further suggest that prevention of CL oxidation by modification of CL composition may support the beneficial action of antioxidants during hypoxia/reoxygenation in the presence of elevated Ca(2+) concentrations.

Carboxyl-terminal receptor domains control the differential dephosphorylation of somatostatin receptors by protein phosphatase 1 isoforms.

We have recently identified protein phosphatase 1ß (PP1ß) as G protein-coupled receptor (GPCR) phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ) as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1ß, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

Phosphorylation of threonine 333 regulates trafficking of the human sst5 somatostatin receptor.

The frequent overexpression of the somatostatin receptors sst2 and sst5 in neuroendocrine tumors provides the molecular basis for therapeutic application of novel multireceptor somatostatin analogs. Although the phosphorylation of the carboxyl-terminal region of the sst2 receptor has been studied in detail, little is known about the agonist-induced regulation of the human sst5 receptor. Here, we have generated phosphosite-specific antibodies for the carboxyl-terminal threonines 333 (T333) and 347 (T347), which enabled us to selectively detect either the T333-phosphorylated or the T347-phosphorylated form of sst5. We show that agonist-mediated phosphorylation occurs at T333, whereas T347 is constitutively phosphorylated in the absence of agonist. We further demonstrate that the multireceptor somatostatin analog pasireotide and the sst5-selective ligand L-817,818 but not octreotide or KE108 were able to promote a detectable T333 phosphorylation. Interestingly, BIM-23268 was the only sst5 agonist that was able to stimulate T333 phosphorylation to the same extent as natural somatostatin. Agonist-induced T333 phosphorylation was dose-dependent and selectively mediated by G protein-coupled receptor kinase 2. Similar to that observed for the sst2 receptor, phosphorylation of sst5 occurred within seconds. However, unlike that seen for the sst2 receptor, dephosphorylation and recycling of sst5 were rapidly completed within minutes. We also identify protein phosphatase 1γ as G protein-coupled receptor phosphatase for the sst5 receptor. Together, we provide direct evidence for agonist-selective phosphorylation of carboxyl-terminal T333. In addition, we identify G protein-coupled receptor kinase 2-mediated phosphorylation and protein phosphatase 1γ-mediated dephosphorylation of T333 as key regulators of rapid internalization and recycling of the human sst5 receptor.

Metabolic engineering of cobalamin (vitamin B12) production in Bacillus megaterium.

Cobalamin (vitamin B(12)) production in Bacillus megaterium has served as a model system for the systematic evaluation of single and multiple directed molecular and genetic optimization strategies. Plasmid and genome-based overexpression of genes involved in vitamin B(12) biosynthesis, including cbiX, sirA, modified hemA, the operons hemAXCDBL and cbiXJCDETLFGAcysG(A)cbiYbtuR, and the regulatory gene fnr, significantly increased cobalamin production. To reduce flux along the heme branch of the tetrapyrrole pathway, an antisense RNA strategy involving silencing of the hemZ gene encoding coproporphyrinogen III oxidase was successfully employed. Feedback inhibition of the initial enzyme of the tetrapyrrole biosynthesis, HemA, by heme was overcome by stabilized enzyme overproduction. Similarly, the removal of the B(12) riboswitch upstream of the cbiXJCDETLFGAcysG(A)cbiYbtuR operon and the recombinant production of three different vitamin B(12) binding proteins (glutamate mutase GlmS, ribonucleotide triphosphate reductase RtpR and methionine synthase MetH) partly abolished B(12)-dependent feedback inhibition. All these strategies increased cobalamin production in B. megaterium. Finally, combinations of these strategies enhanced the overall intracellular vitamin B(12) concentrations but also reduced the volumetric cellular amounts by placing the organism under metabolic stress.

Softening of the actin cytoskeleton by inhibition of myosin II.

We have investigated the mechanical properties of fibroblast cells after adding the myosin inhibitor blebbistatin and the Rho-kinase inhibitor Y-27632 by atomic force microscopy (AFM). We have observed a decrease in the elastic modulus from a value of around 20 kPa down to a value around 8 kPa on a time scale of around 30-60 min when applying the myosin inhibitor blebbistatin, whereas the Y-27632 did not show any prominent mechanical effects. From topographic images, we can conclude that, after adding blebbistatin, actin filaments are not visible any more, whereas Y-27632 did not show any prominent effects in cell morphology. This study shows that tension generated by myosin contributes to the cellular stiffness and thus can be observed by measuring the elastic modulus of cells.

The effect of visible speech in the perceptual rating of pathological voices.

OBJECTIVE: To test a simple method for improving consistency among raters for the perceptual evaluation of pathological voice quality by providing visible speech (spectrogram) as additional information because, to date, the interrater variability still limits the widespread clinical use of the best available rating system. DESIGN: Experimental comparison between 2 different ways (with and without the addition of visible speech) of perceptual rating by trained professionals of recorded pathological voices. Furthermore, the correlation between acoustical (jitter, shimmer, and noise-harmonic ratio) and perceptual parameters was investigated in both rating conditions. SUBJECTS: Six experts evaluated 70 recorded pathological voices using the GIRBAS (grade, instability, roughness, breathiness, asthenicity, and strain) scale in 2 separate sessions: first, conventionally, without visible speech as additional information, and several months later, with visible speech as additional information. MAIN OUTCOME MEASURES: The kappa interrater agreement and the correlation coefficient between GIRBAS scores and acoustic measures. RESULTS: We found a significant effect of visible speech on the agreement between the raters. The interrater agreement according to kappa statistics was significantly stronger with the addition of visible speech than without for rating grade, roughness, and breathiness. The correlation between acoustical and perceptual parameters showed no significant effect of visible speech. CONCLUSIONS: The addition of visible speech to the perceptual evaluation of pathological voices is an interesting clinical asset to enhance its reliability. The addition of visible speech to the clinical setting is feasible, since affordable computer programs are currently available that can provide the spectrogram in quasi-real time while conversing with the patient. The acoustical analysis might be applied in addition to perceptual rating in a multidimensional approach to assess voice quality.

Stabilin-1 localizes to endosomes and the trans-Golgi network in human macrophages and interacts with GGA adaptors.

Stabilin-1 and stabilin-2 constitute a novel family of fasciclin domain-containing hyaluronan receptor homologues recently described by us. Whereas stabilin-1 is expressed in sinusoidal endothelial cells and in macrophages in vivo, stabilin-2 is absent from the latter. In the present study, we analyzed the subcellular distribution of stabilin-1 in primary human macrophages. Using flow cytometry, expression of stabilin-1 was demonstrated on the surface of interleukin-4/dexamethasone-stimulated macrophages (MPhi2). By immunofluorescence and confocal microscopy, we established that stabilin-1 is preferentially localized in early endosome antigen-1-positive early/sorting endosomes and in recycling endosomes identified by transferrin endocytosis. Association of stabilin-1 was infrequently seen with p62 lck ligand-positive late endosomes and with CD63-positive lysosomes but not in lysosome-associated membrane protein-1-positive lysosomes. Stabilin-1 was also found in the trans-Golgi network (TGN) but not in Golgi stack structures. Glutathione S-transferase pull-down assay revealed that the cytoplasmic tail of stabilin-1 but not stabilin-2 binds to recently discovered Golgi-localized, gamma-ear-containing, adenosine 5'-diphosphate-ribosylation factor-binding (GGA) adaptors GGA1, GGA2, and GGA3 long, mediating traffic between Golgi and endosomal/lysosomal compartments. Stabilin-1 did not bind to GGA3 short, which lacks a part of the Vps27p/Hrs/STAM domain. Deletion of DDSLL and LL amino acid motifs resulted in decreased binding of stabilin-1 with GGAs. A small portion of stabilin-1 colocalized with GGA2 and GGA3 in the TGN in MPhi2. Treatment with brefeldin A resulted in accumulation of stabilin-1 in the TGN. Our results suggest that stabilin-1 is involved in the GGA-mediated sorting processes at the interface of the biosynthetic and endosomal pathways; similarly to other GGA-interacting proteins, stabilin-1 may thus function in endocytic and secretory processes of human macrophages.

Identification and functional analysis of enzymes required for precorrin-2 dehydrogenation and metal ion insertion in the biosynthesis of sirohaem and cobalamin in Bacillus megaterium.

In Bacillus megaterium, the hemAXBCDL genes were isolated and were found to be highly similar to the genes from Bacillus subtilis that are required for the conversion of glutamyl-tRNA into uroporphyrinogen III. Overproduction and purification of HemC (porphobilinogen deaminase) and -D (uroporphyrinogen III synthase) allowed these enzymes to be used for the in vitro synthesis of uroporphyrinogen III from porphobilinogen. A second smaller cluster of three genes (termed sirABC) was also isolated and found to encode the enzymes that catalyse the transformation of uroporphyrinogen III into sirohaem on the basis of their ability to complement a defined Escherichia coli (cysG) mutant. The functions of SirC and -B were investigated by direct enzyme assay, where SirC was found to act as a precorrin-2 dehydrogenase, generating sirohydrochlorin, and SirB was found to act as a ferrochelatase responsible for the final step in sirohaem synthesis. CbiX, a protein found encoded within the main B. megaterium cobalamin biosynthetic operon, shares a high degree of similarity with SirB and acts as the cobaltochelatase associated with cobalamin biosynthesis by inserting cobalt into sirohydrochlorin. CbiX contains an unusual histidine-rich region in the C-terminal portion of the protein, which was not found to be essential in the chelation process. Sequence alignments suggest that SirB and CbiX share a similar active site to the cobaltochelatase, CbiK, from Salmonella enterica.