Circadian changes in Drosophila motor terminals

In Drosophila melanogaster, as in most other higher organisms, a circadian clock controls the rhythmic distribution of rest/sleep and locomotor activity. Here we report that the morphology of Drosophila flight neuromuscular terminals changes between day and night, with a rhythm in synaptic bouton size that continues in constant darkness, but is abolished during aging. Furthermore, arrhythmic mutations in the clock genes timeless and period also disrupt this circadian rhythm. Finally, these clock mutants also have an opposing effect on the nonrhythmic phenotype of neuronal branching, with tim mutants showing a dramatic hyperbranching morphology and per mutants having fewer branches than wild-type flies. These unexpected results reveal further circadian as well as nonclock related pleiotropic effects for these classic behavioral mutants.     

Hepatitis C virus induces proteolytic cleavage of sterol regulatory element binding proteins and stimulates their phosphorylation via oxidative stress

Hepatic steatosis is a common histological feature of chronic hepatitis C. Hepatitis C virus (HCV) gene expression has been shown to alter host cell cholesterol/lipid metabolism and thus induce hepatic steatosis. Since sterol regulatory element binding proteins (SREBPs) are major regulators of lipid metabolism, we sought to determine whether genotype 2a-based HCV infection induces the expression and posttranslational activation of SREBPs. HCV infection stimulates the expression of genes related to lipogenesis. HCV induces the proteolytic cleavage of SREBPs. HCV core and NS4b derived from genotype 3a are also individually capable of inducing the proteolytic processing of SREBPs. Further, we demonstrate that HCV stimulates the phosphorylation of SREBPs. Our studies show that HCV-induced oxidative stress and subsequent activation of the phosphatidylinositol 3-kinase (PI3-K)-Akt pathway and inactivation (phosphorylation) of PTEN (phosphatase and tensin homologue) mediate the transactivation of SREBPs. HCV-induced SREBP-1 and -2 activities were sensitive to antioxidant (pyrrolidine dithiocarbamate), Ca(2+) chelator 1,2-bis(aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-tetra(acetoxymethyl) ester (BAPTA-AM), and PI3-K inhibitor (LY294002). Collectively, these studies provide insight into the mechanisms of hepatic steatosis associated with HCV infection.     

Phenotypic and genetic diversity of coexisting Listonella anguillarum, Vibrio harveyi and Vibrio chagassi recovered from skin haemorrhages of diseased sand smelt, Atherina boyeri, in the Gulf of Trieste (NE Adriatic Sea)

Aims: This study identified and characterized coexisting Vibrios associated with haemorrhagic skin lesion bearing sand smelt fishes (Atherina boyeri) in north‐eastern Adriatic Sea. Methods and Results: Bacteria were isolated from external skin lesions of four samples, and representative morphotypes grown on thiosulfate–citrate–bile salt–sucrose agar were isolated. In total 25 isolates, presumptively assigned to Vibrio genus, were biochemically characterized and were grouped in 10 phenotypic profiles. Phenotypes were heterogeneously distributed among the diseased sand smelt analysed; only one phenotype was recovered from all the samples. Sequencing of 16S rRNA was performed to identify representatives of all phenotypes. Phylogenetic analysis using the neighbour‐joining method revealed six isolates clustered within the Vibrio harveyi group, three clustered with known Vibrio chagasii strains and three clustered with Listonella anguillarum. Conclusions: Vibrios with a broad phenotypic variability were found in the external lesions of diseased A. boyeri. In total three species of Vibrio were identified: V. harveyi showed the wider phenotypical and ribotypical heterogeneity while L. anguillarum shared similar biochemical characteristics with typical strains. Significance and Impact of the study: Previously unreported coexistence of potential pathogenic species colonizing diseased A. boyeri has ecological as well as epidemiological significance.     

The Eye in the Sky: Combined Use of Unmanned Aerial Systems and GPS Data Loggers for Ecological Research and Conservation of Small Birds

Technological advances for wildlife monitoring have expanded our ability to study behavior and space use of many species. But biotelemetry is limited by size, weight, data memory and battery power of the attached devices, especially in animals with light body masses, such as the majority of bird species. In this study, we describe the combined use of GPS data logger information obtained from free-ranging birds, and environmental information recorded by unmanned aerial systems (UASs). As a case study, we studied habitat selection of a small raptorial bird, the lesser kestrel Falco naumanni, foraging in a highly dynamic landscape. After downloading spatio-temporal information from data loggers attached to the birds, we programmed the UASs to fly and take imagery by means of an onboard digital camera documenting the flight paths of those same birds shortly after their recorded flights. This methodology permitted us to extract environmental information at quasi-real time. We demonstrate that UASs are a useful tool for a wide variety of wildlife studies.     

Effects of synthetic cohesin-containing scaffold protein architecture on binding dockerin-enzyme fusions on the surface of Lactococcus lactis

Background

In the last years, the biotechnological production of platform chemicals for fuel components has become a major focus of interest. Although ligno-cellulosic material is considered as suitable feedstock, the almost inevitable pretreatment of this recalcitrant material may interfere with the subsequent fermentation steps. In this study, the fungus Ustilago maydis was used to produce itaconic acid as platform chemical for the synthesis of potential biofuels such as 3-methyltetrahydrofuran. No studies, however, have investigated how pretreatment of ligno-cellulosic biomass precisely influences the subsequent fermentation by U. maydis. Thus, this current study aims to first characterize U. maydis in shake flasks and then to evaluate the influence of three exemplary pretreatment methods on the cultivation and itaconic acid production of this fungus. Cellulose enzymatically hydrolysed in seawater and salt-assisted organic-acid catalysed cellulose were investigated as substrates. Lastly, hydrolysed hemicellulose from fractionated beech wood was applied as substrate.

Results

U. maydis was characterized on shake flask level regarding its itaconic acid production on glucose. Nitrogen limitation was shown to be a crucial condition for the production of itaconic acid. For itaconic acid concentrations above 25 g/L, a significant product inhibition was observed. Performing experiments that simulated influences of possible pretreatment methods, U. maydis was only slightly affected by high osmolarities up to 3.5 osmol/L as well as of 0.1 M oxalic acid. The production of itaconic acid was achieved on pretreated cellulose in seawater and on the hydrolysed hemicellulosic fraction of pretreated beech wood.

Conclusion

The fungus U. maydis is a promising producer of itaconic acid, since it grows as single cells (yeast-like) in submerged cultivations and it is extremely robust in high osmotic media and real seawater. Moreover, U. maydis can grow on the hemicellulosic fraction of pretreated beech wood. Thereby, this fungus combines important advantages of yeasts and filamentous fungi. Nevertheless, the biomass pretreatment does indeed affect the subsequent itaconic acid production. Although U. maydis is insusceptible to most possible impurities from pretreatment, high amounts of salts or residues of organic acids can slow microbial growth and decrease the production. Consequently, the pretreatment step needs to fit the prerequisites defined by the actual microorganisms applied for fermentation.     

Lipoteichoic acid is an important microbe-associated molecular pattern of Lactobacillus rhamnosus GG

Background

Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).

Result

The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.

Conclusions

These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

A genetic validation study reveals a role of vitamin D metabolism in the response to interferon-alfa-based therapy of chronic hepatitis C

Background

To perform a comprehensive study on the relationship between vitamin D metabolism and the response to interferon-α-based therapy of chronic hepatitis C.

Methodology/Principal Findings

Associations between a functionally relevant polymorphism in the gene encoding the vitamin D 1α-hydroxylase (CYP27B1-1260 rs10877012) and the response to treatment with pegylated interferon-α (PEG-IFN-α) and ribavirin were determined in 701 patients with chronic hepatitis C. In addition, associations between serum concentrations of 25-hydroxyvitamin D₃ (25[OH]D₃) and treatment outcome were analysed. CYP27B1-1260 rs10877012 was found to be an independent predictor of sustained virologic response (SVR) in patients with poor-response IL28B genotypes (15% difference in SVR for rs10877012 genotype AA vs. CC, p = 0.02, OR = 1.52, 95% CI = 1.061–2.188), but not in patients with favourable IL28B genotype. Patients with chronic hepatitis C showed a high prevalence of vitamin D insufficiency (25[OH]D₃<20 ng/mL) during all seasons, but 25(OH)D₃ serum levels were not associated with treatment outcome.

Conclusions/Significance

Our study suggests a role of bioactive vitamin D (1,25[OH]₂D₃, calcitriol) in the response to treatment of chronic hepatitis C. However, serum concentration of the calcitriol precursor 25(OH)D₃ is not a suitable predictor of treatment outcome.     

The selectivity of canary HVC neurons for the Bird's Own Song: rate coding, temporal coding, or both?

The neuronal selectivity observed in the avian song system for the Bird's Own Song progressively emerged as an extraordinary fruitful model to investigate the neural code underlying the recognition of complex stimuli and the occurrence of learned behaviors. In adult zebra finch, neurons from the HVC (used as a proper name) show very selective auditory responses, firing more to presentation of the Bird's Own Song (BOS) than to reverse BOS or other conspecific songs. However, as adult zebra finches always produce the same stereotyped song, the presence of such highly selective neurons in birds with larger repertoire still remains an open question. Data presented here show that neurons selective for the BOS can be found in adult canary, a seasonal breeding bird which display a large repertoire. More precisely, we found that a large proportion of neurons (29/36) exhibits higher responses to presentation of the forward than to the reverse BOS, and that 22% of the cells were identified as selective on the basis of the d' value. For a cell that was extensively studied, we evaluated to what extent temporal stimulus-related structure predicts the acoustic stimulus using linear or non-linear artificial neural networks (ANN). These analyses indicated that the temporal structure contained in spike trains characterizes more accurately the stimulus than the firing rate. The limitations of applying ANN analyses to electrophysiological data are discussed and potential solutions to increase the confidence in these analysis are proposed.     

Quantitative PCR in EBV-infected renal transplant patients

In this study we investigated the levels of Epstein Barr virus (EBV) DNA by quantitative polymerase chain reaction (Q-PCR) in serum, whole blood and peripheral blood mononuclear cells (PBMC) from anti-EA IgG seropositive or anti-EA IgG seronegative EBV infected renal transplant recipients. We compared serological data with the viral load to monitor the risk of developing post-transplant lymphoproliferative disorders (PTLD). All patients were asymptomatic and none of them developed PTLD at the time of the study. EBV DNA quantitation for each patient varied in whole blood and PBMC samples probably due to different numbers of mononuclear cells present in samples from which DNA was extracted (whole blood vs. purified PBMC). In 92% of the serum samples EBV DNA was undetectable probably due to absence of free genomes since the number of DNA copies detected in samples from whole blood and PBMC does not reach very high levels. The correlation between the presence of EA-antibody, considered serological evidence of EBV reactivation, and the viral load showed that 60% of EA-positive patients had quantifiable EBV DNA, whereas in 40% of EA-positive patients EBV DNA was undetectable, showing serological reactivity but no viral replication. Of the remaining EA-negative patients, EBV DNA could be detected in 71% of them, whereas 29% did not show EBV DNA, indicating no EBV replication. In conclusion, our results confirm that the presence of serum IgG anti-EA antibody is not a reliable marker of active EBV infection whereas the evaluation of the viral load in blood samples is a useful diagnostic tool to monitor and to better understand the course of EBV infection in immunocompromised renal transplant patients at risk of developing PTLD.