Construction and Rescue of a Molecular Clone of Deformed Wing Virus (DWV)

European honey bees are highly important in crop pollination, increasing the value of global agricultural production by billions of dollars. Current knowledge about virulence and pathogenicity of Deformed wing virus (DWV), a major factor in honey bee colony mortality, is limited. With this study, we close the gap between field research and laboratory investigations by establishing a complete in vitro model for DWV pathogenesis. Infectious DWV was rescued from a molecular clone of a DWV-A genome that induces DWV symptoms such as crippled wings and discoloration. The expression of DWV proteins, production of infectious virus progeny, and DWV host cell tropism could be confirmed using newly generated anti-DWV monoclonal antibodies. The recombinant RNA fulfills Koch’s postulates circumventing the need of virus isolation and propagation of pure virus cultures. In conclusion, we describe the development and application of a reverse genetics system for the study of DWV pathogenesis.     

Biophysical characterization of the interaction between hepatic glucokinase and its regulatory protein: impact of physiological and pharmacological effectors

Glucokinase (GK) is a key enzyme of glucose metabolism in liver and pancreatic beta-cells, and small molecule activators of GK (GKAs) are under evaluation for the treatment of type 2 diabetes. In liver, GK activity is controlled by the GK regulatory protein (GKRP), which forms an inhibitory complex with the enzyme. Here, we performed isothermal titration calorimetry and surface plasmon resonance experiments to characterize GK-GKRP binding and to study the influence that physiological and pharmacological effectors of GK have on the protein-protein interaction. In the presence of fructose-6-phosphate, GK-GKRP complex formation displayed a strong entropic driving force opposed by a large positive enthalpy; a negative change in heat capacity was observed (Kd = 45 nm, DeltaH = 15.6 kcal/mol, TDeltaS = 25.7 kcal/mol, DeltaCp = -354 cal mol(-1) K(-1)). With k(off) = 1.3 x 10(-2) s(-1), the complex dissociated quickly. The thermodynamic profile suggested a largely hydrophobic interaction. In addition, effects of pH and buffer demonstrated the coupled uptake of one proton and indicated an ionic contribution to binding. Glucose decreased the binding affinity between GK and GKRP. This decrease was potentiated by an ATP analogue. Prototypical GKAs of the amino-heteroaryl-amide type bound to GK in a glucose-dependent manner and impaired the association of GK with GKRP. This mechanism might contribute to the antidiabetic effects of GKAs.     

A multilocus phylogeny of Malagasy scincid lizards elucidates the relationships of the fossorial genera Androngo and Cryptoscincus

A phylogeny for 29 species of scincine lizards from Madagascar, based on 3693 bp of six mitochondrial and five nuclear genes, revealed multiple parallel evolution of adaptations for a burrowing life, and unexpected relationships of the monotypic genera Androngo and Cryptoscincus. Androngo trivittatus was sister to Pygomeles braconnieri, and Cryptoscincus minimus was deeply nested within the genus Paracontias, all of these being fossorial taxa of elongated bodies and partly or fully reduced limbs. To account for these results, we place Cryptoscincus as a junior synonym of Paracontias, and discuss possible taxonomic consequences that may affect the status of Androngo, once additional data become available.     

Cell recovery comparison between plasma depletion/reduction- and red cell reduction-processing of umbilical cord blood

Background aimsLimited cell dose has hampered the use of cord blood transplantation (CBT) in adults. One method of minimizing nucleated cell loss in cord blood (CB) processing is to deplete or reduce plasma but not red blood cells - plasma depletion/reduction (PDR).MethodsThe nucleated cell loss of PDR was studied, and determined to be less than 0.1% in the discarded supernatant plasma fraction in validation experiments. After testing and archival sampling, the median nucleated cell recovery for PDR processing was 90%, and median CD34⁺ cell recovery 88%. In a CB bank inventory of 12 339 products with both pre- and post-processing total nucleated cells (TNC), PDR processing resulted in median post-processing TNC recoveries of 90.0% after testing and archival samples removal. Using the same 10 CB units divided into two halves, we compared directly the recovery of PDR against hydroxyethyl starch red cell reduction (RCR) for TNC, CD34⁺ cells and colony-forming units (CFU-GM, CFU-E, CFU-GEMM and total CFU) after parallel processing. We also compared the loss of very small embryonic-like stem cells (VSEL).ResultsWe demonstrated significantly higher recoveries using PDR for TNC (124%), CD34⁺ cells (121%), CFU-GM (225%), CFU-GEMM (201%), total CFU (186%) and VSEL (187%). The proportion of high TNC products was compared between 10 912 PDR and 38 819 RCR CB products and found to be 200% higher for products that had TNC ≥150 × 10⁷ (P = 0.0001) for the PDR inventory.ConclusionsOur data indicate that PDR processing of CB provides a significantly more efficient usage of this valuable and scarce resource.     

Diatom communities of acidic mountain streams in Poland

A comparison has been made of the species composition of diatom communities developing in acidic Polish mountain streams which flow over calcium-poor substrates: sandstones in the Silesian Beskid (section of the Western Carpathians), the witokrzyskie Mts, and over granite in the Karkonosze range (in the Sudetic Mts). The number of taxa and diversity of the diatom assemblages decreased along a decreasing pH gradient. The correlation between pH and the number of taxa was positive and significant (r ² = 0.69, p < 0.005). A small number of species (< 20) and low diversity were found in the communities developing in strongly acidic streams such as in the witokrzyskie Mts with pH 4.1–5.2, and in the Silesian Beskid with pH 3.5–4.0. In the stream of the Karkonosze Mts, with pH 5.2–6.0, the communities were characterized by their greater number of species and higher diversity.Acidobiontic and acidophilous diatoms were generally dominant. The pH-indiferent forms were less abundant, and their proportion increased above pH 5.0. Eunotia exigua, E. paludosa var. trinacria, E. tenella and Pinnularia subcapitata dominated in streams with the lowest pH, while E. exigua, E. sudetica and Achnanthes kryophila predominated in a stream with water pH above 5.2. Eunotia exigua, a common acidobiontic species was present in all the examined communities, and was a strong dominant in waters of pH 5.0. A corresponding decrease in abundance of E. exigua was observed with an increase in pH.     

Phenotypic detection of clonotypic B cells in multiple myeloma by specific immunoglobulin ligands reveals their rarity in multiple myeloma

In multiple myeloma, circulating "clonotypic" B cells, that express the immunoglobulin rearrangement of the malignant plasma cell clone, can be indirectly detected by PCR. Their role as potential "feeder" cells for the malignant plasma cell pool remains controversial. Here we established for the first time an approach that allows direct tracking of such clonotypic cells by labeling with patient-specific immunoglobulin ligands in 15 patients with myeloma. Fifty percent of patients showed evidence of clonotypic B cells in blood or bone marrow by PCR. Epitope-mimicking peptides from random libraries were selected on each patient's individual immunoglobulin and used as ligands to trace cells expressing the idiotypic immunoglobulin on their surface. We established a flow cytometry and immunofluorescence protocol to track clonotypic B cells and validated it in two independent monoclonal B cell systems. Using this method, we found clonotypic B cells in only one out of 15 myeloma patients. In view of the assay's validated sensitivity level of 10(-3), this surprising data suggests that the abundance of such cells has been vastly overestimated in the past and that they apparently represent a very rare population in myeloma. Our novel tracing approach may open perspectives to isolate and analyze clonotypic B cells and determine their role in myeloma pathobiology.     

Pattern of expression of vascular endothelial growth factor and its receptors in the ovine choroid plexus during long and short photoperiods

Vascular endothelial growth factor (VEGF-A) plays an important role in maintaining cerebrospinal fluid (CSF) homeostasis and the function of the choroid plexuses (CPs). The objective of the study was to determine the expression of vascular endothelial growth factor (VEGF-A), tyrosine kinase receptors Flt-1 and KDR and KDR co-receptor neuropilin 1 (NRP-1) in ovine CPs during different photoperiods. CPs were collected from the lateral brain ventricles from ovariectomized, estradiol-treated ewes during long day (LD; 16L:8D, n?=?5) and short day (SD; 8L:16D, n?=?5) photoperiods. We analyzed mRNA expression levels of two VEGF-A isoforms, VEGF-A ₁₂₀ and VEGF-A ₁₆₄ and our results indicate that VEGF-A ₁₆₄ was the predominant isoform. Expression levels of VEGF-A and Flt-1 were similar during the SD and LD photoperiods. There were significant increases in KDR mRNA and protein expression (p?<?0.05) and NRP-1 mRNA expression (p?<?0.05) during SD. These data show that expression of KDR and its co-receptor NRP-1 are up-regulated by short photoperiod and that this effect is not dependent on ovarian steroids. Our results suggest that the VEGF-A-system may be involved in photoperiodic plasticity of CP capillaries and may therefore be responsible for photoperiodic changes in the CSF turnover rate in ewes.     

Physical,Behavioural and Genetic Predictors of Adult Hypertension: The Findings of the Kaunas Cardiovascular Risk Cohort Study

Background

The roots of adult hypertension go back to childhood. This study aimed to examine the independent effects of physical, behavioural and genetic factors identified in childhood and mid-adulthood for prediction of adult hypertension.

Methods

The study subjects were participants of the Kaunas Cardiovascular Risk Cohort study started in 1977 (n = 1082, age 12–13 years). In 2012, a total of 507 individuals (63.9% of eligible sample) participated in the 35-year follow-up survey. Health examination involved measurements of blood pressure (BP), anthropometric parameters, and interview about health behaviours. Subjects were genotyped for AGT (M235T), ACE (I/D, rs4340), ADM (rs7129220), and CACNB2 (rs12258967) genes polymorphisms. A genetic risk score was calculated as the sum of the number of risk alleles at each of four single nucleotide polymorphisms.

Results

AGT TT genotype male carriers had the highest mean values of systolic BP in childhood. In females, ADM genotype AA was associated with the highest values of systolic and diastolic BP, while CACNB2 genotype CC carriers had the highest values of diastolic BP in childhood. Systolic and diastolic BP in childhood, gain in BMI from childhood to adulthood, and risky alcohol consumption predicted hypertension in middle-aged men. In women, genetic risk score together with diastolic BP in childhood and gain in BMI were significant predictors of adult hypertension. The comparison of four nested logistic regression models showed that the prediction of hypertension improved significantly after the addition of BMI gain. Genetic risk score had a relatively weak effect on the improvement of the model performance in women.

Conclusions

BP in childhood and the gain in BMI from childhood to adulthood were significant predictors of adult hypertension in both genders. Genetic risk score in women and risky alcohol consumption in men were independently related with the risk of adult hypertension.     

Diatoms: unicellular surrogates for macroalgal community structure in streams?

As wholesale biodiversity assessment is often impractical, the use of surrogates that reflect the assemblage structure and diversity of other taxa has attracted increased attention. We sampled 47 boreal streams for diatoms and macroalgae and examined their assemblage patterns along major environmental gradients. Our main intention was to examine whether diatoms might be useful surrogates for macroalgae in boreal streams. We also assessed whether taxon richness and community composition provided similar insights into the patterns of cross-taxon concordance. According to canonical correspondence analysis, diatom distribution was most strongly related to water pH, conductivity, latitude and longitude, and macroalgal distribution to water pH and iron content, latitude and bed instability. In Mantel’s test, diatoms and macroalgae showed significant cross-taxon concordance. However, there was no significant correlation between taxon richness of the two algal groups, likely reflecting their differing responses to environmental variables. We found evidence that although diatoms and macroalgae are partly controlled by different environmental factors, they are segregated rather similarly along latitude and a few environmental gradients such as water pH and iron content. We conclude that, at least at broad geographical extents and in small streams, diatoms reflect the structure of the macroalgal community and are therefore useful surrogates for cost-effective biomonitoring of algal communities in streams.     

Innate immunity orchestrates the mobilization and homing of hematopoietic stem/progenitor cells by engaging purinergic signaling—an update

Purinergic Signalling - Bone marrow (BM) as an active hematopoietic organ is highly sensitive to changes in body microenvironments and responds to external physical stimuli from the surrounding...