Diet of bluegill Lepomis macrochirus in a South African reservoir during winter and summer

Alien fishes are considered a major threat to aquatic biodiversity in South Africa, yet relatively little regional information on their biology and ecology is available for many of these species. Seasonal changes in the diet of the bluegill Lepomis macrochirus in Howieson’s Poort Dam, Grahamstown, were assessed during summer and winter in 2014–2015, using stomach content analysis. In winter, juvenile and adult fish diets were dominated by crustacean zooplankton and insects, respectively. In summer, juvenile fish fed on crustaceans and insects, whereas adults consumed mostly fish eggs, indicating a potential impact by these invasive fish on native fish through oophagy.     

Substrate specificity of recombinant dengue 2 virus NS2B-NS3 protease: influence of natural and unnatural basic amino acids on hydrolysis of synthetic fluorescent substrates

A recombinant dengue 2 virus NS2B-NS3 protease (NS means non-structural virus protein) was compared with human furin for the capacity to process short peptide substrates corresponding to seven native substrate cleavage sites in the dengue viral polyprotein. Using fluorescence resonance energy transfer peptides to measure kinetics, the processing of these substrates was found to be selective for the Dengue protease. Substrates containing two or three basic amino acids (Arg or Lys) in tandem were found to be the best, with Abz-AKRRSQ-EDDnp being the most efficiently cleaved. The hydrolysis of dipeptide substrates Bz-X-Arg-MCA where X is a non-natural basic amino acid were also kinetically examined, the best substrates containing aliphatic basic amino acids. Our results indicated that proteolytic processing by dengue NS3 protease, tethered to its activating NS2B co-factor, was strongly inhibited by Ca2+ and kosmotropic salts of the Hofmeister's series, and significantly influenced by substrate modifications between S4 and S6'. Incorporation of basic non-natural amino acids in short peptide substrates had significant but differential effects on Km and k(cat), suggesting that further dissection of their influences on substrate affinity might enable the development of effective dengue protease inhibitors.     

"Silent" sites in Drosophila genes are not neutral: evidence of selection among synonymous codons

The patterns of synonymous codon usage in 91 Drosophila melanogaster genes have been examined. Codon usage varies strikingly among genes. This variation is associated with differences in G+C content at silent sites, but (unlike the situation in mammalian genes) these differences are not correlated with variation in intron base composition and so are not easily explicable in terms of mutational biases. Instead, those genes with high G+C content at silent sites, resulting from a strong "preference" for a particular subset of the codons that are mostly C- ending, appear to be the more highly expressed genes. This suggests that G+C content is reduced in sequences where selective constraints are weaker, as indeed seen in a pseudogene. These and other data discussed are consistent with the effects of translational selection among synonymous codons, as seen in unicellular organisms. The existence of selective constraints on silent substitutions, which may vary in strength among genes, has implications for the use of silent molecular clocks.      

Stimulation of Cultured Melanocytes in Medium Containing a Serum Substitute: Ultroser-G

Melanocyte cultures were established and maintained routinely in Ham's F-10 medium containing 12-O-tetradecanoyl-phorbol-13-acetate (TPA), isobutylmethylxanthine (IBMX), cholera toxin (CT) and fetal calf serum (FCS). Three serum substitutes (Ultroser-G, Nutridoma-Hu and Nutricyte-H) were tested in order to obtain a medium without FCS having a more constant composition. Melanocyte proliferation was examined in long-term culture experiments by in situ cell counts at different periods of time. Only with Ultroser-G (1-2%) was the proliferation of melanocytes maintained without both FCS and CT, whereas the addition of the other two serum substitutes resulted in stabilization of melanocyte densities in the cultures up to 28 days. In the medium containing 1% Ultroser-G and IBMX without TPA minimal or no increases in melanocyte density were found. Addition of basic fibroblast growth factor (bFGF, 1 ng/ml) to the medium without TPA resulted in a partial restimulation of growth in different experiments. In this system with 1% Ultroser-G and 1 ng/ml bFGF, IBMX could also be replaced by other factors (dbcAMP LTC4 and a purified form of α-melanocyte stimulating hormone). The culture medium with 1% Ultroser-G containing TPA and IBMX is now used for routine melanocyte culture. In this medium TPN/IBMX can easily be replaced by bFGF/dbcAMP with optimal growth stimulation. The combination bFGF/α-MSH and other more physiological stimulators offers an alternative to study responses of melanocytes in culture with respect to proliferation, metabolism, and phenotype.     

Effect of intragenic rearrangement and changes in the 3' consensus sequence on NSP1 expression and rotavirus replication

The nonpolyadenylated mRNAs of rotavirus are templates for the synthesis of protein and the segmented double-stranded RNA (dsRNA) genome. During serial passage of simian SA11 rotaviruses in cell culture, two variants emerged with gene 5 dsRNAs containing large (1.1 and 0.5 kb) sequence duplications within the open reading frame (ORF) for NSP1. Due to the sequence rearrangements, both variants encoded only C-truncated forms of NSP1. Comparison of these and other variants encoding defective NSP1 with their corresponding wild-type viruses indicated that the inability to encode authentic NSP1 results in a small-plaque phenotype. Thus, although nonessential, NSP1 probably plays an active role in rotavirus replication in cell culture. In determining the sequences of the gene 5 dsRNAs of the SA11 variants and wild-type viruses, it was unexpectedly found that their 3' termini ended with 5'-UGAACC-3' instead of the 3' consensus sequence 5'-UGACC-3', which is present on the mRNAs of nearly all other group A rotaviruses. Cell-free assays indicated that the A insertion into the 3' consensus sequence interfered with its ability to promote dsRNA synthesis and to function as a translation enhancer. The results provide evidence that the 3' consensus sequence of the gene 5 dsRNAs of SA11 rotaviruses has undergone a mutation causing it to operate suboptimally in RNA replication and in the expression of NSP1 during the virus life cycle. Indeed, just as rotavirus variants which encode defective NSP1 appear to have a selective advantage over those encoding wild-type NSP1 in cell culture, it may be that the atypical 3' end of SA11 gene 5 has been selected for because it promotes the expression of lower levels of NSP1 than the 3' consensus sequence.     

OPTIMAS-DW: A comprehensive transcriptomics,metabolomics, ionomics,proteomics and phenomics data resource for maize

Background

The wild barley Hordeum chilense fulfills some requirements for being a useful tool to investigate the endosperm yellow pigment content (YPC) in the Triticeae including its diploid constitution, the availability of genetic resources (addition and deletion stocks and a high density genetic map) and, especially, its high seed YPC not silenced in tritordeums (amphiploids derived from H. chilense and wheat). Thus, the aim of this work was to test the utility of the H. chilense genome for investigating the YPC in the Triticeae.

Results

Twelve genes related to endosperm carotenoid content and/or YPC in grasses (Dxr, Hdr [synonym ispH], Ggpps1, Psy2, Psy3, Pds, Zds, e-Lcy, b-Lcy, Hyd3, Ccd1 and Ppo1) were identified, and mapped in H. chilense using rice genes to identify orthologs from barley, wheat, sorghum and maize. Macrocolinearity studies revealed that gene positions were in agreement in H. vulgare and H. chilense. Additionally, three main regions associated with YPC were identified in chromosomes 2H^ch, 3H^ch and 7H^ch in H. chilense, the former being the most significant one.

Conclusions

The results obtained are consistent with previous findings in wheat and suggest that Ggpps1, Zds and Hyd3 on chromosome 2H^ch may be considered candidate genes in wheat for further studies in YPC improvement. Considering the syntenic location of carotenoid genes in H. chilense, we have concluded that the H^ch genome may constitute a valuable tool for YPC studies in the Triticeae.

     

Moving instead of asking? Performance-based tests and BASFI-questionnaire measure different aspects of physical function in ankylosing spondylitis

Introduction

Ankylosing Spondylitis (AS) is characterised by limitations in physical function. The Bath Ankylosing Spondylitis Functional Index (BASFI) is considered to be the gold-standard to assess physical function in AS patients. However, the BASFI questionnaire is a self-reported outcome measure and susceptible to subjective interpretation (under- or over-estimation). More objective outcome measures, like performance-based tests, could provide an objective outcome measurement for the evaluation of limitations in physical function. Therefore, the primary aim of this study was to determine the association between performance-based measures and the BASFI questionnaire.

Methods

In this cross-sectional study 126 AS patients completed the BASFI questionnaire and eight performance-based tests based on BASFI-items. Each test received three scores: one for performance (time or points) and a score for exertion and pain experienced during performance (using modified Borg-scale and VAS 0-100 mm, respectively). Linear regression analyses were used to assess the associations between the BASFI questionnaire and performance-based tests.

Results

The univariable association between performance and BASFI-score was moderate with a R-square of 0.31 and Beta of 0.56 (p's < 0.05). In a multivariable analysis, the association between performance, exertion and pain on the one hand and BASFI-score on the other was assessed; R-square increased to 0.54: the Beta's for exertion and pain during performance were 0.38 and 0.26, respectively; the Beta for performance decreased to 0.19 (p's < 0.05).

Conclusions

This study demonstrates that alongside actual performance, patients seem to incorporate exertion and pain in their assessment of perceived physical function on the BASFI questionnaire. Performance-based tests could provide an objective outcome measurement for the evaluation of physical function and give relevant new information in addition to the BASFI questionnaire.     

Designing a strategy to implement cost-effective blood transfusion management in elective hip and knee arthroplasties: A study protocol

ABSTRACT: BACKGROUND: Total hip and knee arthroplasties are two of the most commonly performed procedures in orthopedic surgery. Different blood-saving measures (BSMs) are used to reduce the often-needed allogenic blood transfusions in these procedures. A recent large randomized controlled trial showed it is not cost effective to use the BSMs of erythropoietin and perioperative autologous blood salvage in elective primary hip and knee arthroplasties. Despite dissemination of these study results, medical professionals keep using these BSMs. To actually change practice, an implementation strategy is needed that is based on a good understanding of target groups and settings and the psychological constructs that predict behavior of medical professionals. However, detailed insight into these issuses is lacking. Therefore, this study aims to explore which groups of professionals should be targeted at which settings, as well as relevant barriers and facilitators that should be taken into acount in the strategy to implement evidence-based, cost-effective blood transfusion management and to de-implement BSMs. METHODS: The study consists of three phases. First, a questionnaire survey among all Dutch orthopedic hospital departments and independent treatment centers (n?=?99) will be conducted to analyze current blood management practice. Second, semistructured interviews will be held among 10 orthopedic surgeons and 10 anesthesiologists to identify barriers and facilitators that are relevant for the uptake of cost-effective blood transfusion management. Interview questions will be based on the Theoretical Domains Interview framework. The interviews will be followed by a questionnaire survey among 800 medical professionals in orthopedics and anesthesiology (400 professionals per discipline) in which the identified barriers and facilitators will be ranked by frequency and importance. Finally, an implementation strategy will be developed based on the results from the previous phases, using principles of intervention mapping and an expert panel. DISCUSSION: The developed strategy for cost-effective blood transfusion management by de-implementing BSMs is likely to reduce costs for elective hip and knee arthroplasties. In addition, this study will lead to generalized knowledge regarding relevant factors for the de-implementation of non-cost-effective interventions and insight in the differences between implementation and de-implementation strategies.     

Correlation between catalysis and tertiary structure arrangement in an archaeal halophilic subtilase

Nep (Natrialba magadii extracellular protease) is a halolysin-like peptidase secreted by the haloalkaliphilic archaeon N. magadii that exhibits optimal activity and stability in salt-saturated solutions. In this work, the effect of salt on the function and structure of Nep was investigated. In absence of salt, Nep became unfolded and aggregated, leading to the loss of activity. The enzyme did not recover its structural and functional properties even after restoring the ideal conditions for catalysis. At salt concentrations higher than 1 M (NaCl), Nep behaved as monomers in solution and its enzymatic activity displayed a nonlinear concave-up dependence with salt concentration resulting in a 20-fold activation at 4 M NaCl. Although transition from a high to a low-saline environment (3–1 M NaCl) did not affect its secondary structure contents, it diminished the enzyme stability and provoked large structural rearrangements, changing from an elongated shape at 3 M NaCl to a compact conformational state at 1 M NaCl. The thermodynamic analysis of peptide hydrolysis by Nep suggests a significant enzyme reorganization depending on the environmental salinity, which supports in solution SAXS and DLS studies. Moreover, solvent kinetic isotopic effect (SKIE) data indicates the general acid-base mechanism as the rate-limiting step for Nep catalysis, like classical serine-peptidases. All these data correlate the Nep conformational states with the enzymatic behavior providing a further understanding on the stability and structural determinants for the functioning of halolysins under different salinities.     

Rheumatoid factor and anti-citrullinated protein antibody positivity,but not level,are associated with increased mortality in patients with rheumatoid arthritis: results from two large independent cohorts

Introduction

This study aimed to investigate rheumatoid factor (RF) and anti-citrullinated protein antibody (ACPA) status and levels as predictors of mortality in two large cohorts of patients with early inflammatory arthritis (EIA).

Methods

Data from the Norfolk Arthritis Register (NOAR) and Leiden Early Arthritis Clinic (EAC) cohorts were used. At baseline, patients had demographic data and smoking status recorded; RF, ACPA and inflammatory markers were measured in the local laboratories. Patients were flagged with national death registers until death or censor date. Antibody status was stratified as negative, low or high positive by RF and ACPA levels individually. In addition, patients were grouped as seronegative, RF positive, ACPA positive or double antibody (RF and ACPA) positive. Cox regression models explored associations between antibody status and mortality adjusting for age, sex, smoking status, inflammatory markers and year of enrolment.

Results

A total of 4962 patients were included, 64% were female. Median age at onset was 56 (NOAR) and 54 (EAC) years. In NOAR and EAC respectively, 35% and 42% of patients were ACPA/RF positive. When antibody status was stratified as negative, low or high positive, there were no consistent findings between the two cohorts. Double antibody positivity was associated with excess mortality in both cohorts compared to seronegative patients: NOAR and EAC respective adjusted HR (95% confidence interval) 1.35 (1.09 to 1.68) and 1.58 (1.16 to 2.15).

Conclusions

Patients with EIA who are seropositive for both RF and ACPA have increased mortality compared to those who are single positive or seronegative. Antibody level in seropositive patients was not consistently associated with excess mortality.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0483-3) contains supplementary material, which is available to authorized users.