In vivo and in vitro protective role of proline

Accumulation of proline in response to environmental stresses seems tobe widespread among plants. To elucidate the role of proline in plantresponses,in vivo and in vitro, we studied theeffect of proline on catalase (CAT; EC 1.11.1.6), peroxidase (POD; EC 1.11.1.7)and polyphenol oxidase (PPO; EC 1.14.18.1). In vivo, thesethree enzymes were activated by proline, while CAT and POD were activated andPPO was inactivated by NaCl. In vitro, CAT and POD wereactivated and PPO was inactivated by proline. Proline appeared to protect thesethree enzyme activities. The significance of these findings with regard toenvironmental stress-induced proline accumulation in vivois discussed. The ability of proline to activate the enzymes may suggest alimited conformational change. These results are important for characterisationof metabolic responses to environmental stresses and can be used as a stressindicator.     

Spectrofluorimetric analysis of 7-hydroxycoumarin binding to bovine phenol sulfotransferase

Phenol sulfotransferases (SULT1s, EC 2.8.2.1) catalyze sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to the hydroxyl oxygen of aromatic acceptor substrates. Previous work with the bovine SULT1A1 has utilized the highly fluorescent substrate 7-hydroxycoumarin (7-HC, umbelliferone) as an acceptor substrate [Biochem. Biophys. Res. Commun. 261 (1999) 815]. Here we report that adenosine-3',5'-bisphosphate (PAP)-dependent binding of 7-HC to bSULT1A1 can be observed due to the appearance of a 400-420-nm shoulder in the emission spectrum, using an excitation wavelength of 280 nm. This emission was observed by placing 7-HC in ethanol, which is consistent with bSULT1A1 phenol binding site hydrophobicity. Titrations with 7-HC indicate a K(d) for 7-HC of 0.58 microM and substoichiometric binding to the homodimeric enzyme. The bSULT1A1:PAP:7-HC complex could be disrupted with pentachlorophenol (PCP), titrations with which indicated 0.5 equivalents per enzyme subunit. Titrations of enzyme plus 7-HC with PAP also indicated 0.5 equivalents per enzyme subunit. These results suggest a model of homodimeric bSULT1A1 in which subunit interactions favor half-site reactivity in the formation of a dead end complex.     

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.     

The effects of 11-ketotestosterone on occupation of downstream location and seawater in the New Zealand shortfinned eel, Anguilla australis

The androgen 11-ketotestosterone (11KT) is associated with the physiological and morphological changes that occur during the transformation of sedentary ('yellow') freshwater eels (Anguilla spp.) into their migratory form ('silver') prior to their spawning migration in the ocean. In this study, we investigate the possible role of 11KT in modulating behaviors consistent with downstream migration; i.e., downstream and salinity preference in the New Zealand shortfinned eel (A. australis). Unlike silvering, 11KT did not induce preference for downstream locations, scored as presence at the downstream ends of 35 m raceways. Likewise, there was no evidence for increased salinity preference in 11KT-treated yellow eels, scored as preference for sea water over fresh water in a choice experiment. However, the 11KT treatment induced higher frequency of movements between fresh water and sea water, which may indicate restlessness.     

Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging (MALDI MSI) for Monitoring of Drug Response in Primary Cancer Spheroids

Malignant ascites is a fluid, which builds up in the abdomen and contains cancer cells in the form of single cells or multicellular clusters called spheroids. Malignant ascites has been observed in patients suffering from ovarian, cervical, gastric, colorectal, pancreatic, endometrial, or primary liver cancer. The spheroids are believed to play a major role in chemo resistance and metastasis of the cancer. To ease the discomfort of patients, malignant ascites (MA) is often drained from the abdomen using a procedure called paracentesis. MA retrieved via this minimal invasive procedure is a great source for cancer spheroids, which can be used for testing chemotherapeutic drugs and drug combinations. Herein, the existing workflow is adapted to make concurrent monitoring of drug accumulation, drug response, and drug metabolites feasible using primary spheroids or spheroids grown without a scaffolding matrix. To achieve this, those spheroids are embedded in matrigel, before fixing them with formalin. This makes it possible to process, store, and ship samples at room temperature. This new approach might be used to choose the best targeted therapy for each patient and thereby facilitate personalized medicine.     

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.     

Preparation of a novel hydrophobic affinity cryogel for adsorption of lipase and its utilization as a chromatographic adsorbent for fast protein liquid chromatography

In this study, we have prepared a hydrophobic cryogel for the chromatographic separation of lipase from its aqueous solutions including single protein and protein mixture and also Yarrowia lipolytica cell extract. N‐methacryloyl‐(l )‐phenylalanine methyl ester was used as a monomer to provide the hydrophobic character to the prepared cryogels. The highest adsorption capacity was observed at pH 5.0 at 0.5 mL min^?1 flow rate. The chromatographic separation of lipase was achieved from a binary mixture of lipase:bovine serum albumin (BSA) and lipase:lysozyme, and was also achieved from triple‐mixture of lipase:lysozyme:BSA by using fast protein liquid chromatography. Finally, lipase purification was performed from Yarrowia lipolytica cell extract used as a natural source. These studies have shown that the hydrophobic cryogel has good chromatographic performance for the separation and purification of lipase not only from aqueous solution, but also from cell extract as a natural source of lipase. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 30:376–382, 2014     

Genetic mapping and developmental timing of transmission ratio distortion in a mouse interspecific backcross

Background

Sheep carcasses with yellow fat are sporadically observed at Norwegian slaughter houses. This phenomenon is known to be inherited as a recessive trait, and is caused by accumulation of carotenoids in adipose tissue. Two enzymes are known to be important in carotenoid degradation in mammals, and are therefore potential candidate genes for this trait. These are beta-carotene 15,15'-monooxygenase 1 (BCMO1) and the beta-carotene oxygenase 2 (BCO2).

Results

In the present study the coding region of the BCMO1 and the BCO2 gene were sequenced in yellow fat individuals and compared to the corresponding sequences from control animals with white fat. In the yellow fat individuals a nonsense mutation was found in BCO2 nucleotide position 196 (c.196C>T), introducing a stop codon in amino acid position 66. The full length protein consists of 575 amino acids. In spite of a very low frequency of this mutation in the Norwegian AI-ram population, 16 out of 18 yellow fat lambs were found to be homozygous for this mutation.

Conclusion

In the present study a nonsense mutation (c.196C>T) in the beta-carotene oxygenase 2 (BCO2) gene is found to strongly associate with the yellow fat phenotype in sheep. The existence of individuals lacking this mutation, but still demonstrating yellow fat, suggests that additional mutations may cause a similar phenotype in this population. The results demonstrate a quantitatively important role for BCO2 in carotenoid degradation, which might indicate a broad enzyme specificity for carotenoids. Animals homozygous for the mutation are not reported to suffer from any negative health or development traits, pointing towards a minor role of BCO2 in vitamin A formation. Genotyping AI rams for c.196C>T can now be actively used in selection against the yellow fat trait.