Non-native hydrophobic interactions detected in unfolded apoflavodoxin by paramagnetic relaxation enhancement

Transient structures in unfolded proteins are important in elucidating the molecular details of initiation of protein folding. Recently, native and non-native secondary structure have been discovered in unfolded A. vinelandii flavodoxin. These structured elements transiently interact and subsequently form the ordered core of an off-pathway folding intermediate, which is extensively formed during folding of this α–β parallel protein. Here, site-directed spin-labelling and paramagnetic relaxation enhancement are used to investigate long-range interactions in unfolded apoflavodoxin. For this purpose, glutamine-48, which resides in a non-native α-helix of unfolded apoflavodoxin, is replaced by cysteine. This replacement enables covalent attachment of nitroxide spin-labels MTSL and CMTSL. Substitution of Gln-48 by Cys-48 destabilises native apoflavodoxin and reduces flexibility of the ordered regions in unfolded apoflavodoxin in 3.4 M GuHCl, because of increased hydrophobic interactions in the unfolded protein. Here, we report that in the study of the conformational and dynamic properties of unfolded proteins interpretation of spin-label data can be complicated. The covalently attached spin-label to Cys-48 (or Cys-69 of wild-type apoflavodoxin) perturbs the unfolded protein, because hydrophobic interactions occur between the label and hydrophobic patches of unfolded apoflavodoxin. Concomitant hydrophobic free energy changes of the unfolded protein (and possibly of the off-pathway intermediate) reduce the stability of native spin-labelled protein against unfolding. In addition, attachment of MTSL or CMTSL to Cys-48 induces the presence of distinct states in unfolded apoflavodoxin. Despite these difficulties, the spin-label data obtained here show that non-native contacts exist between transiently ordered structured elements in unfolded apoflavodoxin

The implication of identifying JAK2V617F in myeloproliferative neoplasms and myelodysplastic syndromes with bone marrow fibrosis

The myeloproliferative neoplasms (MPN) and myelodysplastic syndromes (MDS) occasionally demonstrate overlapping morphological features including hypercellularity, mild/nonspecific dysplastic changes and variable bone marrow fibrosis. Thus, when the associated bone marrow fibrosis results in a suboptimal specimen for morphological evaluation, the descriptive diagnosis “fibrotic marrow with features indeterminate for MDS versus MPN” is often applied. The JAK2V617F mutation was recently shown to be frequently identified in MPN, but it is rarely present in other myeloid disorders. However, the diagnostic utility of JAK2V617F screening in hypercellular bone marrow specimens with fibrosis has not been previously investigated. Using a real-time polymerase chain reaction melting-curve assay capable of detecting JAK2V617F in archived fixed materials, we retrospectively studied JAK2V617F in 45 cases with fibrotic hypercellular bone marrow at initial presentation, including 19 cases initially described as “with features indeterminate for MDS versus MPN”. These 19 cases were reclassified into more specific categories of MDS (n = 14) or MPN (n = 5) based on the availability of subsequent clinical data and/or bone marrow examinations. The JAK2V617F allele was identified in 17 out of 18 BCR/ABL gene-negative MPN cases with marrow fibrosis, whereas only wild-type alleles were identified in the remaining non-MPN cases. Importantly, JAK2V617F alleles were seen in all five cases of “with features indeterminate for MDS versus MPN” at initial presentation that were later determined to be MPN, but they were absent in the 14 cases later determined to be MDS. Our results suggest that JAK2V617F allele evaluation can be a useful ancillary test for discriminating MDS from MPN in specimens with bone marrow fibrosis

SNP Array Karyotyping Allows for the Detection of Uniparental Disomy and Cryptic Chromosomal Abnormalities in MDS/MPD-U and MPD

We applied single nucleotide polymorphism arrays (SNP-A) to study karyotypic abnormalities in patients with atypical myeloproliferative syndromes (MPD), including myeloproliferative/myelodysplastic syndrome overlap both positive and negative for the JAK2 V617F mutation and secondary acute myeloid leukemia (AML). In typical MPD cases (N = 8), which served as a control group, those with a homozygous V617F mutation showed clear uniparental disomy (UPD) of 9p using SNP-A. Consistent with possible genomic instability, in 19/30 MDS/MPD-U patients, we found additional lesions not identified by metaphase cytogenetics. In addition to UPD9p, we also have detected UPD affecting other chromosomes, including 1 (2/30), 11 (4/30), 12 (1/30) and 22 (1/30). Transformation to AML was observed in 8/30 patients. In 5 V617F+ patients who progressed to AML, we show that SNP-A can allow for the detection of two modes of transformation: leukemic blasts evolving from either a wild-type jak2 precursor carrying other acquired chromosomal defects, or from a V617F+ mutant progenitor characterized by UPD9p. SNP-A-based detection of cryptic lesions in MDS/MPD-U may help explain the clinical heterogeneity of this disorder

Evaluating the effectiveness of psychosocial resilience training for heart health, and the added value of promoting physical activity: a cluster randomized trial of the READY program

Background: Depression and poor social support are significant risk factors for coronary heart disease (CHD), and stress and anxiety can trigger coronary events. People experiencing such psychosocial difficulties are more likely to be physically inactive, which is also an independent risk factor for CHD. Resilience training can target these risk factors, but there is little research evaluating the effectiveness of such programs. This paper describes the design and measures of a study to evaluate a resilience training program (READY) to promote psychosocial well-being for heart health, and the added value of integrating physical activity promotion

The C allele of JAK2 rs4495487 is an additional candidate locus that contributes to myeloproliferative neoplasm predisposition in the Japanese population

<p>Abstract</p> <p>Background</p> <p>Polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) are myeloproliferative neoplasms (MPNs) characterized in most cases by a unique somatic mutation, <it>JAK2 </it>V617F. Recent studies revealed that <it>JAK2 </it>V617F occurs more frequently in a specific <it>JAK2 </it>haplotype, named <it>JAK2 </it>46/1 or GGCC haplotype, which is tagged by rs10974944 (C/G) and/or rs12343867 (T/C). This study examined the impact of single nucleotide polymorphisms (SNPs) of the <it>JAK2 </it>locus on MPNs in a Japanese population.</p> <p>Methods</p> <p>We sequenced 24 <it>JAK2 </it>SNPs in Japanese patients with PV. We then genotyped 138 MPN patients (33 PV, 96 ET, and 9 PMF) with known <it>JAK2 </it>mutational status and 107 controls for a novel SNP, in addition to two SNPs known to be part of the 46/1 haplotype (rs10974944 and rs12343867). Associations with risk of MPN were estimated by odds ratios and their 95% confidence intervals using logistic regression.</p> <p>Results</p> <p>A novel locus, rs4495487 (T/C), with a mutated T allele was significantly associated with PV. Similar to rs10974944 and rs12343867, rs4495487 in the <it>JAK2 </it>locus is significantly associated with <it>JAK2</it>-positive MPN. Based on the results of SNP analysis of the three <it>JAK2 </it>locus, we defined the "GCC genotype" as having at least one minor allele in each SNP (G allele in rs10974944, C allele in rs4495487, and C allele in rs12343867). The GCC genotype was associated with increased risk of both <it>JAK2 </it>V617F-positive and <it>JAK2 </it>V617F-negative MPN. In ET patients, leukocyte count and hemoglobin were significantly associated with <it>JAK2 </it>V617F, rather than the GCC genotype. In contrast, none of the <it>JAK2 </it>V617F-negative ET patients without the GCC genotype had thrombosis, and splenomegaly was frequently seen in this subset of ET patients. PV patients without the GCC genotype were significantly associated with high platelet count.</p> <p>Conclusions</p> <p>Our results indicate that the C allele of <it>JAK2 </it>rs4495487, in addition to the 46/1 haplotype, contributes significantly to the occurrence of <it>JAK2 </it>V617F-positive and <it>JAK2 </it>V617F-negative MPNs in the Japanese population. Because lack of the GCC genotype represents a distinct clinical-hematological subset of MPN, analyzing <it>JAK2 </it>SNPs and quantifying <it>JAK2 </it>V617F mutations will provide further insights into the molecular pathogenesis of MPN.</p

The diagnosis of BCR/ABL-negative chronic myeloproliferative diseases (CMPD): a comprehensive approach based on morphology, cytogenetics, and molecular markers

Recent years showed significant progress in the molecular characterization of the chronic myeloproliferative disorders (CMPD) which are classified according to the WHO classification of 2001 as polycythemia vera (PV), chronic idiopathic myelofibrosis (CIMF), essential thrombocythemia (ET), CMPD/unclassifiable (CMPD-U), chronic neutrophilic leukemia, and chronic eosinophilic leukemia (CEL)/hypereosinophilic syndrome, all to be delineated from BCR/ABL-positive chronic myeloid leukemia (CML). After 2001, the detection of the high frequency of the JAK2V617F mutation in PV, CIMF, and ET, and of the FIP1L1–PDGFRA fusion gene in CEL further added important information in the diagnosis of CMPD. These findings also enhanced the importance of tyrosine kinase mutations in CMPD and paved the way to a more detailed classification and to an improved definition of prognosis using also novel minimal residual disease (MRD) markers. Simultaneously, the broadening of therapeutic strategies in the CMPD, e.g., due to reduced intensity conditioning in allogeneic hematopoietic stem cell transplantation and the introduction of tyrosine kinase inhibitors in CML, in CEL, and in other ABL and PDGRFB rearrangements, increased the demands to diagnostics. Therefore, today, a multimodal diagnostic approach combining cytomorphology, cytogenetics, and individual molecular methods is needed in BCR/ABL-negative CMPD. A stringent diagnostic algorithm for characterization, choice of treatment, and monitoring of MRD will be proposed in this review

Body mass index as a predictor of healthy and disease-free life expectancy between ages 50 and 75 : a multicohort study

BACKGROUND: While many studies have shown associations between obesity and increased risk of morbidity and mortality, little comparable information is available on how body mass index (BMI) impacts health expectancy. We examined associations of BMI with healthy and chronic disease-free life expectancy in four European cohort studies. METHODS: Data were drawn from repeated waves of cohort studies in England, Finland, France and Sweden. BMI was categorized into four groups from normal weight (18.5-24.9 kg m(-2)) to obesity class II (>= 35 kg m(-2)). Health expectancy was estimated with two health indicators: sub-optimal self-rated health and having a chronic disease (cardiovascular disease, cancer, respiratory disease and diabetes). Multistate life table models were used to estimate sex-specific healthy life expectancy and chronic disease-free life expectancy from ages 50 to 75 years for each BMI category. RESULTS: The proportion of life spent in good perceived health between ages 50 and 75 progressively decreased with increasing BMI from 81% in normal weight men and women to 53% in men and women with class II obesity which corresponds to an average 7-year difference in absolute terms. The proportion of life between ages 50 and 75 years without chronic diseases decreased from 62 and 65% in normal weight men and women and to 29 and 36% in men and women with class II obesity, respectively. This corresponds to an average 9 more years without chronic diseases in normal weight men and 7 more years in normal weight women between ages 50 and 75 years compared to class II obese men and women. No consistent differences were observed between cohorts. CONCLUSIONS: Excess BMI is associated with substantially shorter healthy and chronic disease-free life expectancy, suggesting that tackling obesity would increase years lived in good health in populations.Peer reviewe