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Find video protocols related to scientific articles indexed in Pubmed.
The relationship between depression and frailty syndrome: a systematic review.
Aging Ment Health
PUBLISHED: 10-17-2014
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Objectives: Frailty is a geriatric syndrome characterised by the clinical presentation of identifiable physical alterations such as loss of muscle mass and strength, energy and exercise tolerance, and decreased physiological reserve. Frailty and depressive symptoms are common issues facing older adults and may be associated. It is not clear if the depression facilitates the appearance of frailty syndrome or vice versa or these two coexist independently in the same individuals. Method: We performed searches in several databases (Embase, PubMed, CINAHL, Scopus, and PsycINFO) papers published between November 2003 to February 2014 about frailty syndrome and depression in people aged 65 and older published and the reference lists of from the articles retrieved were pearled in order to identify any which may have been missed in the initial search. Two independent reviewers extracted descriptive information on the prevalence and co-occurrence of frailty and depression in older individuals and of frailty criteria among depressed patients. Results: Depression and frailty occur in a significant proportion of frail older individuals. Common pathophysiological alterations and biomarkers in the two syndromes have been recently described. Conclusion: Studies on the causal relationship between the two syndromes are clearly necessary in the future.
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Probing the Effect of Force on HIV-1 Receptor CD4.
ACS Nano
PUBLISHED: 10-14-2014
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Cell-surface proteins are central for the interaction of cells with their surroundings and are also associated with numerous diseases. These molecules are exposed to mechanical forces, but the exact relation between force and the functions and pathologies associated with cell-surface proteins is unclear. An important cell-surface protein is CD4, the primary receptor of HIV-1. Here we show that mechanical force activates conformational and chemical changes on CD4 that may be important during viral attachment. We have used single-molecule force spectroscopy and analysis on HIV-1 infectivity to demonstrate that the mechanical extension of CD4 occurs in a time-dependent manner and correlates with HIV-1 infectivity. We show that Ibalizumab, a monoclonal antibody that blocks HIV-1, prevents the mechanical extension of CD4 domains 1 and 2. Furthermore, we demonstrate that thiol/disulfide exchange in CD4 requires force for exposure of cryptic disulfide bonds. This mechanical perspective provides unprecedented information that can change our understanding on how viruses interact with their hosts.
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Altered thiol chemistry in human amyotrophic lateral sclerosis-linked mutants of superoxide dismutase 1.
J. Biol. Chem.
PUBLISHED: 08-04-2014
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Neurodegenerative diseases share a common characteristic, the presence of intracellular or extracellular deposits of protein aggregates in nervous tissues. Amyotrophic Lateral Sclerosis (ALS) is a severe and fatal neurodegenerative disorder, which affects preferentially motoneurons. Changes in the redox state of superoxide dismutase 1 (SOD1) are associated with the onset and development of familial forms of ALS. In human SOD1 (hSOD1), a conserved disulfide bond and two free cysteine residues can engage in anomalous thiol/disulfide exchange resulting in non-native disulfides, a hallmark of ALS that is related to protein misfolding and aggregation. Because of the many competing reaction pathways, traditional bulk techniques fall short at quantifying individual thiol/disulfide exchange reactions. Here, we adapt recently developed single-bond chemistry techniques to study individual disulfide isomerization reactions in hSOD1. Mechanical unfolding of hSOD1 leads to the formation of a polypeptide loop held by the disulfide. This loop behaves as a molecular jump rope that brings reactive Cys-111 close to the disulfide. Using force-clamp spectroscopy, we monitor nucleophilic attack of Cys-111 at either sulfur of the disulfide and determine the selectivity of the reaction. Disease-causing mutations G93A and A4V show greatly altered reactivity patterns, which may contribute to the progression of familial ALS.
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Abdominal Obesity and Metabolic Syndrome Burden in Adolescents-Penn State Children Cohort Study.
J Clin Densitom
PUBLISHED: 06-24-2014
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To investigate the association between abdominal obesity and metabolic syndrome (MetS) burden in a population-based sample of adolescents, we used data from 421 adolescents who completed the follow-up examination in the Penn State Children Cohort study. Dual-energy x-ray absorptiometry (DXA) was used to assess abdominal obesity, as measured by android/gynoid fat ratio (A/G ratio), android/whole body fat proportion (A/W proportion), visceral (VAT) and subcutaneous fat (SAT) areas. Continuous metabolic syndrome score (cMetS), calculated as the sum of the age and sex-adjusted standardized residual (Z-score) of five established MetS components, was used to assess the MetS burden. Linear regression models were used to analyze the impact of DXA measures on cMetS components. All models were adjusted for age, race, sex, and general obesity. We found abdominal obesity is significantly associated with increased cMetS. With 1 standard deviation (SD) increase in A/G ratio, A/W proportion, VAT area, and SAT area, cMetS increased by 1.34 (SE=0.17), 1.25 (SE=0.19), 1.67 (SE=0.17), and 1.84 (SE=0.20) units, respectively. At individual component level, strongest association was observed between abdominal obesity and insulin resistance (IR) than lipid-based or blood pressure-based components. VAT and SAT had a stronger impact on IR than android ratio-based DXA measurements. In conclusion, abdominal obesity is associated with higher MetS burden in adolescent population. The association between abdominal obesity and IR measure is the strongest, suggesting the key impact of abdominal obesity on IR in adolescents MetS burden.
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Correlates of suicide ideation and attempts in children and adolescents with eating disorders.
Eat Disord
PUBLISHED: 05-19-2014
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This is the first study determining correlates of suicide behavior in children with eating disorders using multiple sleep, psychological, and demographic variables. Mothers rated suicide ideation and attempts in 90 children ages 7-18 with bulimia nervosa or anorexia nervosa. Suicide ideation was more prevalent in children with bulimia nervosa (43%) than children with anorexia nervosa (20%). All children with bulimia nervosa who experienced ideation attempted suicide, whereas only 3% of children with anorexia nervosa attempted suicide. Correlates of ideation were externalizing behavior problems and sleep disturbances. Correlates of attempts were bulimia nervosa, self-induced vomiting, nightmares, and physical or sexual abuse. These problems should be assessed and targeted for intervention because of their association with suicide behavior.
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The circadian pattern of cardiac autonomic modulation and obesity in adolescents.
Clin. Auton. Res.
PUBLISHED: 05-15-2014
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To assess the impact of obesity and population attributes on the circadian pattern of cardiac autonomic modulation (CAM) in a population-based sample of adolescents.
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Whole-genome sequencing and comprehensive molecular profiling identify new driver mutations in gastric cancer.
Nat. Genet.
PUBLISHED: 04-18-2014
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Gastric cancer is a heterogeneous disease with diverse molecular and histological subtypes. We performed whole-genome sequencing in 100 tumor-normal pairs, along with DNA copy number, gene expression and methylation profiling, for integrative genomic analysis. We found subtype-specific genetic and epigenetic perturbations and unique mutational signatures. We identified previously known (TP53, ARID1A and CDH1) and new (MUC6, CTNNA2, GLI3, RNF43 and others) significantly mutated driver genes. Specifically, we found RHOA mutations in 14.3% of diffuse-type tumors but not in intestinal-type tumors (P < 0.001). The mutations clustered in recurrent hotspots affecting functional domains and caused defective RHOA signaling, promoting escape from anoikis in organoid cultures. The top perturbed pathways in gastric cancer included adherens junction and focal adhesion, in which RHOA and other mutated genes we identified participate as key players. These findings illustrate a multidimensional and comprehensive genomic landscape that highlights the molecular complexity of gastric cancer and provides a road map to facilitate genome-guided personalized therapy.
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Insomnia symptoms, objective sleep duration and hypothalamic-pituitary-adrenal activity in children.
Eur. J. Clin. Invest.
PUBLISHED: 03-12-2014
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Insomnia symptoms are the most common parent-reported sleep complaints in children; however, little is known about the pathophysiology of childhood insomnia symptoms, including their association with hypothalamic-pituitary-adrenal (HPA) axis activation. The objective of this study is to examine the association between parent-reported insomnia symptoms, objective short sleep duration and cortisol levels in a population-based sample of school-aged children.
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Evaluation of the effect of D-002, a mixture of beeswax alcohols, on osteoarthritis symptoms.
Korean J. Intern. Med.
PUBLISHED: 02-27-2014
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Nonsteroidal anti-inflammatory drugs relieve osteoarthritis (OA) symptoms but cause adverse effects. D-002, a mixture of beeswax alcohols, is effective against experimental OA. A pilot study found that D-002 (50 mg/day) for 8 weeks improves OA symptoms. The aim of this study was to investigate the effects of D-002 (50 to 100 mg/day) administered for 6 weeks on OA symptoms.
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Patterns of somatic alterations between matched primary and metastatic colorectal tumors characterized by whole-genome sequencing.
Genomics
PUBLISHED: 02-25-2014
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Colorectal cancer (CRC) patients have poor prognosis after formation of distant metastasis. Understanding the molecular mechanisms by which genetic changes facilitate metastasis is critical for the development of targeted therapeutic strategies aimed at controlling disease progression while minimizing toxic side effects. A comprehensive portrait of somatic alterations in CRC and the changes between primary and metastatic tumors has yet to be developed. We performed whole genome sequencing of two primary CRC tumors and their matched liver metastases. By comparing to matched germline DNA, we catalogued somatic alterations at multiple scales, including single nucleotide variations, small insertions and deletions, copy number aberrations and structural variations in both the primary and matched metastasis. We found that the majority of these somatic alterations are present in both sites. Despite the overall similarity, several de novo alterations in the metastases were predicted to be deleterious, in genes including FBXW7, DCLK1 and FAT2, which might contribute to the initiation and progression of distant metastasis. Through careful examination of the mutation prevalence among tumor cells at each site, we also proposed distinct clonal evolution patterns between primary and metastatic tumors in the two cases. These results suggest that somatic alterations may play an important role in driving the development of colorectal cancer metastasis and present challenges and opportunities when considering the choice of treatment.
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Clinical features of prefrail older individuals and emerging peripheral biomarkers: a systematic review.
Arch Gerontol Geriatr
PUBLISHED: 02-17-2014
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Frailty is a geriatric syndrome characterized by the clinical presentation of identifiable physical alterations such as loss of muscle mass and strength, energy and exercise tolerance, and decreased physiological reserve. Individuals with one or two of these alterations are defined as prefrail. The clinical features of prefrail older individuals have been investigated to a lesser extent compared to the frail population, even though this intermediate stage may provide insights into the mechanisms involved in the physical decline associated with aging and it is considered to be potentially reversible. We performed searches in the Medline, Embase, Scopus, Cinahl, and Cochrane databases from January 1995 to July 2013 for papers about the identification of prefrail people aged 65 and older published either in English or Spanish, and the reference lists of from the articles retrieved were pearled in order to identify any which may have been missed in the initial search. Two independent reviewers extracted descriptive information on frailty criteria and outcomes from the selected papers: of the 277 articles retrieved from the searches and 25 articles retrieved from pearling, 84 met the study inclusion criteria. The prevalence of prefrailty ranges between 35% and 50% in individuals aged over 60, is more common in women, and the age and the number of comorbidities in these individuals is similar to their frail counterparts. Weakness is the most prevalent symptom in prefrail individuals although there are some sex differences. Some serum biomarkers seem to discriminate prefrail from non-frail individuals but further research would be required to confirm this.
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Cognitive-emotional hyperarousal in the offspring of parents vulnerable to insomnia: a nuclear family study.
J Sleep Res
PUBLISHED: 02-14-2014
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Cognitive-emotional hyperarousal is believed to be a predisposing factor for insomnia; however, there is limited information on the association of familial vulnerability to insomnia and cognitive-emotional hyperarousal. The aim of this study was to estimate the heritability of stress-related insomnia and examine whether parental vulnerability to stress-related insomnia is associated with cognitive-emotional hyperarousal in their offspring. We studied a volunteer sample of 135 nuclear families comprised of 270 middle-aged (51.5 ± 5.4 years) fathers and mothers and one of their biological offspring (n = 135, 20.2 ± 1.1 years). We measured vulnerability to stress-related insomnia (i.e. Ford Insomnia Response to Stress Test: FIRST), perceived stress, depression and anxiety in all participants, and arousability, presleep cognitive and somatic arousal, coping and personality in the offspring. We found a heritability estimate of 29% for FIRST scores. High FIRST parents had three to seven times the odds of having offspring highly vulnerable to stress-related insomnia. Offspring of high FIRST parents showed higher arousability, presleep cognitive arousal and emotion-oriented coping. Furthermore, high FIRST mothers contributed to offspring's higher anxiety and lower task-oriented coping, while high FIRST fathers contributed to offspring's higher presleep somatic arousal and conscientiousness. Vulnerability to stress-related insomnia is significantly heritable. Parents vulnerable to stress-related insomnia have offspring with cognitive-emotional hyperarousal who rely upon emotion-oriented coping. These data give support to the notion that arousability and maladaptive coping are key factors in the aetiology of insomnia.
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Reversible binding of solvent to naked PbII centers in unusual homoleptic alkynyl-based Pt2Pb2 clusters.
Chemistry
PUBLISHED: 02-02-2014
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We report a series of luminescent sandwich-type clusters [Pt2 Pb2 (C?CR)8] (R=Tol, 1; C6 H4 OMe-3, 2; C6 H4 OMe-4, 3) with a dynamic Pt2 Pb2 metallic core, which is key to their intriguing stimuli-responsive photophysical properties. The solvent-free solids 1-3 display an orange emission ascribed to charge transfer from Pt-alkynyl fragments to a delocalized orbital with mixed Pt2 Pb2 /C?CR nature, with a predominant lead contribution and Pb???Pb bonding character ((3) MLCCT/(3) IL). They exhibit mechanical, color, and luminescence changes that are reversible and perceivable with the naked eye, which are attributed to small inter- and intramolecular structural modifications induced by gentle grinding. Interestingly, 1 and 2 also exhibit remarkable and fast reversible vapochromic responses to donor solvent vapors (acetone, THFMe-2: yellow; NCMe: green, vs. dry solids: orange). The structures of 1(acetone)2 ?2(Me2 CO), 2(acetone)3 , and 2(THFMe-2)2 allow the vapochromic responses to be ascribed to the fast creation/disruption of solvate clusters [Pt2 Pb2 (C?CR)8 Sx] (x?2), with concomitant electronic and geometrical modifications within the Pt2 Pb2 core, which are easily accessible through a slight change in the stereochemical activity of the lone pair. The binding of one (or two) solvent molecules to Pb(2+) increases the Pb???Pb separation in the metallic core, causing a destabilization of the target orbital and larger energy gaps of the transitions. All the solvates exhibit remarkable rigidochromism upon a decrease in temperature, which is also associated with the gradual increase in the transannular Pb???Pb separation, as revealed by X-ray crystallography of 1(acetone)2 at different temperatures. Investigation of the crystal lattice of 1?CH2 Cl2 and 3?2?CH2 Cl2 further suggests that the lack of vapor stimuli response of complex 3 could be attributed to the presence of competitive additional secondary intermolecular Pb???O(OMe) contacts, which give rise to a more compact network built up from extended chains of clusters.
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Pyogenic granuloma, an unusual presentation of peripubertal vaginal bleeding. Case report and review of the literature.
J. Pediatr. Endocrinol. Metab.
PUBLISHED: 01-28-2014
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Abstract Pyogenic granuloma, also named lobular capillary hemangioma, is a common proliferative vascular lesion known as a benign condition despite its rapid growth. It may appear in any cutaneous or mucosal surface but is usually restricted to the oral cavity. It is characterized by a friable mulberry-like lesion that can be sessile or pedunculated. Bleeding is usually its first clinical manifestation. Locations on respiratory, digestive and genital tracts are uncommon and sporadic. We describe the occurrence of an intravaginal pyogenic granuloma in a peripubertal girl with recurrent vaginal bleeding. This is the first reported case of a genital tract lobular capillary hemangioma in pediatric age to our knowledge. Therefore, we suggest this entity in the differential diagnosis of an unclear peripubertal vaginal bleeding.
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S-glutathionylation of cryptic cysteines enhances titin elasticity by blocking protein folding.
Cell
PUBLISHED: 01-24-2014
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The giant elastic protein titin is a determinant factor in how much blood fills the left ventricle during diastole and thus in the etiology of heart disease. Titin has been identified as a target of S-glutathionylation, an end product of the nitric-oxide-signaling cascade that increases cardiac muscle elasticity. However, it is unknown how S-glutathionylation may regulate the elasticity of titin and cardiac tissue. Here, we show that mechanical unfolding of titin immunoglobulin (Ig) domains exposes buried cysteine residues, which then can be S-glutathionylated. S-glutathionylation of cryptic cysteines greatly decreases the mechanical stability of the parent Ig domain as well as its ability to fold. Both effects favor a more extensible state of titin. Furthermore, we demonstrate that S-glutathionylation of cryptic cysteines in titin mediates mechanochemical modulation of the elasticity of human cardiomyocytes. We propose that posttranslational modification of cryptic residues is a general mechanism to regulate tissue elasticity.
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Decoding complex patterns of genomic rearrangement in hepatocellular carcinoma.
Genomics
PUBLISHED: 01-11-2014
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Elucidating the molecular basis of hepatocellular carcinoma (HCC) is crucial to developing targeted diagnostics and therapies for this deadly disease. The landscape of somatic genomic rearrangements (GRs), which can lead to oncogenic gene fusions, remains poorly characterized in HCC. We have predicted 4314 GRs including large-scale insertions, deletions, inversions and translocations based on the whole-genome sequencing data for 88 primary HCC tumor/non-tumor tissues. We identified chromothripsis in 5 HCC genomes (5.7%) recurrently affecting chromosomal arms 1q and 8q. Albumin (ALB) was found to harbor GRs, deactivating mutations and deletions in 10% of cohort. Integrative analysis identified a pattern of paired intra-chromosomal translocations flanking focal amplifications and asymmetrical patterns of copy number variation flanking breakpoints of translocations. Furthermore, we predicted 260 gene fusions which frequently result in aberrant over-expression of the 3' genes in tumors and validated 18 gene fusions, including recurrent fusion (2/88) of ABCB11 and LRP2.
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Insomnia and its impact on physical and mental health.
Curr Psychiatry Rep
PUBLISHED: 11-06-2013
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In contrast to the association of insomnia with mental health, its association with physical health has remained largely unexplored until recently. Based on findings that insomnia with objective short sleep duration is associated with activation of both limbs of the stress system and other indices of physiological hyperarousal, which should adversely affect physical and mental health, we have recently demonstrated that this insomnia phenotype is associated with a significant risk of cardiometabolic and neurocognitive morbidity and mortality. In contrast, insomnia with normal sleep duration is associated with sleep misperception and cognitive-emotional arousal, but not with signs of physiological hyperarousal or cardiometabolic or neurocognitive morbidity. Interestingly, both insomnia phenotypes are associated with mental health, although most likely through different pathophysiological mechanisms. We propose that objective measures of sleep duration may become part of the routine evaluation and diagnosis of insomnia, and that these two insomnia phenotypes may respond differentially to biological versus psychological treatments.
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Insomnia with Short Sleep Duration: Nosological, Diagnostic, and Treatment Implications.
Sleep Med Clin
PUBLISHED: 09-28-2013
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The diagnosis of insomnia is based solely on subjective complaints. This has contributed to the low reliability and validity of the current nosology of insomnia as well as to its lack of firm association with clinically relevant outcomes such as cardiometabolic and neurocognitive morbidity. We review evidence that insomnia with objective short sleep duration is associated with physiological hyperarousal, higher risk for hypertension, diabetes, neurocognitive impairment, and mortality as well as with a persistent course. It also appears that objective short sleep duration in poor sleepers is a biological marker of genetic predisposition to chronic insomnia. In contrast, insomnia with objective normal sleep duration is associated with cognitive-emotional and cortical arousal and sleep misperception but not with signs of physiological hyperarousal or medical complications. Thus, short sleep duration in insomnia may be a reliable marker of the biological severity and medical impact of the disorder. We propose that (a) objective measures of sleep be included in the diagnosis of insomnia and its subtypes, (b) objective measures of sleep obtained in the home environment of the patient would become part of the routine assessment and diagnosis of insomnia in a clinicians office setting, and (c) insomnia with short sleep duration may respond better to biological treatments, whereas insomnia with normal sleep duration may respond primarily to psychological therapies.
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Conformational plasticity of the essential membrane-associated mannosyltransferase PimA from mycobacteria.
J. Biol. Chem.
PUBLISHED: 08-20-2013
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Phosphatidyl-myo-inositol mannosyltransferase A (PimA) is an essential glycosyltransferase (GT) that initiates the biosynthetic pathway of phosphatidyl-myo-inositol mannosides, lipomannan, and lipoarabinomannan, which are key glycolipids/lipoglycans of the mycobacterial cell envelope. PimA belongs to a large family of peripheral membrane-associated GTs for which the understanding of the molecular mechanism and conformational changes that govern substrate/membrane recognition and catalysis remains a major challenge. Here we used single molecule force spectroscopy techniques to study the mechanical and conformational properties of PimA. In our studies, we engineered a polyprotein containing PimA flanked by four copies of the well characterized I27 protein, which provides an unambiguous mechanical fingerprint. We found that PimA exhibits weak mechanical stability albeit displaying ?-sheet topology expected to unfold at much higher forces. Notably, PimA unfolds following heterogeneous multiple step mechanical unfolding pathways at low force akin to molten globule states. Interestingly, the ab initio low resolution envelopes obtained from small angle x-ray scattering of the unliganded PimA and the PimA·GDP complexed forms clearly demonstrate that not only the "open" and "closed" conformations of the GT-B enzyme are largely present in solution, but in addition, PimA experiences remarkable flexibility that undoubtedly corresponds to the N-terminal "Rossmann fold" domain, which has been proved to participate in protein-membrane interactions. Based on these results and on our previous experimental data, we propose a model wherein the conformational transitions are important for the mannosyltransferase to interact with the donor and acceptor substrates/membrane.
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Nanomechanics of HaloTag tethers.
J. Am. Chem. Soc.
PUBLISHED: 08-19-2013
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The active site of the Haloalkane Dehydrogenase (HaloTag) enzyme can be covalently attached to a chloroalkane ligand providing a mechanically strong tether, resistant to large pulling forces. Here we demonstrate the covalent tethering of protein L and I27 polyproteins between an atomic force microscopy (AFM) cantilever and a glass surface using HaloTag anchoring at one end and thiol chemistry at the other end. Covalent tethering is unambiguously confirmed by the observation of full length polyprotein unfolding, combined with high detachment forces that range up to ?2000 pN. We use these covalently anchored polyproteins to study the remarkable mechanical properties of HaloTag proteins. We show that the force that triggers unfolding of the HaloTag protein exhibits a 4-fold increase, from 131 to 491 pN, when the direction of the applied force is changed from the C-terminus to the N-terminus. Force-clamp experiments reveal that unfolding of the HaloTag protein is twice as sensitive to pulling force compared to protein L and refolds at a slower rate. We show how these properties allow for the long-term observation of protein folding-unfolding cycles at high forces, without interference from the HaloTag tether.
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The value of neutrophil and lymphocyte count in frail older women.
Exp. Gerontol.
PUBLISHED: 08-12-2013
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Increasing evidence suggests that systemic inflammation is associated with many pathophysiological processes including frailty in older adults. We evaluated the relationships between white blood cell subtypes, geriatric assessment, and frailty syndrome and in particular, how they correlate with individual frailty criteria (involuntary loss of weight, low energy or exhaustion, slow mobility, muscle weakness, and low physical activity) in frail older women. There was a significant and positive correlation between the frailty score and neutrophil count, but a significantly negative correlation was found when this score was compared to the lymphocyte count. These associations were significant only for two frailty criteria: poor muscular strength and low physical activity. Further investigation into the role of white blood cell subtypes in ageing and its associated adverse outcomes in older adults is warranted, in particular in the loss of muscular strength and for poor physical activity.
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Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.
Genome Res.
PUBLISHED: 06-20-2013
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Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.
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Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy.
Nat Protoc
PUBLISHED: 06-06-2013
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Here we describe a protocol for using force-clamp spectroscopy to precisely quantify the effect of force on biochemical reactions. A calibrated force is used to control the exposure of reactive sites in a single polyprotein substrate composed of repeated domains. The use of polyproteins allows the identification of successful single-molecule recordings from unambiguous mechanical unfolding fingerprints. Biochemical reactions are then measured directly by detecting the length changes of the substrate held at a constant force. We present the layout of a force-clamp spectrometer along with protocols to design and conduct experiments. These experiments measure reaction kinetics as a function of applied force. We show sample data of the force dependency of two different reactions, protein unfolding and disulfide reduction. These data, which can be acquired in just a few days, reveal mechanistic details of the reactions that currently cannot be resolved by any other technique.
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Facial follicular cysts: a case of lichen planus follicularis tumidus?
J. Cutan. Pathol.
PUBLISHED: 04-30-2013
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Lichen planus follicularis tumidus (LPFT) represents an uncommon variety of lichen planus (LP). Clinically, it presents with prominent purplish lesions or white-pigmented yellowish cysts and comedones. Histopathologically, it is similar to lichen planopilaris, and it is additionally characterized by follicles and cysts surrounded by a lichenoid lymphocytic infiltrate. The most common location is the retroauricular region, and it may be associated with other variants of LP. Herein, we describe the case of a 50-year-old woman with a history of lower limb hypertrophic LP who subsequently presented with multiple pink, tumid, pruritic plaques with white-yellow cysts and comedones extensively affecting the bilateral face. Histopathologic examination revealed a lichenoid infiltrate surrounding the follicles and cysts. We diagnosed LPFT and began treatment with topical corticosteroids, antihistamines, systemic corticosteroids and oral acitretin without improvement. Subsequently, the patient had an acceptable response to cyclosporine at doses of 5?mg/kg/day with remission of itching and tumidity but with residual cysts and comedones remaining. To date, the literature contains only 16 cases of LPFT. To our knowledge, this is the most severe case and is the only one with cessation of disease activity in response to cyclosporine.
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Conservation of protein structure over four billion years.
Structure
PUBLISHED: 04-10-2013
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Little is known about the evolution of protein structures and the degree of protein structure conservation over planetary time scales. Here, we report the X-ray crystal structures of seven laboratory resurrections of Precambrian thioredoxins dating up to approximately four billion years ago. Despite considerable sequence differences compared with extant enzymes, the ancestral proteins display the canonical thioredoxin fold, whereas only small structural changes have occurred over four billion years. This remarkable degree of structure conservation since a time near the last common ancestor of life supports a punctuated-equilibrium model of structure evolution in which the generation of new folds occurs over comparatively short periods and is followed by long periods of structural stasis.
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Phycocyanobilin promotes PC12 cell survival and modulates immune and inflammatory genes and oxidative stress markers in acute cerebral hypoperfusion in rats.
Toxicol. Appl. Pharmacol.
PUBLISHED: 04-04-2013
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Since the inflammatory response and oxidative stress are involved in the stroke cascade, we evaluated here the effects of Phycocyanobilin (PCB, the C-Phycocyanin linked tetrapyrrole) on PC12 cell survival, the gene expression and the oxidative status of hypoperfused rat brain. After the permanent bilateral common carotid arteries occlusion (BCCAo), the animals were treated with saline or PCB, taking samples 24h post-surgery. Global gene expression was analyzed with GeneChip Rat Gene ST 1.1 from Affymetrix; the expression of particular genes was assessed by the Fast SYBR Green RT-PCR Master Mix and Bioplex methods; and redox markers (MDA, PP, CAT, SOD) were evaluated spectrophotometrically. The PCB treatment prevented the H2O2 and glutamate induced PC12 cell injury assessed by the MTT assay, and modulated 190 genes (93 up- and 97 down-regulated) associated to several immunological and inflammatory processes in BCCAo rats. Furthermore, PCB positively modulated 19 genes mostly related to a detrimental pro-inflammatory environment and counteracted the oxidative imbalance in the treated BCCAo animals. Our results support the view of an effective influence of PCB on major inflammatory mediators in acute cerebral hypoperfusion. These results suggest that PCB has a potential to be a treatment for ischemic stroke for which further studies are needed.
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Subjective and objective sleep and self-harm behaviors in young children: a general population study.
Psychiatry Res
PUBLISHED: 03-27-2013
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Significant association between sleep disturbances and suicidal ideation and/or attempts is reported in adults and adolescents. However, there is paucity of studies exploring the association between sleep and self-harm behaviors (SHB) in young children and are limited to only subjective sleep measures. We examined the association between SHB and both subjective and objective sleep in a population-based sample of 5-12 yr old. Parents of every student in 3 local school (K-5) districts (n=7312) was sent a screening questionnaire. Randomly selected children from this sample underwent a comprehensive history, physical examination, a 9-h overnight polysomnogram and completed several questionnaires. Among the final sample (n=693), 27 children had SHB with adjusted prevalence of 3%. There was no difference in age, gender, obesity, or socioeconomic status in subjects with or without SHB. Significantly more children with SHB had subjective sleep difficulty and depression. Difficulty maintaining sleep and frequent nightmares were associated with SHB independent of depression or demographics. Polysomnographic %REM-sleep was significantly higher in the SHB group after adjusting for demographics and depression. These data indicate that parent reported sleep disturbances are independently associated with SHB. It is possible that higher REM-sleep is a non-invasive biomarker for risk of self-harm behaviors in young children.
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Prevalence of insomnia symptoms in a general population sample of young children and preadolescents: gender effects.
Sleep Med.
PUBLISHED: 03-20-2013
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Our population-based study examined the prevalence of insomnia symptoms and its sociodemographic, subjective, and polysomnographic (PSG) sleep risk factors in young and preadolescent children.
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Effects of D-002, a mixture of high molecular weight beeswax alcohols, on patients with nonalcoholic fatty liver disease.
Korean J. Intern. Med.
PUBLISHED: 03-14-2013
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Nonalcoholic fatty liver disease (NAFLD) is intimately related to insulin resistance and ranges from a benign course to liver fibrosis and cirrhosis. NAFLD management mainly involves dietary modification and weight loss. Although no fully successful pharmacological intervention is available, alternative therapies to treat NAFLD have shown promising results. Experimental studies have shown that D-002, a mixture of beeswax alcohols with antioxidant effects, is hepatoprotective. The aim of this study was to investigate the efficacy and safety of D-002 in patients with NALFD.
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Insomnia with objective short sleep duration: the most biologically severe phenotype of the disorder.
Sleep Med Rev
PUBLISHED: 02-16-2013
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Until recently, the association of chronic insomnia with significant medical morbidity was not established and its diagnosis was based solely on subjective complaints. We present evidence that insomnia with objective short sleep duration is the most biologically severe phenotype of the disorder, as it is associated with cognitive-emotional and cortical arousal, activation of both limbs of the stress system, and a higher risk for hypertension, impaired heart rate variability, diabetes, neurocognitive impairment, and mortality. Also, it appears that objective short sleep duration is a biological marker of genetic predisposition to chronic insomnia. In contrast, insomnia with objective normal sleep duration is associated with cognitive-emotional and cortical arousal and sleep misperception but not with signs of activation of both limbs of the stress system or medical complications. Furthermore, the first phenotype is associated with unremitting course, whereas the latter is more likely to remit. We propose that short sleep duration in insomnia is a reliable marker of the biological severity and medical impact of the disorder. Objective measures of sleep obtained in the home environment of the patient would become part of the routine assessment of insomnia patients in a clinicians office setting. We speculate that insomnia with objective short sleep duration has primarily biological roots and may respond better to biological treatments, whereas insomnia with objective normal sleep duration has primarily psychological roots and may respond better to psychological interventions alone.
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Phylogeny of Tetillidae (Porifera, Demospongiae, Spirophorida) based on three molecular markers.
Mol. Phylogenet. Evol.
PUBLISHED: 02-15-2013
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Tetillidae are spherical to elliptical cosmopolitan demosponges. The family comprises eight genera: namely, Acanthotetilla Burton, 1959, Amphitethya Lendenfeld, 1907, CinachyraSollas, 1886, CinachyrellaWilson, 1925, Craniella Schmidt, 1870, Fangophilina Schmidt, 1880, Paratetilla Dendy, 1905, and Tetilla Schmidt, 1868. These genera are characterized by few conflicting morphological characters, resulting in an ambiguity of phylogenetic relationships. The phylogeny of tetillid genera was investigated using the cox1, 18S rRNA and 28S rRNA (C1-D2 domains) genes in 88 specimens (8 genera, 28 species). Five clades were identified: (i) Cinachyrella, Paratetilla and Amphitethya species, (ii) Cinachyrella levantinensis, (iii) Tetilla, (iv) Craniella, Cinachyra and Fangophilina and (v) Acanthotetilla. Consequently, the phylogenetic analysis supports the monophyly of Tetilla, a genus lacking any known morphological synapomorphy. Acanthotetilla is also recovered. In contrast, within the first clade, species of the genera Paratetilla and Amphitethya were nested within Cinachyrella. Similarly, within the fourth clade, species of the genera Cinachyra and Fangophilina were nested within Craniella. As previously postulated by taxonomists, the loss of ectodermal specialization (i.e., a cortex) has occurred several times independently. Nevertheless, the presence or absence of a cortex and its features carry a phylogenetic signal. Surprisingly, the common view that assumes close relationships among sponges with porocalices (i.e., surface depressions) is refuted.
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Elasticity, structure, and relaxation of extended proteins under force.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-13-2013
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Force spectroscopies have emerged as a powerful and unprecedented tool to study and manipulate biomolecules directly at a molecular level. Usually, protein and DNA behavior under force is described within the framework of the worm-like chain (WLC) model for polymer elasticity. Although it has been surprisingly successful for the interpretation of experimental data, especially at high forces, the WLC model lacks structural and dynamical molecular details associated with protein relaxation under force that are key to the understanding of how force affects protein flexibility and reactivity. We use molecular dynamics simulations of ubiquitin to provide a deeper understanding of protein relaxation under force. We find that the WLC model successfully describes the simulations of ubiquitin, especially at higher forces, and we show how protein flexibility and persistence length, probed in the force regime of the experiments, are related to how specific classes of backbone dihedral angles respond to applied force. Although the WLC model is an average, backbone model, we show how the protein side chains affect the persistence length. Finally, we find that the diffusion coefficient of the proteins end-to-end distance is on the order of 10(8) nm(2)/s, is position and side-chain dependent, but is independent of the length and independent of the applied force, in contrast with other descriptions.
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Plasma and urine metabolic fingerprinting of type 1 diabetic children.
Electrophoresis
PUBLISHED: 02-01-2013
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Type 1 diabetes mellitus is one of the most common chronic disorders of childhood. The metabolic control is lost due to the lack of insulin, which is the main treatment for the disease. Nevertheless, long-term complications appear even under good glycemic control. Metabolomics, an emerging strategy, can help in diagnosis, prognosis, and monitoring of metabolic disorders. The objective of the present study was to investigate the alterations in plasma (by LC-MS) and urine (CE-MS) of type 1 diabetic children that were under insulin treatment and good glycemic control. Even without remarkable biochemical differences between the two groups (diabetic and control) except for glucose level and glycosilated hemoglobin, metabolomic tools were able to capture subtle metabolic differences. The main changes in plasma were associated to lipidic metabolism (nonesterified fatty acids, lysophospholipids, and other derivatives of fatty acids), and some markers of the differential activity of the gut microflora were also found (bile acids, p-cresol sulfate). In urine, changes associated to protein and amino acid metabolism were found (amino acids, their metabolites and derivatives), and among them one advanced glycation end product (carboxyethylarginine) and one early glycation end product (fructosamine) were excreted in higher proportion in the diabetic group.
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The Antitumor Peptide CIGB-552 Increases COMMD1 and Inhibits Growth of Human Lung Cancer Cells.
J Amino Acids
PUBLISHED: 01-16-2013
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We have demonstrated that the peptide L-2 designed from an alanine scanning of the Limulus-derived LALF32-51 region is a potential candidate for the anticancer therapy and its cell-penetrating capacity is an associated useful property. By the modification in the primary structure of L-2, a second-generation peptide (CIGB-552) was developed. However, the molecular mechanism underlying its cytotoxic activity remains partially unknown. In this study, it was shown that CIGB-552 increases the levels of COMMD1, a protein involved in copper homeostasis, sodium transport, and the NF-?B signaling pathway. We found that CIGB-552 induces ubiquitination of RelA and inhibits the antiapoptotic activity regulated by NF-?B, whereas the knockdown of COMMD1 blocks this effect. We also found that CIGB-552 decreases the antioxidant capacity and induces the peroxidation of proteins and lipids in the tumor cells. Altogether, this study provides new insights into the mechanism of action of the peptide CIGB-552, which could be relevant in the design of future anticancer therapies.
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Protein folding under mechanical forces: a physiological view.
Physiology (Bethesda)
PUBLISHED: 01-03-2013
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Mechanical forces regulate the function of numerous proteins relevant to physiology. The functions and folding of proteins have been under scrutiny for decades, but it was not until recently that mechanical forces have been considered. Here, we review different techniques for studying protein folding, highlighting their physiological significance.
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The BioSample Database (BioSD) at the European Bioinformatics Institute.
Nucleic Acids Res.
PUBLISHED: 11-16-2011
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The BioSample Database (http://www.ebi.ac.uk/biosamples) is a new database at EBI that stores information about biological samples used in molecular experiments, such as sequencing, gene expression or proteomics. The goals of the BioSample Database include: (i) recording and linking of sample information consistently within EBI databases such as ENA, ArrayExpress and PRIDE; (ii) minimizing data entry efforts for EBI database submitters by enabling submitting sample descriptions once and referencing them later in data submissions to assay databases and (iii) supporting cross database queries by sample characteristics. Each sample in the database is assigned an accession number. The database includes a growing set of reference samples, such as cell lines, which are repeatedly used in experiments and can be easily referenced from any database by their accession numbers. Accession numbers for the reference samples will be exchanged with a similar database at NCBI. The samples in the database can be queried by their attributes, such as sample types, disease names or sample providers. A simple tab-delimited format facilitates submissions of sample information to the database, initially via email to biosamples@ebi.ac.uk.
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Familial glucocorticoid deficiency due to compound heterozygosity of two novel MC2R mutations.
J. Pediatr. Endocrinol. Metab.
PUBLISHED: 08-10-2011
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Familial glucocorticoid deficiency (FGD) is a rare autosomal recessive disorder characterized by isolated glucocorticoid deficiency. Mutations in the ACTH receptor (melanocortin 2 receptor, MC2R) or the MC2R accessory protein (MRAP) cause FGD types 1 and 2, respectively. A 2-year-old adopted Chinese girl presented with hypertonic seizures associated with hypoglycemia, skin hyperpigmentation, muscle weakness and mild jaundice. Hormonal analyses revealed high ACTH, low serum cortisol along with normal blood electrolytes. On hydrocortisone supplementation, the disease symptoms disappeared and the child recovered, although further episodes occurred with infection. To date, her physical and neurocognitive development progress is normal. A clinical diagnosis of FGD was given. We undertook MC2R and MRAP mutation screening. Two novel MC2R mutations were identified: p.D107G localized in the transmembrane region, predicted to be trafficking-competent but is unable to bind to ACTH, and p.R145C, situated in the second intracellular loop, predicted to be trafficking-defective.
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Direct quantification of the attempt frequency determining the mechanical unfolding of ubiquitin protein.
J. Biol. Chem.
PUBLISHED: 07-16-2011
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Understanding protein dynamics requires a comprehensive knowledge of the underlying potential energy surface that governs the motion of each individual protein molecule. Single molecule mechanical studies have provided the unprecedented opportunity to study the individual unfolding pathways along a well defined coordinate, the end-to-end length of the protein. In these experiments, unfolding requires surmounting an energy barrier that separates the native from the extended state. The calculation of the absolute value of the barrier height has traditionally relied on the assumption of an attempt frequency, ?(‡). Here we used single molecule force-clamp spectroscopy to directly determine the value of ?(‡) for mechanical unfolding by measuring the unfolding rate of the small protein ubiquitin at varying temperatures. Our experiments demonstrate a significant effect of the temperature on the mechanical rate of unfolding. By extrapolating the unfolding rate in the absence of force for different temperatures, varying within the range spanning from 5 to 45 °C, we measured a value for the activation barrier of ?G(‡) = 71 ± 5 kJ/mol and an exponential prefactor ?(‡) ?4 × 10(9) s(-1). Although the measured prefactor value is 3 orders of magnitude smaller than the value predicted by the transition state theory (?6 × 10(12) s(-1)), it is 400-fold higher than that encountered in analogous experiments studying the effect of temperature on the reactivity of a protein-embedded disulfide bond (?10(7) M(-1) s(-1)). This approach will allow quantitative characterization of the complete energy landscape of a folding polypeptide from highly extended states, of capital importance for proteins with elastic function.
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[Relation between myocardial infarction and circadian rhythm in patients attended in a prehospital emergency service].
Med Clin (Barc)
PUBLISHED: 06-19-2011
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The aim of this study is to analyze the presence of circadian rhythm in the time of onset of symptoms of acute myocardial infarction treated by a prehospital emergency system and the influence of modifiable cardiovascular risk factors and non-modifiable as modulators of that circadian rhythm.
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Effects of D-003 (10 mg/day) on bone mineral density of the lumbar spine and femoral neck in postmenopausal women: a randomized, double-blinded study.
Korean J. Intern. Med.
PUBLISHED: 06-01-2011
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Increased osteoclast activity is a pivotal finding in osteoporosis. This increase is mediated via the mevalonate-to-cholesterol pathway, which is involved in producing the intermediates required for osteoclast activity. D-003, a mixture of high molecular weight sugarcane wax acids, has been shown to inhibit cholesterol synthesis prior to mevalonate production, resulting in a reduction of bone loss and resorption in ovariectomized rats. Moreover, previous studies have demonstrated that short-term D-003 treatment reduces urinary excretion of deoxypyridinoline/creatinine in postmenopausal women.
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Probing osmolyte participation in the unfolding transition state of a protein.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 05-25-2011
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Understanding the molecular mechanisms of osmolyte protection in protein stability has proved to be challenging. In particular, little is known about the role of osmolytes in the structure of the unfolding transition state of a protein, the main determinant of its dynamics. We have developed an experimental protocol to directly probe the transition state of a protein in a range of osmolyte environments. We use an atomic force microscope in force-clamp mode to apply mechanical forces to the protein I27 and obtain force-dependent rate constants of protein unfolding. We measure the distance to the unfolding transition state, ?x(u), along a 1D reaction coordinate imposed by mechanical force. We find that for the small osmolytes, ethylene glycol, propylene glycol, and glycerol, ?x(u) scales with the size of the molecule, whereas for larger osmolytes, sorbitol and sucrose, ?x(u) remains the same as that measured in water. These results are in agreement with steered molecular dynamics simulations that show that small osmolytes act as solvent bridges in the unfolding transition state structure, whereas only water molecules act as solvent bridges in large osmolyte environments. These results demonstrate that novel force protocols combined with solvent substitution can directly probe angstrom changes in unfolding transition state structure. This approach creates new opportunities to gain molecular level understanding of the action of osmolytes in biomolecular processes.
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The origins, evolution, and functional potential of alternative splicing in vertebrates.
Mol. Biol. Evol.
PUBLISHED: 05-06-2011
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Alternative splicing (AS) has the potential to greatly expand the functional repertoire of mammalian transcriptomes. However, few variant transcripts have been characterized functionally, making it difficult to assess the contribution of AS to the generation of phenotypic complexity and to study the evolution of splicing patterns. We have compared the AS of 309 protein-coding genes in the human ENCODE pilot regions against their mouse orthologs in unprecedented detail, utilizing traditional transcriptomic and RNAseq data. The conservation status of every transcript has been investigated, and each functionally categorized as coding (separated into coding sequence [CDS] or nonsense-mediated decay [NMD] linked) or noncoding. In total, 36.7% of human and 19.3% of mouse coding transcripts are species specific, and we observe a 3.6 times excess of human NMD transcripts compared with mouse; in contrast to previous studies, the majority of species-specific AS is unlinked to transposable elements. We observe one conserved CDS variant and one conserved NMD variant per 2.3 and 11.4 genes, respectively. Subsequently, we identify and characterize equivalent AS patterns for 22.9% of these CDS or NMD-linked events in nonmammalian vertebrate genomes, and our data indicate that functional NMD-linked AS is more widespread and ancient than previously thought. Furthermore, although we observe an association between conserved AS and elevated sequence conservation, as previously reported, we emphasize that 30% of conserved AS exons display sequence conservation below the average score for constitutive exons. In conclusion, we demonstrate the value of detailed comparative annotation in generating a comprehensive set of AS transcripts, increasing our understanding of AS evolution in vertebrates. Our data supports a model whereby the acquisition of functional AS has occurred throughout vertebrate evolution and is considered alongside amino acid change as a key mechanism in gene evolution.
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Protease power strokes force proteins to unfold.
Cell
PUBLISHED: 05-03-2011
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ATP-dependent proteases degrade proteins in the cytosol of cells. Two recent articles, by Aubin-Tam et al. (2011) and Maillard et al. (2011 [this issue]), use single-molecule optical tweezers to show directly that these molecular machines use the energy derived from ATP hydrolysis to mechanically unfold and translocate its substrates into the proteolytic chamber.
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Direct observation of disulfide isomerization in a single protein.
Nat Chem
PUBLISHED: 04-14-2011
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Photochemical uncaging techniques use light to release active molecules from otherwise inert compounds. Here we expand this class of techniques by demonstrating the mechanical uncaging of a reactive species within a single protein. We proved this novel technique by capturing the regiospecific reaction between a thiol and a vicinal disulfide bond. We designed a protein that includes a caged cysteine and a buried disulfide. The mechanical unfolding of this protein in the presence of an external nucleophile frees the single reactive cysteine residue, which now can cleave the target disulfide via a nucleophilic attack on either one of its two sulfur atoms. This produces two different and competing reaction pathways. We used single-molecule force spectroscopy to monitor the cleavage of the disulfides, which extends the polypeptide by a magnitude unambiguously associated with each reaction pathway. This allowed us to measure, for the first time, the kinetics of disulfide-bond isomerization in a protein.
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Prevalence and risk factors of excessive daytime sleepiness in a community sample of young children: the role of obesity, asthma, anxiety/depression, and sleep.
Sleep
PUBLISHED: 04-05-2011
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We investigated the prevalence and association of excessive daytime sleepiness (EDS) with a wide range of factors (e.g., medical complaints, obesity, objective sleep [including sleep disordered breathing], and parent-reported anxiety/depression and sleep difficulties) in a large general population sample of children. Few studies have researched the prevalence and predictors of EDS in young children, none in a general population sample of children, and the results are inconsistent.
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The Spanish version of the Insomnia Severity Index: a confirmatory factor analysis.
Sleep Med.
PUBLISHED: 03-07-2011
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To examine the psychometric properties of the Spanish version of the Insomnia Severity Index (ISI) and to determine its factor structure with confirmatory factor analysis (CFA).
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Kinetic measurements on single-molecule disulfide bond cleavage.
J. Am. Chem. Soc.
PUBLISHED: 02-22-2011
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We use single-molecule force clamp spectroscopy (SMFCS) to explore the reactivity of tris(2-carboxyethyl)phosphine (TCEP), 1, 4-dl-dithiothreitol (DTT) and hydrosulfide anion (HS(-)) on disulfide bonds within a mechanically stretched polypeptide. The single-bond level bimolecular nucleophilic substitution (S(N)2) events are recorded at a series of precisely controlled temperatures so that the Arrhenius kinetic parameters, that is, the height of the activation energy barrier (E(a)) and the attempting frequency (A) of the chemical reactions, can be determined. The values of A are typically at the order of 10(7) M(-1) s(-1), which is far lower than that predicted by the transition-state theory, in which A is given by k(B)T/h and around 10(12) M(-1) s(-1) at room temperature. Furthermore, E(a) is derived to be 30-40 kJ/mol, which can be lowered by ?6-8% with every 100 pN mechanical force applied. The correlation of the A and E(a) with the molecular structures reveals that the relative magnitude of these two parameters cannot be simply judged from the size of the molecule or the nucleophilicity of the attacking atom. The comparison of the influences on the reaction rate induced by force and temperature indicates an equivalent accelerating effect by every 50 pN or 10 K increment, giving for the first time the relationship between mechanical and thermal effects on a single-molecule S(N)2 chemical reaction.
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[Knowledge of gender-based violence in the population seen in primary care].
Aten Primaria
PUBLISHED: 02-11-2011
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To determine the knowledge and attitudes towards gender-based violence in the Primary Care patient population and their relationship with sociodemographic factors and personal experience.
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Contrasting the individual reactive pathways in protein unfolding and disulfide bond reduction observed within a single protein.
J. Am. Chem. Soc.
PUBLISHED: 02-10-2011
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Identifying the dynamics of individual molecules along their reactive pathways remains a major goal of modern chemistry. For simple chemical reactions, the transition state position is thought to be highly localized. Conversely, in the case of more complex reactions involving proteins, the potential energy surfaces become rougher, resulting in heterogeneous reaction pathways with multiple transition state structures. Force-clamp spectroscopy experimentally probes the individual reaction pathways sampled by a single protein under the effect of a constant stretching force. Herein, we examine the distribution of conformations that populate the transition state of two different reactions; the unfolding of a single protein and the reduction of a single disulfide bond, both occurring within the same single protein. By applying the recently developed static disorder theory, we quantify the variance of the barrier heights, ?(2), governing each distinct reaction. We demonstrate that the unfolding of the I27 protein follows a nonexponential kinetics, consistent with a high value of ?(2) ? 18 (pN nm)(2). Interestingly, shortening of the protein upon introduction of a rigid disulfide bond significantly modulates the disorder degree, spanning from ?(2) ? 8 to ?21 (pN nm)(2). These results are in sharp contrast with the exponential distribution of times measured for an S(N)2 chemical reaction, implying the absence of static disorder ?(2) ? 0 (pN nm)(2). Our results demonstrate the high sensitivity of the force-clamp technique to capture the signatures of disorder in the individual pathways that define two distinct force-induced reactions, occurring within the core of a single protein.
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Single-molecule paleoenzymology probes the chemistry of resurrected enzymes.
Nat. Struct. Mol. Biol.
PUBLISHED: 01-24-2011
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It is possible to travel back in time at the molecular level by reconstructing proteins from extinct organisms. Here we report the reconstruction, based on sequence predicted by phylogenetic analysis, of seven Precambrian thioredoxin enzymes (Trx) dating back between ~1.4 and ~4 billion years (Gyr). The reconstructed enzymes are up to 32 °C more stable than modern enzymes, and the oldest show markedly higher activity than extant ones at pH 5. We probed the mechanisms of reduction of these enzymes using single-molecule force spectroscopy. From the force dependency of the rate of reduction of an engineered substrate, we conclude that ancient Trxs use chemical mechanisms of reduction similar to those of modern enzymes. Although Trx enzymes have maintained their reductase chemistry unchanged, they have adapted over 4 Gyr to the changes in temperature and ocean acidity that characterize the evolution of the global environment from ancient to modern Earth.
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Evaluation of anti-inflammatory and antinociceptive effects of D-002 (beeswax alcohols).
J Nat Med
PUBLISHED: 01-22-2011
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D-002, a mixture of six higher aliphatic alcohols purified from beeswax, displayed anti-inflammatory effects in carrageenan-induced pleurisy and cotton pellet granuloma in rats. The aim of the present study was to confirm the anti-inflammatory properties of D-002 and to explore its potential analgesic effects. Xylene-induced mouse ear oedema was used to assess the anti-inflammatory effect, acetic acid-induced writhing and hot plate responses for the analgesic activity, and the open field and horizontal rotarod tests for motor performance. For anti-inflammatory tests, mice were randomised into a negative vehicle control and five xylene-treated groups: the vehicle, D-002 (25, 50 and 200 mg/kg) and indomethacin 1 mg/kg (reference drug). Treatments were given for 15 days. Effects on oedema formation and myeloperoxidase (MPO) activity were tested. For analgesia and motor performance tests, mice were randomised into a vehicle control and D-002-treated groups (25, 50 and 200 mg/kg). Two sets of experiments were done, which included acute and repeat (15 days) dosing. D-002 (25, 50 and 200 mg/kg) significantly decreased xylene-induced ear oedema (44.7, 60.8 and 76.4%, respectively) and the increase of MPO activity induced by xylene (38.0, 47.0 and 57.0%, respectively), while indomethacin significantly inhibited xylene-induced oedema (59.9%) and MPO activity (57.5%). Single and repeat doses of D-002 (25, 50 and 200 mg/kg) decreased the acetic acid-induced writhing responses by 21.2, 28.2 and 40.1%, for the single doses; 25.2, 35.1 and 43.2%, respectively, for the repeat doses, but did not affect the hot plate, open field and rotarod behaviours. Aspirin 100 mg/kg significantly decreased acetic acid-induced abdominal constrictions and morphine (5 mg/kg) significantly increased the latency of the hot plate response. This study confirmed the anti-inflammatory effects of D-002 and demonstrated its analgesic effects on the acetic acid-induced writhing, but not on the hot plate response, which suggests that the antinociceptive effects of D-002 could be related to its anti-inflammatory activity.
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SAIL--a software system for sample and phenotype availability across biobanks and cohorts.
Bioinformatics
PUBLISHED: 12-17-2010
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The Sample avAILability system-SAIL-is a web based application for searching, browsing and annotating biological sample collections or biobank entries. By providing individual-level information on the availability of specific data types (phenotypes, genetic or genomic data) and samples within a collection, rather than the actual measurement data, resource integration can be facilitated. A flexible data structure enables the collection owners to provide descriptive information on their samples using existing or custom vocabularies. Users can query for the available samples by various parameters combining them via logical expressions. The system can be scaled to hold data from millions of samples with thousands of variables.
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Characterization of activity epochs in actimetric registries for infantile colic diagnosis: Identification and feature extraction based on wavelets and symbolic dynamics.
Conf Proc IEEE Eng Med Biol Soc
PUBLISHED: 11-25-2010
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The diagnosis and therapy planning of high prevalence pathologies such as infantile colic can be substantially improved by statistical signal processing of activity/rest registries. Assuming that colic episodes are associated to activity episodes, diagnosis aid systems should be based on preprocessing techniques able to separate real activity from rest epochs, and feature extraction methods to identify meaningful indices with diagnostic capabilities. In this paper, we propose a two step diagnosis aid methodology for infantile colic in children below 3 months old. Identification of activity periods is performed by means of a wavelet based activity filter which does not depend on the acquisition device (as so far proposed methods do). In addition, symbolic dynamic analysis is used for extraction of discriminative indices from the activity time series. Results on real data yielded 100% sensitivity and 80% specificity in a study group composed of 46 cases and 10 control subjects.
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High-nuclearity Pt-Tl-Fe complexes: structural, electrochemistry, and spectroelectrochemistry studies.
Inorg Chem
PUBLISHED: 11-17-2010
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A series of heteropolynuclear Pt-Tl-Fe complexes have been synthesized and structurally characterized. The final structures strongly depend on the geometry of the precursor and the Pt/Tl ratio used. Thus, the anionic heteroleptic cis-configured [cis-Pt(C(6)F(5))(2)(C?CFc)(2)](2-) and [Pt(bzq)(C?CFc)(2)](-) (Fc = ferrocenyl) complexes react with Tl(+) to form discrete octanuclear (PPh(3)Me)(2)[{trans,cis,cis-PtTl(C(6)F(5))(2)(C?CFc)(2)}(2)] (1), [PtTl(bzq)(C?CFc)(2)](2) (5; bzq = benzoquinolate), and decanuclear [trans,cis,cis-PtTl(2)(C(6)F(5))(2)(C?CFc)(2)](2) (3) derivatives, stabilized by both Pt(II)···Tl(I) and Tl(I)···?(2)(alkynyl) bonds. By contrast, Q(2)[trans-Pt(C(6)F(5))(2)(C?CFc)(2)] (Q = NBu(4)) reacts with Tl(+) to give the one-dimensional (1-D) anionic [(NBu(4)){trans,trans,trans-PtTl(C(6)F(5))(2)(C?CFc)(2)}](n) (2) and neutral [trans,trans,trans-PtTl(2)(C(6)F(5))(2)(C?CFc)(2)](n) (4) polymeric chains based on [PtFc(2)](2-) platinate fragments and Tl(+) (2) or [Tl···Tl](2+) (4) units, respectively, connected by Pt(II)···Tl(I) and secondary weak ?-?(1) (2) or ?(2) (4) alkynyl···Tl(I) bonding. The formation of 1-4 is reversible, and thus treatment of neutral 3 and 4 with PPh(3)MeBr causes the precipitation of TlBr, returning toward the formation of the anionic 1 and 2 (Q = PPh(3)Me). Two slightly different pseudopolymorphs were found for 2, depending on the crystallization solvent. Finally, the reaction of the homoleptic [Pt(C?CFc)(4)](2-) with 2 equiv of Tl(+) affords the tetradecanuclear sandwich type complex [Pt(2)Tl(4)(C?CFc)(8)] (6). Electrochemical, spectroelectrochemical, and theoretical studies have been carried out to elucidate the effect produced by the interaction of the Tl(+) with the Pt-C?CFc fragments. The cyclic voltammetry (CV) and differential pulse voltammetry (DPV) of 1-5 reveal that, in general, neutralization of the anionic fragments increases the stability of the fully oxidized species and gives higher E(1/2) (Fc) values than those observed in their precursors, increasing with the number of Pt-Tl bonding interactions. However, the electronic communication between Fc groups is reduced or even lost upon Tl(+) coordination, as confirmed by electrochemical (CVs and DPVs voltammograms, 1-5) and spectroelectrochemical (UV-vis-NIR, 2-4) studies. Complexes 2 and 4 still display some electronic interaction between the Fc groups, supported by the presence of an IVCT band in their UV-vis-NIR spectra of oxidized species and additional comparative DFT calculations with the precursor [trans-Pt(C(6)F(5))(2)(C?CFc)(2)](2-) and complex 3.
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Sleep misperception and chronic insomnia in the general population: role of objective sleep duration and psychological profiles.
Psychosom Med
PUBLISHED: 10-26-2010
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To examine the role of objective sleep duration, a novel marker in phenotyping insomnia, and psychological profiles on sleep misperception in a large, general population sample. Sleep misperception is considered by some investigators a common characteristic of chronic insomnia, whereas others propose it as a separate diagnosis. The frequency and the determinants of sleep misperception in general population samples are unknown.
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Waters role in the force-induced unfolding of ubiquitin.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 10-25-2010
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In atomic force spectroscopic studies of the elastomeric protein ubiquitin, the ?-strands 1-5 serve as the force clamp. Simulations show how the rupture force in the force-induced unfolding depends on the kinetics of water molecule insertion into positions where they can eventually form hydrogen bonding bridges with the backbone hydrogen bonds in the force-clamp region. The intrusion of water into this region is slowed down by the hydrophobic shielding effect of carbonaceous groups on the surface residues of ?-strands 1-5, which thereby regulates water insertion prior to hydrogen bond breakage. The experiments show that the unfolding of the mechanically stressed protein is nonexponential due to static disorder. Our simulations show that different numbers and/or locations of bridging water molecules give rise to a long-lived distribution of transition states and static disorder. We find that slowing down the translational (not rotational) motions of the water molecules by increasing the mass of their oxygen atoms, which leaves the force field and thereby the equilibrium structure of the solvent unchanged, increases the average rupture force; however, the early stages of the force versus time behavior are very similar for our "normal" and fictitious "heavy" water models. Finally, we construct six mutant systems to regulate the hydrophobic shielding effect of the surface residues in the force-clamp region. The mutations in the two termini of ?-sheets 1-5 are found to determine a preference for different unfolding pathways and change mutants average rupture force.
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Hopping around an entropic barrier created by force.
Biochem. Biophys. Res. Commun.
PUBLISHED: 10-11-2010
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We use Langevin dynamics to investigate the role played by the recently discovered force-induced entropic energy barrier on the two-state hopping phenomena that has been observed in single RNA, DNA and protein molecules placed under a stretching force. Simple considerations about the free energy of a molecule readily show that the application of force introduces an entropic barrier separating the collapsed state of the molecule, from a force-driven extended conformation. A notable characteristic of the force induced barrier is its long distances to transition state, up to tens of nanometers, which renders the kinetics of crossing this barrier highly sensitive to an applied force. Langevin dynamics across such force induced barriers readily demonstrates the hopping behavior observed for a variety of single molecules placed under force. Such hopping is frequently interpreted as a manifestation of two-state folding/unfolding reactions observed in bulk experiments. However, given that such barriers do not exist at zero force these reactions do not take place at all in bulk.
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Transsphenoidal surgery in patients with acromegaly: operative strategies for overcoming technically challenging anatomical variations.
Neurosurg Focus
PUBLISHED: 10-05-2010
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In addition to difficulties with anesthetic and medical management, transsphenoidal operations in patients with longstanding acromegaly are associated with inherent intraoperative challenges because of anatomical variations that occur frequently in these patients. The object of this study was to review the overall safety profile and anatomical/technical challenges associated with transsphenoidal surgery in patients with acromegaly.
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Insomnia with short sleep duration and mortality: the Penn State cohort.
Sleep
PUBLISHED: 09-23-2010
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Because insomnia with objective short sleep duration is associated with increased morbidity, we examined the effects of this insomnia subtype on all-cause mortality.
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Metabolomic approach to the nutraceutical effect of rosemary extract plus ?-3 PUFAs in diabetic children with capillary electrophoresis.
J Pharm Biomed Anal
PUBLISHED: 07-02-2010
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Type 1 diabetes mellitus is a major endocrine disorder, affecting approximately 5% of the worlds population. It not only leads to hyperglycaemia but also causes many complications, and numerous studies have demonstrated that oxidative stress contributes to these complications. As a new strategy to improve the oxidative damage in diabetes, interest has grown in the usage of natural antioxidants, even more in the long term. Among them, Rosmarinus officinalis (rosemary) has been widely accepted as one of the species with the highest antioxidant activity. In addition, omega-3 polyunsaturated fatty acids were efficient in delaying and decreasing cardiovascular risk factors associated with diabetes. Type 1 diabetic children and the corresponding controls were enrolled in the assay. The aim was evaluating the effect of a special additive containing rosemary extract, vitamin E and PUFAs added to their standard diet through the meat. In the analytical point of view, a metabolomic approach with CE-UV was used to detect possible differences in urine of diabetic children as compared to controls. After the application of the appropriate multivariate statistical tools, clear differences could be observed between treated and non-treated diabetic children and some of the metabolites associated could be identified. This was specially challenging as most of the clinical biochemical parameters measured by target analysis showed no differences between the groups.
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Probing static disorder in Arrhenius kinetics by single-molecule force spectroscopy.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 06-08-2010
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The widely used Arrhenius equation describes the kinetics of simple two-state reactions, with the implicit assumption of a single transition state with a well-defined activation energy barrier DeltaE, as the rate-limiting step. However, it has become increasingly clear that the saddle point of the free-energy surface in most reactions is populated by ensembles of conformations, leading to nonexponential kinetics. Here we present a theory that generalizes the Arrhenius equation to include static disorder of conformational degrees of freedom as a function of an external perturbation to fully account for a diverse set of transition states. The effect of a perturbation on static disorder is best examined at the single-molecule level. Here we use force-clamp spectroscopy to study the nonexponential kinetics of single ubiquitin proteins unfolding under force. We find that the measured variance in DeltaE shows both force-dependent and independent components, where the force-dependent component scales with F(2), in excellent agreement with our theory. Our study illustrates a novel adaptation of the classical Arrhenius equation that accounts for the microscopic origins of nonexponential kinetics, which are essential in understanding the rapidly growing body of single-molecule data.
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Role of conservative mutations in protein multi-property adaptation.
Biochem. J.
PUBLISHED: 05-08-2010
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Protein physicochemical properties must undergo complex changes during evolution, as a response to modifications in the organism environment, the result of the proteins taking up new roles or because of the need to cope with the evolution of molecular interacting partners. Recent work has emphasized the role of stability and stability-function trade-offs in these protein adaptation processes. In the present study, on the other hand, we report that combinations of a few conservative, high-frequency-of-fixation mutations in the thioredoxin molecule lead to largely independent changes in both stability and the diversity of catalytic mechanisms, as revealed by single-molecule atomic force spectroscopy. Furthermore, the changes found are evolutionarily significant, as they combine typically hyperthermophilic stability enhancements with modulations in function that span the ranges defined by the quite different catalytic patterns of thioredoxins from bacterial and eukaryotic origin. These results suggest that evolutionary protein adaptation may use, in some cases at least, the potential of conservative mutations to originate a multiplicity of evolutionarily allowed mutational paths leading to a variety of protein modulation patterns. In addition the results support the feasibility of using evolutionary information to achieve protein multi-feature optimization, an important biotechnological goal.
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Rhomboidal heterometallic alkynyl based Pt2Cd2 clusters: structural, photophysical, and theoretical studies.
Inorg Chem
PUBLISHED: 04-13-2010
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Reactions between [Pt(C[triple bond]CR)(4)](2-) (R = Tol a, C(6)H(4)OMe-4 b, C(6)H(4)OMe-3 c) and Cd(2+) depend on the media and the alkynyl substituent, leading to the formation of yellow tetranuclear solvate complexes [Pt(C[triple bond]CR)(4)Cd(acetone)](2) 1a,b(acetone)(2) and [Pt(C[triple bond]CC(6)H(4)OMe-3)(4)Cd(dmso)](2) 1c(dmso)(2) or white polymeric solvate-free species [Pt(C[triple bond]CR)(4)Cd](x) 1a-c. Treatment of 1a,b(acetone)(2) or 1a-c with N-donor ligands affords a series of tetranuclear clusters [Pt(C[triple bond]CR)(4)CdL](2) (L = py; 2a-c. R = Tol; L = NC(5)H(4)CH(3)-4 3, NC(5)H(4)CF(3)-4 4, pzH 5). X-ray crystallographic studies reveal that, in the tolyl complexes (2a, 4, and 5), the Cd-L(2+) unit is closely bonded to one Pt-C(alpha)(acetylide) bond (Pt-Cd = 2.7, Cd-C(alpha) approximately 2.48 A), and the resulting "Pt(C[triple bond]CTol)(4)CdL" unit dimerizes by two additional eta(2)-Cd-acetylide and a weaker Pt...Cd bonding interaction leading to a planar unsymmetrical rhomboidal metal core. By contrast, the m-methoxyphenyl derivatives (2c, 1c(dmso)(2)) form symmetrical Pt(2)Cd(2) cores, with each Cd bonded (coordination number, C.N. = 5) to the incoming ligand (pyridine 2c, dmso 1c(dmso)(2)) and four Pt-C(alpha) bonds (Pt-Cd approximately 2.85; Cd-C(alpha) 2.470(10)-2.551(5) A) of different Pt(II) fragments. Evidence of ligand dissociation was found for the solvate (1a,b(acetone)(2), 1c(dmso)(2)) and NC(5)H(4)CF(3)-4 (4) derivatives by NMR and UV-vis absorption spectra. All tetranuclear aggregates exhibit bright blue to green luminescence in the solid state. Time-dependent density functional theory (TD-DFT) calculations were performed to shed light on the nature of the electronic transitions. In the solvate 1a,b(acetone)(2) and 1c(dmso)(2), emissions have been assigned to a platinum-alkynyl to cadmium charge transfer ((3)MLMCT), mixed with some intraligand (3)IL(C[triple bond]CR) character. In the imine derivatives 2-5, they are suggested to come from an excited state of large Pt(d)/piC[triple bond]CR-->pi*(imine) MLLCT character, mixed with some Pt(d)/pi(C[triple bond]CR)-->Pt(2)Cd(2)/pi*C[triple bond]CR (MLMCT) contribution.
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Single-molecule force spectroscopy approach to enzyme catalysis.
J. Biol. Chem.
PUBLISHED: 04-09-2010
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Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.
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Cognitive-emotional hyperarousal as a premorbid characteristic of individuals vulnerable to insomnia.
Psychosom Med
PUBLISHED: 04-05-2010
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To examine whether cognitive-emotional hyperarousal is a premorbid characteristic of middle-aged and young good sleepers vulnerable to stress-related insomnia.
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Collapse dynamics of single proteins extended by force.
Biophys. J.
PUBLISHED: 02-17-2010
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Single-molecule force spectroscopy has opened up new approaches to the study of protein dynamics. For example, an extended protein folding after an abrupt quench in the pulling force was shown to follow variable collapse trajectories marked by well-defined stages that departed from the expected two-state folding behavior that is commonly observed in bulk. Here, we explain these observations by developing a simple approach that models the free energy of a mechanically extended protein as a combination of an entropic elasticity term and a short-range potential representing enthalpic hydrophobic interactions. The resulting free energy of the molecule shows a force-dependent energy barrier of magnitude, DeltaE =epsilon(F - F(c))(3/2), separating the enthalpic and entropic minima that vanishes at a critical force F(c). By solving the Langevin equation under conditions of a force quench, we generate folding trajectories corresponding to the diffusional collapse of an extended polypeptide. The predicted trajectories reproduce the different stages of collapse, as well as the magnitude and time course of the collapse trajectories observed experimentally in ubiquitin and I27 protein monomers. Our observations validate the force-clamp technique as a powerful approach to determining the free-energy landscape of proteins collapsing and folding from extended states.
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Isopeptide bonds block the mechanical extension of pili in pathogenic Streptococcus pyogenes.
J. Biol. Chem.
PUBLISHED: 02-05-2010
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In the early stages of an infection, pathogenic bacteria use long fibrous structures known as pili as adhesive anchors for attachment to the host cells. These structures also play key roles in colony and biofilm formation. In all those processes, pili must withstand large mechanical forces. The pili of the nasty gram-positive human pathogen Streptococcus pyogenes are assembled as single, micrometer long tandem modular proteins of covalently linked repeats of pilin proteins. Here we use single molecule force spectroscopy techniques to study the mechanical properties of the major pilin Spy0128. In our studies, we engineer polyproteins containing repeats of Spy0128 flanked by the well characterized I27 protein which provides an unambiguous mechanical fingerprint. We find that Spy0128 is an inextensible protein, even when pulled at forces of up to 800 pN. We also found that this remarkable mechanical resilience, unique among the modular proteins studied to date, results from the strategically located intramolecular isopeptide bonds recently identified in the x-ray structure of Spy0128. Removal of the isopeptide bonds by mutagenesis readily allowed Spy0128 domains to unfold and extend, albeit at relatively high forces of 172 pN (N-terminal domain) or 250 pN (C-terminal domain). Our results show that in contrast to the elastic roles played by large tandem modular proteins such as titin and fibronectin, the giant pili of S. pyogenes evolved to abrogate mechanical extensibility, a property that may be crucial in the pathogenesis of this most virulent bacterium and, therefore, become the target of new therapeutic approaches against its infections.
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The role of different hyaluronic acids in the articular cartilage of rabbit.
Open Orthop J
PUBLISHED: 01-19-2010
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To elucidate if the differences found in the physico-chemical and rheological behaviour of Hyaluronic Acids result in different in vivo activity. For this purpose two Hyaluronic Acids (HA), HA-1 and HA-2, with similar molecular weight but different percentage of concentration variation, were compared through an osteoarthritis model.
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