The metal-binding properties of the blue crab copper specific CuMT-2: a crustacean metallothionein with two cysteine triplets

Most crustacean metallothioneins (MTs) contain 18 Cys residues and bind six divalent metal ions. The copper-specific CuMT-2 (MTC) of the blue crab Callinectes sapidus with 21Cys residues, of which six are organized in two uncommon Cys-Cys-Cys sequences, represents an exception. However, its metal-binding properties are unknown. By spectroscopic and spectrometric techniques we show that all 21 Cys residues of recombinant MTC participate in the binding of Cu(I), Zn(II), and Cd(II) ions, indicating that both Cys triplets act as ligands. The fully metallated M8 II-MTC (MisZn, Cd) form possesses high- and low-affinity metal binding sites, as evidenced by the formation of Zn6-MTC and Cd7-MTC species from M8 II-MTC after treatment with Chelex 100. The NMR characterization of Cd7-MTC suggests the presence of a two-domain structure, each domain containing one Cys triplet and encompassing either the three-metal or the four-metal thiolate cluster. Whereas the metal-Cys connectivities in the three-metal cluster located in the N-terminal domain (residues 1-31) reveal a Cd3Cys9 cyclohexane-like structure, the presence of dynamic processes in the C-terminal domain (residues 32-64) precluded the determination of the organization of the four-metal cluster. Absorption and circular dichroism features accompanying the stepwise binding of Cu(I) to MTC suggest that all 21Cys are involved in the binding of eight to nine Cu(I) ions (Cu8-9-MTC). The subsequent generation of Cu12-MTC involves structural changes consistent with a decrease in the Cu(I) coordination number. Overall, the metal-binding properties of MTC reported here contribute to a better understanding of the role of Cys triplets in MT

Unintentional weight loss: Clinical characteristics and outcomes in a prospective cohort of 2677 patients.

Background Whereas there are numerous studies on unintentional weight loss (UWL), these have been limited by small sample sizes, short or variable follow‐up, and focus on older patients. Although some case series have revealed that malignancies escaping early detection and uncovered subsequently are exceptional, reported follow-ups have been too short or unspecified and necropsies seldom made. Our objective was to examine the etiologies, characteristics, and long-term outcome of UWL in a large cohort of outpatients. Methods We prospectively enrolled patients referred to an outpatient diagnosis unit for evaluation of UWL as a dominant or isolated feature of disease. Eligible patients underwent a standard baseline evaluation with laboratory tests and chest X-ray. Patients without identifiable causes 6 months after presentation underwent a systematic follow-up lasting for 60 further months. Subjects aged ≥65 years without initially recognizable causes underwent an oral cavity examination, a videofluoroscopy or swallowing study, and a depression and cognitive assessment. Results Overall, 2677 patients (mean age, 64.4 [14.7] years; 51% males) were included. Predominant etiologies were digestive organic disorders (nonmalignant in 17% and malignant in 16%). Psychosocial disorders explained 16% of cases. Oral disorders were second to nonhematologic malignancies as cause of UWL in patients aged ≥65 years. Although 375 (14%) patients were initially diagnosed with unexplained UWL, malignancies were detected in only 19 (5%) within the first 28 months after referral. Diagnosis was established at autopsy in 14 cases. Conclusion This investigation provides new information on the relevance of follow-up in the long-term clinical outcome of patients with unexplained UWL and on the role of age on this entity. Although unexplained UWL seldom constitutes a short-term medical alert, malignancies may be undetectable until death. Therefore, these patients should be followed up regularly (eg yearly visits) for longer than reported periods, and autopsies pursued when facing unsolved deaths

Time to diagnosis and associated costs of an outpatient vs inpatient setting in the diagnosis of lymphoma: a retrospective study of a large cohort of major lymphoma subtypes in Spain

Background: Mainly because of the diversity of clinical presentations, diagnostic delays in lymphoma can be excessive. The time spent in primary care before referral to the specialist may be relatively short compared with the interval between hospital appointment and diagnosis. Although studies have examined the diagnostic intervals and referral patterns of patients with lymphoma, the time to diagnosis of outpatient compared to inpatient settings and the costs incurred are unknown. Methods: We performed a retrospective study at two academic hospitals to evaluate the time to diagnosis and associated costs of hospital-based outpatient diagnostic clinics or conventional hospitalization in four representative lymphoma subtypes. The frequency, clinical and prognostic features of each lymphoma subtype and the activities of the two settings were analyzed. The costs incurred during the evaluation were compared by microcosting analysis. Results: A total of 1779 patients diagnosed between 2006 and 2016 with classical Hodgkin, large B-cell, follicular, and mature nodal peripheral T-cell lymphomas were identified. Clinically aggressive subtypes including large B-cell and peripheral T-cell lymphomas were more commonly diagnosed in inpatients than in outpatients (39.1 vs 31.2% and 18.9 vs 13.5%, respectively). For each lymphoma subtype, inpatients were older and more likely than outpatients to have systemic symptoms, worse performance status, more advanced Ann Arbor stages, and high-risk prognostic scores. The admission time for diagnosis (i.e. from admission to excisional biopsy) of inpatients was significantly shorter than the time to diagnosis of outpatients (12.3 [3.3] vs 16.2 [2.7] days; P < .001). Microcosting revealed a mean cost of (sic)4039.56 (513.02) per inpatient and of (sic)1408.48 (197.32) per outpatient, or a difference of (sic)2631.08 per patient. Conclusions: Although diagnosis of lymphoma was quicker with hospitalization, the outpatient approach seems to be cost-effective and not detrimental. Despite the considerable savings with the latter approach, there may be hospitalization-associated factors which may not be properly managed in an outpatient unit (e.g. aggressive lymphomas with severe symptoms) and the cost analysis did not account for this potentially added value. While outcomes were not analyzed in this study, the impact on patient outcome of an outpatient vs inpatient diagnostic setting may represent a challenging future research

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Genetic analysis of high bone mass cases from the BARCOS cohort of spanish postmenopausal women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Genetic Analysis of High Bone Mass Cases from the BARCOS Cohort of Spanish Postmenopausal Women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM

Genetic analysis of high bone mass cases from the BARCOS cohort of spanish postmenopausal women

The aims of the study were to establish the prevalence of high bone mass (HBM) in a cohort of Spanish postmenopausal women (BARCOS) and to assess the contribution of LRP5 and DKK1 mutations and of common bone mineral density (BMD) variants to a HBM phenotype. Furthermore, we describe the expression of several osteoblast-specific and Wnt-pathway genes in primary osteoblasts from two HBM cases. A 0.6% of individuals (10/1600) displayed Z-scores in the HBM range (sum Z-score >4). While no mutation in the relevant exons of LRP5 was detected, a rare missense change in DKK1 was found (p.Y74F), which cosegregated with the phenotype in a small pedigree. Fifty-five BMD SNPs from Estrada et al. [NatGenet 44:491-501,2012] were genotyped in the HBM cases to obtain risk scores for each individual. In this small group of samples, Z-scores were found inversely related to risk scores, suggestive of a polygenic etiology. There was a single exception, which may be explained by a rare penetrant genetic variant, counterbalancing the additive effect of the risk alleles. The expression analysis in primary osteoblasts from two HBM cases and five controls suggested that IL6R, DLX3, TWIST1 and PPARG are negatively related to Z-score. One HBM case presented with high levels of RUNX2, while the other displayed very low SOX6. In conclusion, we provide evidence of lack of LRP5 mutations and of a putative HBM-causing mutation in DKK1. Additionally, we present SNP genotyping and expression results that suggest additive effects of several genes for HBM