Monitoring SARS-CoV-2 genetic variability: A post-market surveillance workflow for combined bioinformatic and laboratory evaluation of commercial RT-PCR assay performance.

OBJECTIVE: The speed at which Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) is mutating has made it necessary to frequently assess how these genomic changes impact the performance of diagnostic real-time polymerase chain reaction (RT-PCR) assays. Herein, we describe a generic three-step workflow to assess the effect of genomic mutations on inclusivity and sensitivity of RT-PCR assays. METHODS: Sequences collected from the Global Initiative on Sharing All Influenza Data (GISAID) were mapped to a SARS-CoV-2 reference genome to evaluate the position and prevalence of mismatches in the oligonucleotide-binding sites of the QIAstat-Dx, an RT-PCR panel designed to detect SARS-CoV-2. The frequency of mutations and their impact on melting temperature were assessed, and sequences flagged by risk-based criteria were examined in vitro. RESULTS: Out of 8,900,393 SARS-CoV-2 genome sequences analyzed, only 173 (0.0019%) genomes contained potentially critical mutations for the QIAstat-Dx; follow-up in-vitro testing confirmed no impact on the assays' performance. CONCLUSIONS: The current study demonstrates that SARS-CoV-2 genetic variants do not affect the performance of the QIAstat-Dx device. It is recommended that manufacturers incorporate this workflow into obligatory post-marketing surveillance activities, as this approach could potentially enhance genetic monitoring of their product.

Evaluation of the QIAstat-Dx Meningitis/Encephalitis Panel, a multiplex PCR platform for the detection of community-acquired meningoencephalitis.

Rapid identification of the causative pathogens of central nervous system infections is essential for providing appropriate management and improving patient outcomes. The performance of QIAstat-Dx Meningitis/Encephalitis (ME) Panel-a multiplex PCR testing platform-in detecting pathogens implicated in meningitis and/or encephalitis was evaluated using BioFire FilmArray ME Panel as a comparator method. This multicenter study analyzed 585 retrospective residual cerebrospinal fluid specimens and 367 contrived specimens. The QIAstat-Dx ME Panel showed positive percent agreement (PPA) values of 100% for Neisseria meningitidis, Streptococcus agalactiae, Escherichia coli K1, Listeria monocytogenes, and Cryptococcus gattii/neoformans on clinical samples compared to the BioFire FilmArray ME Panel. The PPA values observed for Haemophilus influenzae and Streptococcus pneumoniae were 80% and 88.24%, respectively. Negative percent agreement (NPA) values were >99.0% for each of the six bacterial targets and one fungal target tested with clinical samples. One viral target, herpes simplex virus 1, exhibited a PPA value of 100.0%, while the remaining viral targets-human parechovirus, herpes simplex virus 2, human herpes virus 6, and varicella zoster virus-were >90.0%, with the exception of enterovirus, which had a PPA value of 77.8%. The QIAstat-Dx ME Panel detected five true-positive and four true-negative cases compared to BioFire FilmArray ME Panel. The NPA values for all viral pathogens were >99.0%. Overall, the QIAstat-Dx ME Panel showed comparable performance to the BioFire FilmArray ME Panel as a rapid diagnostic tool for community-acquired meningitis and encephalitis.

Systematic Review on the Correlation Between SARS-CoV-2 Real-Time PCR Cycle Threshold Values and Epidemiological Trends.

BACKGROUND: The ability to proactively predict the epidemiological dynamics of infectious diseases such as coronavirus disease 2019 (COVID-19) would facilitate efficient public health responses and may help guide patient management. Viral loads of infected people correlate with infectiousness and, therefore, could be used to predict future case rates. AIM: In this systematic review, we determine whether there is a correlation between severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) real-time reverse-transcription polymerase chain reaction (RT-PCR) cycle threshold (Ct) values (a proxy for viral load) and epidemiological trends in patients diagnosed with COVID-19, and whether Ct values are predictive of future cases. METHODS: A PubMed search was conducted on August 22 2022, based on a search strategy of studies reporting correlations between SARS-CoV-2 Ct values and epidemiological trends. RESULTS: Data from 16 studies were relevant for inclusion. RT-PCR Ct values were measured from national (n = 3), local (n = 7), single-unit (n = 5), or closed single-unit (n = 1) samples. All studies retrospectively examined the correlation between Ct values and epidemiological trends, and seven evaluated their prediction model prospectively. Five studies used the temporal reproduction number (Rt) as the measure of the population/epidemic growth rate. Eight studies reported a prediction time in the negative cross-correlation between Ct values and new daily cases, with seven reporting a prediction time of ~1-3 weeks, and one reporting 33 days. CONCLUSION: Ct values are negatively correlated with epidemiological trends and may be useful in predicting subsequent peaks in variant waves of COVID-19 and other circulating pathogens.

Ct values as a diagnostic tool for monitoring SARS-CoV-2 viral load using the QIAstat-Dx® Respiratory SARS-CoV-2 Panel.

OBJECTIVES: Qualitative real-time polymerase chain reaction tests are not designed to provide quantitative or semiquantitative results because cycle threshold (Ct) values are not normalized to standardized controls of known concentration. The aim of this study was to characterize SARS-CoV-2 viral loads based on Ct values, using the QIAstat-Dx® Respiratory SARS-CoV-2 Panel. METHODS: Different lineages of SARS-CoV-2 clinical samples and the World Health Organization international standard were used to assess the linearity of the QIAstat-Dx Respiratory SARS-CoV-2 Panel. Limit of detection for the different lineages was characterized. RESULTS: Comparable efficiencies and linearity for all samples resulted in R2 ≥0.99, covering a dynamic range of 1,000,000-100 copies/mL for the SARS-CoV-2 assay, showing linear correlation between Ct values and viral load down to 300 copies/mL. CONCLUSION: The SARS-CoV-2 Ct values provided by the QIAstat-Dx® Respiratory SARS-CoV-2 Panel could be used as a surrogate for viral load given the linear correlation between Ct values and viral concentration down to limit of detection. This panel allows to obtain reproducible Ct values for SARS-CoV-2 ribonucleic acid downstream of the sample collection, reducing the sample-to-Ct workflow variability. Ct values can help provide a reliable assessment and comparison of viral loads in patients when tested with the QIAstat-Dx Respiratory SARS-CoV-2 Panel.

RRM1 variants cause a mitochondrial DNA maintenance disorder via impaired de novo nucleotide synthesis.

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Multiple assays in a real-time RT-PCR SARS-CoV-2 panel can mitigate the risk of loss of sensitivity by new genomic variants during the COVID-19 outbreak.

OBJECTIVES: In this study, five SARS-CoV-2 PCR assay panels were evaluated against the accumulated genetic variability of the virus to assess the effect on sensitivity of the individual assays. DESIGN OR METHODS: As of week 21, 2020, the complete set of available SARS-CoV-2 genomes from GISAID and GenBank databases were used in this study. SARS-CoV-2 primer sequences from publicly available panels (WHO, CDC, NMDC, and HKU) and QIAstat-Dx were included in the alignment, and accumulated genetic variability affecting any oligonucleotide annealing was annotated. RESULTS: A total of 11,627 (34.38%) genomes included single mutations affecting annealing of any PCR assay. Variations in 8,773 (25.94%) genomes were considered as high risk, whereas additional 2,854 (8.43%) genomes presented low frequent single mutations and were predicted to yield no impact on sensitivity. In case of the QIAstat-Dx SARS-CoV-2 Panel, 99.11% of the genomes matched with a 100% coverage all oligonucleotides, and critical variations were tested in vitro corroborating no loss of sensitivity. CONCLUSIONS: This analysis stresses the importance of targeting more than one region in the viral genome for SARS-CoV-2 detection to mitigate the risk of loss of sensitivity due to the unknown mutation rate during this SARS-CoV-2 outbreak.

Bioavailability and cytosolic kinases modulate response to deoxynucleoside therapy in TK2 deficiency.

BACKGROUND: TK2 is a nuclear gene encoding the mitochondrial matrix protein thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial nucleotide salvage pathway. Deficiency of TK2 activity causes mitochondrial DNA (mtDNA) depletion, which in humans manifests predominantly as a mitochondrial myopathy with onset typically in infancy and childhood. We previously showed that oral treatment of the Tk2 H126N knock-in mouse model (Tk2-/-) with the TK2 substrates, deoxycytidine (dCtd) and thymidine (dThd), delayed disease onset and prolonged median survival by 3-fold. Nevertheless, dCtd + dThd treated Tk2-/- mice showed mtDNA depletion in brain as early as postnatal day 13 and in virtually all other tissues at age 29 days. METHODS: To enhance mechanistic understanding and efficacy of dCtd + dThd therapy, we studied the bioavailability of dCtd and dThd in various tissues as well as levels of the cytosolic enzymes, TK1 and dCK that convert the deoxynucleosides into dCMP and dTMP. FINDINGS: Parenteral treatment relative to oral treatment produced higher levels of dCtd and dThd and improved mtDNA levels in liver and heart, but did not ameliorate molecular defects in brain or prolong survival. Down-regulation of TK1 correlated with temporal- and tissue-specificity of response to dCtd + dThd. Finally, we observed in human infant and adult muscle expression of TK1 and dCK, which account for the long-term efficacy to dCtd + dThd therapy in TK2 deficient patients. INTERPRETATIONS: These data indicate that the cytosolic pyrimidine salvage pathway enzymes TK1 and dCK are critical for therapeutic efficacy of deoxynucleoside therapy for Tk2 deficiency. FUND: National Institutes of Health P01HD32062.

Cardiomyopathy and altered integrin-actin signaling in Fhl1 mutant female mice.

X-linked scapuloperoneal myopathy (X-SM), one of Four-and-a-half LIM 1 (FHL1) related diseases, is an adult-onset slowly progressive myopathy, often associated with cardiomyopathy. We previously generated a knock-in mouse model that has the same mutation (c.365 G > C, p.W122S) as human X-SM patients. The mutant male mouse developed late-onset slowly progressive myopathy without cardiomyopathy. In this study, we observed that heterozygous (Het) and homozygous (Homo) female mice did not show alterations of skeletal muscle function or histology. In contrast, 20-month-old mutant female mice showed signs of cardiomyopathy on echocardiograms with increased systolic diameter [wild-type (WT): 2.74 ± 0.22 mm, mean ± standard deviation (SD); Het: 3.13 ± 0.11 mm, P < 0.01; Homo: 3.08 ± 0.37 mm, P < 0.05) and lower fractional shortening (WT: 31.1 ± 4.4%, mean ± SD; Het: 22.7 ± 2.5%, P < 0.01; Homo: 22.4 ± 6.9%, P < 0.01]. Histological analysis of cardiac muscle revealed frequent extraordinarily large rectangular nuclei in mutant female mice that were also observed in human cardiac muscle from X-SM patients. Western blot demonstrated decreased Fhl1 protein levels in cardiac muscle, but not in skeletal muscle, of Homo mutant female mice. Proteomic analysis of cardiac muscle from 20-month-old Homo mutant female mice indicated abnormalities of the integrin signaling pathway (ISP) in association with cardiac dysfunction. The ISP dysregulation was further supported by altered levels of a subunit of the ISP downstream effectors Arpc1a in Fhl1 mutant mice and ARPC1A in X-SM patient muscles. This study reveals the first mouse model of FHL1-related cardiomyopathy and implicates ISP dysregulation in the pathogenesis of FHL1 myopathy.

Three-Dimensional Analysis of Mitochondrial Crista Ultrastructure in a Patient with Leigh Syndrome by In Situ Cryoelectron Tomography.

Mitochondrial diseases produce profound neurological dysfunction via mutations affecting mitochondrial energy production, including the relatively common Leigh syndrome (LS). We recently described an LS case caused by a pathogenic mutation in USMG5, encoding a small supernumerary subunit of mitochondrial ATP synthase. This protein is integral for ATP synthase dimerization, and patient fibroblasts revealed an almost total loss of ATP synthase dimers. Here, we utilize in situ cryoelectron tomography (cryo-ET) in a clinical case-control study of mitochondrial disease to directly study mitochondria within cultured fibroblasts from a patient with LS and a healthy human control subject. Through tomographic analysis of patient and control mitochondria, we find that loss of ATP synthase dimerization due to the pathogenic mutation causes profound disturbances of mitochondrial crista ultrastructure. Overall, this work supports the crucial role of ATP synthase in regulating crista architecture in the context of human disease.

USMG5 Ashkenazi Jewish founder mutation impairs mitochondrial complex V dimerization and ATP synthesis.

Leigh syndrome is a frequent, heterogeneous pediatric presentation of mitochondrial oxidative phosphorylation (OXPHOS) disease, manifesting with psychomotor retardation and necrotizing lesions in brain deep gray matter. OXPHOS occurs at the inner mitochondrial membrane through the integrated activity of five protein complexes, of which complex V (CV) functions in a dimeric form to directly generate adenosine triphosphate (ATP). Mutations in several different structural CV subunits cause Leigh syndrome; however, dimerization defects have not been associated with human disease. We report four Leigh syndrome subjects from three unrelated Ashkenazi Jewish families harboring a homozygous splice-site mutation (c.87 + 1G>C) in a novel CV subunit disease gene, USMG5. The Ashkenazi population allele frequency is 0.57%. This mutation produces two USMG5 transcripts, wild-type and lacking exon 3. Fibroblasts from two Leigh syndrome probands had reduced wild-type USMG5 mRNA expression and undetectable protein. The mutation did not alter monomeric CV expression, but reduced both CV dimer expression and ATP synthesis rate. Rescue with wild-type USMG5 cDNA in proband fibroblasts restored USMG5 protein, increased CV dimerization and enhanced ATP production rate. These data demonstrate that a recurrent USMG5 splice-site founder mutation in the Ashkenazi Jewish population causes autosomal recessive Leigh syndrome by reduction of CV dimerization and ATP synthesis.

Deoxycytidine and Deoxythymidine Treatment for Thymidine Kinase 2 Deficiency.

OBJECTIVE: Thymidine kinase 2 (TK2), a critical enzyme in the mitochondrial pyrimidine salvage pathway, is essential for mitochondrial DNA (mtDNA) maintenance. Mutations in the nuclear gene, TK2, cause TK2 deficiency, which manifests predominantly in children as myopathy with mtDNA depletion. Molecular bypass therapy with the TK2 products, deoxycytidine monophosphate (dCMP) and deoxythymidine monophosphate (dTMP), prolongs the life span of Tk2-deficient (Tk2-/- ) mice by 2- to 3-fold. Because we observed rapid catabolism of the deoxynucleoside monophosphates to deoxythymidine (dT) and deoxycytidine (dC), we hypothesized that: (1) deoxynucleosides might be the major active agents and (2) inhibition of deoxycytidine deamination might enhance dTMP+dCMP therapy. METHODS: To test these hypotheses, we assessed two therapies in Tk2-/- mice: (1) dT+dC and (2) coadministration of the deaminase inhibitor, tetrahydrouridine (THU), with dTMP+dCMP. RESULTS: We observed that dC+dT delayed disease onset, prolonged life span of Tk2-deficient mice and restored mtDNA copy number as well as respiratory chain enzyme activities and levels. In contrast, dCMP+dTMP+THU therapy decreased life span of Tk2-/- animals compared to dCMP+dTMP. INTERPRETATION: Our studies demonstrate that deoxynucleoside substrate enhancement is a novel therapy, which may ameliorate TK2 deficiency in patients. Ann Neurol 2017;81:641-652.

Dietary Glycemic Index and Glycemic Load Are Positively Associated with Risk of Developing Metabolic Syndrome in Middle-Aged and Elderly Adults.

OBJECTIVES: To evaluate how glycemic index (GI) and glycemic load (GL) are associated with the metabolic syndrome (MetS) and its features in middle-aged and elderly adults at high cardiovascular risk. DESIGN: Prospective, longitudinal, population-based cohort. SETTING: PREvención con DIeta MEDiterránea study. PARTICIPANTS: Men and women (N = 6,606) divided into three age groups (<65, 65-74, ≥75). MEASUREMENTS: Energy and nutrient intake was evaluated using a validated 137-item food frequency questionnaire. MetS and its features were defined in accordance with the criteria of the American Heart Association and National Heart, Lung, and Blood Institute. RESULTS: A positive association was observed between GI and MetS prevalence in the youngest and middle age groups for participants without diabetes mellitus, but no relationship was found for those with diabetes mellitus. During the median follow-up of 4.8 years, higher GI and GL were related to greater risk of MetS in the middle age group, independent of the presence of diabetes mellitus. Changes in dietary GI were associated with risk of developing the high fasting glucose component of the MetS in the oldest age category, and changes in dietary GL were associated with risk of developing abdominal obesity, hypertriglyceridemia, low high-density lipoprotein cholesterol, and high blood pressure in the youngest age category. CONCLUSION: Dietary GI and GL have a potential role in the development of MetS and associated clinical features, with particular age-dependent considerations.

Beneficial effect of pistachio consumption on glucose metabolism, insulin resistance, inflammation, and related metabolic risk markers: a randomized clinical trial.

OBJECTIVE: To examine whether a pistachio-rich diet reduces the prediabetes stage and improves its metabolic risk profile. RESEARCH DESIGN AND METHODS: Prediabetic subjects were recruited to participate in this Spanish randomized clinical trial between 20 September 2011 and 4 February 2013. In a crossover manner, 54 subjects consumed two diets, each for 4 months: a pistachio-supplemented diet (PD) and a control diet (CD). A 2-week washout period separated study periods. Diets were isocaloric and matched for protein, fiber, and saturated fatty acids. A total of 55% of the CD calories came from carbohydrates and 30% from fat, whereas for the PD, these percentages were 50 and 35%, respectively (including 57 g/day of pistachios). RESULTS: Fasting glucose, insulin, and HOMA of insulin resistance decreased significantly after the PD compared with the CD. Other cardiometabolic risk markers such as fibrinogen, oxidized LDL, and platelet factor 4 significantly decreased under the PD compared with the CD (P < 0.05), whereas glucagon-like peptide-1 increased. Interleukin-6 mRNA and resistin gene expression decreased by 9 and 6%, respectively, in lymphocytes after the pistachio intervention (P < 0.05, for PD vs. CD). SLC2A4 expression increased by 69% in CD (P = 0.03, for PD vs. CD). Cellular glucose uptake by lymphocytes decreased by 78.78% during the PD (P = 0.01, PD vs. CD). CONCLUSIONS: Chronic pistachio consumption is emerging as a useful nutritional strategy for the prediabetic state. Data suggest that pistachios have a glucose- and insulin-lowering effect, promote a healthier metabolic profile, and reverse certain metabolic deleterious consequences of prediabetes.

Effect of the glycemic index of the diet on weight loss, modulation of satiety, inflammation, and other metabolic risk factors: a randomized controlled trial.

BACKGROUND: Low-glycemic index (GI) diets have been proven to have beneficial effects in such chronic conditions as type 2 diabetes, ischemic heart disease, and some types of cancer, but the effect of low-GI diets on weight loss, satiety, and inflammation is still controversial. OBJECTIVE: We assessed the efficacy of 2 moderate-carbohydrate diets and a low-fat diet with different GIs on weight loss and the modulation of satiety, inflammation, and other metabolic risk markers. DESIGN: The GLYNDIET study is a 6-mo randomized, parallel, controlled clinical trial conducted in 122 overweight and obese adults. Participants were randomly assigned to one of the following 3 isocaloric energy-restricted diets for 6 mo: 1) a moderate-carbohydrate and high-GI diet (HGI), 2) a moderate-carbohydrate and low-GI diet (LGI), and 3) a low-fat and high-GI diet (LF). RESULTS: At weeks 16 and 20 and the end of the intervention, changes in body mass index (BMI; in kg/m(2)) differed significantly between intervention groups. Reductions in BMI were greater in the LGI group than in the LF group, whereas in the HGI group, reductions in BMI did not differ significantly from those in the other 2 groups (LGI: -2.45 ± 0.27; HGI: -2.30 ± 0.27; LF: -1.43 ± 0.27; F = 4.616, P = 0.012; pairwise comparisons: LGI compared with HGI, P = 1.000; LGI compared with LF, P = 0.016; HGI compared with LF, P = 0.061). The decrease in fasting insulin, homeostatic model assessment of insulin resistance, and homeostatic model assessment of ß cell function was also significantly greater in the LGI group than in the LF group (P < 0.05). Despite this tendency for a greater improvement with a low-GI diet, the 3 intervention groups were not observed to have different effects on hunger, satiety, lipid profiles, or other inflammatory and metabolic risk markers. CONCLUSION: A low-GI and energy-restricted diet containing moderate amounts of carbohydrates may be more effective than a high-GI and low-fat diet at reducing body weight and controlling glucose and insulin metabolism. This trial was registered at Current Controlled Trials (www.controlled-trials.com) as ISRCTN54971867.

Dietary intake of vitamin K is inversely associated with mortality risk.

Vitamin K has been related to cardiovascular disease and cancer risk. However, data on total mortality are scarce. The aim of the present study was to assess the association between the dietary intake of different types of vitamin K and mortality in a Mediterranean population at high cardiovascular disease risk. A prospective cohort analysis was conducted in 7216 participants from the PREDIMED (Prevención con Dieta Mediterránea) study (median follow-up of 4.8 y). Energy and nutrient intakes were evaluated using a validated 137-item food frequency questionnaire. Dietary vitamin K intake was calculated annually using the USDA food composition database and other published sources. Deaths were ascertained by an end-point adjudication committee unaware of the dietary habits of participants after they had reviewed medical records and linked up to the National Death Index. Cox proportional hazard models were fitted to assess the RR of mortality. Energy-adjusted baseline dietary phylloquinone intake was inversely associated with a significantly reduced risk of cancer and all-cause mortality after controlling for potential confounders (HR: 0.54; 95% CI: 0.30, 0.96; and HR: 0.64; 95% CI: 0.45, 0.90, respectively). In longitudinal assessments, individuals who increased their intake of phylloquinone or menaquinone during follow-up had a lower risk of cancer (HR: 0.64; 95% CI: 0.43, 0.95; and HR: 0.41; 95% CI: 0.26, 0.64, respectively) and all-cause mortality (HR: 0.57; 95% CI: 0.44, 0.73; and HR: 0.55; 95% CI: 0.42, 0.73, respectively) than individuals who decreased or did not change their intake. Also, individuals who increased their intake of dietary phylloquinone had a lower risk of cardiovascular mortality risk (HR: 0.52; 95% CI: 0.31, 0.86). However, no association between changes in menaquinone intake and cardiovascular mortality was observed (HR: 0.76; 95% CI: 0.44, 1.29). An increase in dietary intake of vitamin K is associated with a reduced risk of cardiovascular, cancer, or all-cause mortality in a Mediterranean population at high cardiovascular disease risk. This trial was registered at http://www.controlled-trials.com as ISRCTN35739639.

Association between serum ferritin and osteocalcin as a potential mechanism explaining the iron-induced insulin resistance.

BACKGROUND: Increased iron stores are associated with increased risk of type 2 diabetes, however, the mechanisms underlying these associations are poorly understood. Because a reduction of circulating osteocalcin levels after iron overload have been demonstrated in cell cultures, and osteocalcin is related to glucose and insulin metabolism, the iron-induced osteocalcin reductions could contribute to explain the role of iron metabolism in the development of type 2 diabetes mellitus. OBJECTIVE: To analyzed the associations between serum total and uncarboxylated osteocalcin and adiponectin concentrations with serum ferritin and soluble transferrin receptor (sTfR) in elderly subjects. DESIGN: We evaluated a total of 423 subjects from the PREDIMED cohort in a population-based cross-sectional analysis. Extensive clinical, nutritional and laboratory measurements, including total and uncarboxylated osteocalcin, adiponectin, ferritin and sTfR were recorded. RESULTS: Serum ferritin was positively correlated with increased glucose and insulin circulating levels but also with HOMA-IR, and was inversely associated with total osteocalcin and adiponectin. A regression analysis revealed that serum ferritin and transferrin receptor levels were significantly associated with a decrease in total and uncarboxylated osteocalcin. Serum sTfR levels were associated with lower uncarboxylated osteocalcin levels in the whole-study subjects and remained significant only in the IFG (impaired fasting glucose) individuals. CONCLUSIONS: We described, for the first time, an inverse association between serum ferritin and sTfR with osteocalcin and extend previous results on adiponectin, thus supporting that factors related to iron metabolism could contribute to the insulin resistance and the development of type 2 diabetes mellitus. TRIAL REGISTRATION: Controlled-Trials.com ISRCTN35739639 .

Reduced serum concentrations of carboxylated and undercarboxylated osteocalcin are associated with risk of developing type 2 diabetes mellitus in a high cardiovascular risk population: a nested case-control study.

CONTEXT AND OBJECTIVE: Because it has been suggested that osteocalcin (OC), an osteoblast-derived hormone, is a new link between bone and glucose metabolism, we tested whether serum carboxylated osteocalcin (cOC) and undercarboxylated osteocalcin (ucOC) levels are independently associated with the development of type 2 diabetes in subjects at high cardiovascular risk. DESIGN, SETTING, AND PARTICIPANTS: A prospective, nested case-control study was conducted using data from the Prevención con Dieta Mediterránea (PREDIMED) study. We included 153 case subjects with newly diagnosed diabetes and 306 individually matched control subjects free of diabetes identified during a mean 5-year follow-up. Conditional logistic regression models were used to estimate matched odds ratios for incident diabetes according to categories of both forms of OC measured by ELISAs. RESULTS: Baseline serum concentrations of both forms of OC were significantly lower in case subjects than in control subjects. In subjects with incident cases of diabetes, concentrations of cOC, but not of ucOC, were inversely and significantly associated with homeostasis model assessment of insulin resistance levels (ß = -0.335) and with fasting glucose concentrations (ß = -0.044) in control subjects, independent of other relevant confounders. In the conditional logistic model that took into account the matching factors, the odds ratios for diabetes incidence in the lowest vs the highest tertile of cOC and ucOC were 2.03 (95% confidence interval, 1.32-3.13) and 1.88 (1.23-2.85), respectively. Further adjustment for family history of diabetes, lifestyle, and other confounding factors did not appreciably change the magnitude of these associations. CONCLUSION: In a population at high cardiovascular risk, low concentrations of serum cOC and ucOC were strongly associated with an increased risk of incident diabetes.

Design and methods of the GLYNDIET study; assessing the role of glycemic index on weight loss and metabolic risk markers.

BACKGROUND: Glycemic index and/or glycemic load have been explored as an alternative for the prevention and/or management of obesity, cardiovascular disease, type 2 diabetes mellitus, and cancer. OBJECTIVE: The purpose of the manuscript is to describe the design and methods used in the GLYNDIET Project, a study designed to simultaneously address the questions related to the exactly role of low glycaemic index carbohydrates has on weight loss. METHODS: This study was designed as a 6-months randomized, parallel, controlled clinical trial aiming to evaluate the effect of the dietary glycemic index on weightloss, satiety, glucose and insulin metabolism, lipid profile, inflammation and other emergent metabolic risk markers. Eligible subjects were community-dwelling men and women aged between 30 and 60 years, with a body mass index between 27 and 35 kg/m2. Subjects were randomly assigned to three different dietary intervention groups (low glycemic index diet, high glycemic index diet or low-fat diet), that were isocaloric, and did not differ in the amount of dietary fibre. Monthly, study subjects were scheduled for control visits where anthropometry, blood pressure, dietary habits, satiety and physical activity were assessed. Blood, urine and subcutaneous adipose tissue samples were collected at baseline and at the end of the study to further molecular and biochemical measurements. DISCUSSION: The GLYNDIET study was designed to determine if there is a greater effectiveness of a carbohydrate restricted diet with low glycemic index compared to an isocaloric diet with carbohydrates of high glycemic index or low-fat diet on weight loss in middle long-term.

Introducción: El índice glucémico y la carga glucémica se han postulado como una alternativa para la prevención y/o el manejo de la obesidad, enfermedades cardiovasculares, diabetes mellitus tipo 2 y cáncer. Objetivo: Describir el diseño y los métodos utilizados en el proyecto GLYNDIET, un estudio diseñado para evaluar el papel del índice glucémico sobre la pérdida de peso corporal, la saciedad, la inflamación y marcadores de riesgo metabólico. Métodos: Ensayo clínico, en paralelo, controlado, aleatorizado y de 6 meses de duración realizado en hombres y mujeres de entre 30 y 60 AÑOs, con un índice de masa corporal de entre 27 y 35 kg/m2. Los sujetos fueron asignados aleatoriamente a una de las 3 intervenciones (dieta con carbohidratos de bajo índice glucémico, dieta con carbohidratos de alto índice glucémico o dieta baja en grasa). Los sujetos fueron citados mensualmente para realizar visitas control en las que se recogían datos a antropométricos, de presión arterial, hábitos dietéticos, sensación de saciedad y grado de actividad física. Al inicio y al final del estudio se recogieron muestras sanguíneas, urinarias y de tejido adiposo subcutáneo mediante biopsia abdominal. Discusión: El estudio GLYNDIET se diseñó con el objetico de determinar si el consumo de una dieta con carbohidratos de bajo índice glucémico muestra una mayor efectividad sobre la pérdida de peso corporal y la modulación de factores de riesgo metabólico en comparación a una dieta con carbohidratos de alto índice glucémico o una dieta baja en grasas.

Design and methods of the GLYNDIET study; assessing the role of glycemic index on weight loss and metabolic risk markers / Diseño y métodos del Estudio GLYNDIET; evaluando el papel del índice glucémico sobre la pérdida de peso corporal y marcadores de riesgo metabólico

Background: Glycemic index and/or glycemic load have been explored as an alternative for the prevention and/or management of obesity, cardiovascular disease, type 2 diabetes mellitus, and cancer. Objective: The purpose of the manuscript is to describe the design and methods used in the GLYNDIET Project, a study designed to simultaneously address the questions related to the exactly role of low glycaemic index carbohydrates has on weight loss. Methods: This study was designed as a 6-months randomized, parallel, controlled clinical trial aiming to evaluate the effect of the dietary glycemic index on weight-loss, satiety, glucose and insulin metabolism, lipid profile, inflammation and other emergent metabolic risk markers. Eligible subjects were community-dwelling men and women aged between 30 and 60 years, with a body mass index between 27 and 35 kg/m2. Subjects were randomly assigned to three different dietary intervention groups (low glycemic index diet, high glycemic index diet or low-fat diet), that were isocaloric, and did not differ in the amount of dietary fibre. Monthly, study subjects were scheduled for control visits where anthropometry, blood pressure, dietary habits, satiety and physical activity were assessed. Blood, urine and subcutaneous adipose tissue samples were collected at baseline and at the end of the study to further molecular and biochemical measurements. Discussion: The GLYNDIET study was designed to determine if there is a greater effectiveness of a carbohydrate restricted diet with low glycemic index compared to an isocaloric diet with carbohydrates of high glycemic index or low-fat diet on weight loss in middle long-term (AU)

Introducción: El índice glucémico y la carga glucémica se han postulado como una alternativa para la prevención y/o el manejo de la obesidad, enfermedades cardiovasculares, diabetes mellitus tipo 2 y cáncer. Objetivo: Describir el diseño y los métodos utilizados en el proyecto GLYNDIET, un estudio diseñado para evaluar el papel del índice glucémico sobre la pérdida de peso corporal, la saciedad, la inflamación y marcadores de riesgo metabólico. Métodos: Ensayo clínico, en paralelo, controlado, aleatorizado y de 6 meses de duración realizado en hombres y mujeres de entre 30 y 60 años, con un índice de masa corporal de entre 27 y 35 kg/m2. Los sujetos fueron asignados aleatoriamente a una de las 3 intervenciones (dieta con carbohidratos de bajo índice glucémico, dieta con carbohidratos de alto índice glucémico o dieta baja en grasa). Los sujetos fueron citados mensualmente para realizar visitas control en las que se recogían datos a antropométricos, de presión arterial, hábitos dietéticos, sensación de saciedad y grado de actividad física. Al inicio y al final del estudio se recogieron muestras sanguíneas, urinarias y de tejido adiposo subcutáneo mediante biopsia abdominal. Discusión: El estudio GLYNDIET se diseñó con el objetico de determinar si el consumo de una dieta con carbohidratos de bajo índice glucémico muestra una mayor efectividad sobre la pérdida de peso corporal y la modulación de factores de riesgo metabólico en comparación a una dieta con carbohidratos de alto índice glucémico o una dieta baja en grasas (AU)