A Flexible, Pooled CRISPR Library for Drug Development Screens.

Functional genomic screening with CRISPR has provided a powerful and precise new way to interrogate the phenotypic consequences of gene manipulation in high-throughput, unbiased analyses. However, some experimental paradigms prove especially challenging and require carefully and appropriately adapted screening approaches. In particular, negative selection (or sensitivity) screening, often the most experimentally desirable modality of screening, has remained a challenge in drug discovery. Here we assess whether our new, modular genome-wide pooled CRISPR library can improve negative selection CRISPR screening and add utility throughout the drug development pipeline. Our pooled library is split into three parts, allowing it to be scaled to accommodate the experimental challenges encountered during drug development, such as target identification using unlimited cell numbers compared with target identification studies for cell populations where cell numbers are limiting. To test our new library, we chose to look for drug-gene interactions using a well-described small molecule inhibitor targeting poly(ADP-ribose) polymerase 1 (PARP1), and in particular to identify genes which sensitise cells to this drug. We simulate hit identification and performance using each library partition and support these findings through orthogonal drug combination cell panel screening. We also compare our data with a recently published CRISPR sensitivity dataset obtained using the same PARP1 inhibitor. Overall, our data indicate that generating a comprehensive CRISPR knockout screening library where the number of guides can be scaled to suit the biological question being addressed allows a library to have multiple uses throughout the drug development pipeline, and that initial validation of hits can be achieved through high-throughput cell panels screens where clinical grade chemical or biological matter exist.

In Vitro Modeling of Bile Acid Processing by the Human Fecal Microbiota.

Bile acids, the products of concerted host and gut bacterial metabolism, have important signaling functions within the mammalian metabolic system and a key role in digestion. Given the complexity of the mega-variate bacterial community residing in the gastrointestinal tract, studying associations between individual bacterial genera and bile acid processing remains a challenge. Here, we present a novel in vitro approach to determine the bacterial genera associated with the metabolism of different primary bile acids and their potential to contribute to inter-individual variation in this processing. Anaerobic, pH-controlled batch cultures were inoculated with human fecal microbiota and treated with individual conjugated primary bile acids (500 µg/ml) to serve as the sole substrate for 24 h. Samples were collected throughout the experiment (0, 5, 10, and 24 h) and the bacterial composition was determined by 16S rRNA gene sequencing and the bile acid signatures were characterized using a targeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) approach. Data fusion techniques were used to identify statistical bacterial-metabolic linkages. An increase in gut bacteria associated bile acids was observed over 24 h with variation in the rate of bile acid metabolism across the volunteers (n = 7). Correlation analysis identified a significant association between the Gemmiger genus and the deconjugation of glycine conjugated bile acids while the deconjugation of taurocholic acid was associated with bacteria from the Eubacterium and Ruminococcus genera. A positive correlation between Dorea and deoxycholic acid production suggest a potential role for this genus in cholic acid dehydroxylation. A slower deconjugation of taurocholic acid was observed in individuals with a greater abundance of Parasutterella and Akkermansia. This work demonstrates the utility of integrating compositional (metataxonomics) and functional (metabonomics) systems biology approaches, coupled to in vitro model systems, to study the biochemical capabilities of bacteria within complex ecosystems. Characterizing the dynamic interactions between the gut microbiota and the bile acid pool enables a greater understanding of how variation in the gut microbiota influences host bile acid signatures, their associated functions and their implications for health.

Bikini versus traditional incision direct anterior approach: is there any difference in soft tissue damage?

INTRODUCTION: The direct anterior approach for total hip replacements has reported advantages of improved early function and muscle preservation. In an effort to improve healing and cosmesis, a change in the orientation of the incision has been proposed. Traditionally, the skin incision is in-line with the tensor fasciae latae muscle belly. The bikini incision is orthogonal to this orientation. The hypothesis was that muscle damage would be increased by using the bikini incision. METHODS: A traditional or bikini incision was performed on 18 cadaveric hips. On each of the 9 specimens, the traditional incision was performed on 1 side, and a bikini incision on the contralateral hip, with an even distribution of right or left side. Blinded anatomists performed the hip dissections, and assessed for muscle damage as well as for damage to the lateral femoral cutaneous nerve. RESULTS: No difference in muscle damage was identified in the tensor fasciae latae between muscle groups. Muscle damage was very minimal to the gluteus medius and minimus. Damage to the lateral femoral cutaneous nerve occurred equally for both the bikini and traditional skin incisions. CONCLUSIONS: The bikini incision for the direct anterior approach to the hip can be performed safely, with no increase in muscle damage or damage to the lateral femoral cutaneous nerve compared to the traditional incision.

Lysophosphatidylcholines to phosphatidylcholines ratio predicts advanced knee osteoarthritis.

OBJECTIVE: To identify novel biomarker(s) for predicting advanced knee OA. METHODS: Study participants were derived from the Newfoundland Osteoarthritis Study and the Tasmania Older Adult Cohort Study. All knee OA cases were patients who underwent total knee replacement (TKR) due to primary OA. Metabolic profiling was performed on fasting plasma. Four thousand and eighteen plasma metabolite ratios that were highly correlated with that in SF in our previous study were generated as surrogates for joint metabolism. RESULTS: The discovery cohort included 64 TKR cases and 45 controls and the replication cohorts included a cross-sectional cohort of 72 TKR cases and 76 controls and a longitudinal cohort of 158 subjects, of whom 36 underwent TKR during the 10-year follow-up period. We confirmed the previously reported association of the branched chain amino acids to histidine ratio with advanced knee OA (P = 9.3 × 10(-7)) and identified a novel metabolic marker-the lysophosphatidylcholines (lysoPCs) to phosphatidylcholines (PCs) ratio-that was associated with advanced knee OA (P = 1.5 × 10(-7)) after adjustment for age, sex and BMI. When the subjects of the longitudinal cohort were categorized into two groups based on the optimal cut-off of the ratio of 0.09, we found the subjects with the ratio ⩾0.09 were 2.3 times more likely to undergo TKR than those with the ratio <0.09 during the 10-year follow-up (95% CI: 1.2, 4.3, P = 0.02). CONCLUSION: We identified the ratio of lysoPCs to PCs as a novel metabolic marker for predicting advanced knee OA. Further studies are required to examine whether this ratio can predict early OA change.

A cost analysis of single-stage bilateral versus two-stage direct anterior total hip arthroplasty.

PURPOSE: The potential cost savings of single-stage bilateral total hip arthroplasty (THA) are unclear, and the risks associated with it are not well defined. We sought to compare the costs and perioperative complications of single-stage bilateral THA via the direct anterior approach (DAA) to a two-stage bilateral protocol. METHODS: We retrospectively reviewed patients who underwent a single- stage bilateral DAA THA and compared them to a two-stage THA group. We conducted a cost analysis from both the hospital perspective and the Ministry of Health (MOH) perspective. RESULTS: 24 patients were included in this study. The 2 groups were similar in age (58.9 vs 63.9 yrs), height (169.2 vs 170.9 cm), weight (80.2 vs 78.6 kg), BMI (27.9 vs 26.3 kg/m2), ASA score (2.2 vs 2.2), and CCI score (2.3 vs 2.9). The mean cost per patient from the hospital perspective for the single-stage group was $10,728.13 (SD = 621.46) compared to $12,670.63 (SD = 519.72) for the two-stage group (Mean Difference = $1,942.50, 95% CI = $1,457.49 to $2,427.51, p<0.001). Similarly, from the MOH perspective, the cost for the single-stage group was $12,552.34 (SD = 644.93) compared to $14,740.58 (SD = 598.07) for the two-stage group (Mean Difference = $2,188.24, 95% CI = $1,661.67 to $2,714.81, p<0.001). There were no significant differences in complication rate between groups. The largest percent of total cost savings from a hospital perspective was attributed to cost of operating room staff and OR set-up (55%). CONCLUSIONS: Our results suggest that single-stage bilateral DAA THA results in significant cost savings compared to two-stage DAA THA.

SMAD3 Is Upregulated in Human Osteoarthritic Cartilage Independent of the Promoter DNA Methylation.

OBJECTIVE: To compare SMAD3 gene expression between human osteoarthritic and healthy cartilage and to examine whether expression is regulated by the promoter DNA methylation of the gene. METHODS: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary osteoarthritis (OA), and from patients with hip fractures as controls. DNA/RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression, and Sequenom EpiTyper was used to assay DNA methylation. Mann-Whitney test was used to compare the methylation and expression levels between OA cases and controls. Spearman rank correlation coefficient was calculated to examine the association between the methylation and gene expression. RESULTS: A total of 58 patients with OA (36 women, 22 men; mean age 64 ± 9 yrs) and 55 controls (43 women, 12 men; mean age 79 ± 10 yrs) were studied. SMAD3 expression was on average 83% higher in OA cartilage than in controls (p = 0.0005). No difference was observed for DNA methylation levels in the SMAD3 promoter region between OA cases and controls. No correlation was found between SMAD3 expression and promoter DNA methylation. CONCLUSION: Our study demonstrates that SMAD3 is significantly overexpressed in OA. This overexpression cannot be explained by DNA methylation in the promoter region. The results suggest that the transforming growth factor-ß/SMAD3 pathway may be overactivated in OA cartilage and has potential in developing targeted therapies for OA.

Genome-wide DNA methylation study of hip and knee cartilage reveals embryonic organ and skeletal system morphogenesis as major pathways involved in osteoarthritis.

BACKGROUND: Evidence suggests that epigenetics plays a role in osteoarthrits (OA). The aim of the study was to describe the genome wide DNA methylation changes in hip and knee OA and identify novel genes and pathways involved in OA by comparing the DNA methylome of the hip and knee osteoarthritic cartilage tissues with those of OA-free individuals. METHODS: Cartilage samples were collected from hip or knee joint replacement patients either due to primary OA or hip fractures as controls. DNA was extracted from the collected cartilage and assayed by Illumina Infinium HumanMethylation450 BeadChip array, which allows for the analysis of >480,000 CpG sites. Student T-test was conducted for each CpG site and those sites with at least 10 % methylation difference and a p value <0.0005 were defined as differentially methylated regions (DMRs) for OA. A sub-analysis was also done for hip and knee OA separately. DAVID v6.7 was used for the functional annotation clustering of the DMR genes. Clustering analysis was done using multiple dimensional scaling and hierarchical clustering methods. RESULTS: The study included 5 patients with hip OA, 6 patients with knee OA and 7 hip cartilage samples from OA-free individuals. The comparisons of hip, knee and combined hip/knee OA patients with controls resulted in 26, 72, and 103 DMRs, respectively. The comparison between hip and knee OA revealed 67 DMRs. The overall number of the sites after considering the overlaps was 239, among which 151 sites were annotated to 145 genes. One-fifth of these genes were reported in previous studies. The functional annotation clustering of the identified genes revealed clusters significantly enriched in skeletal system morphogenesis and development. The analysis revealed significant difference among OA and OA-free cartilage, but less different between hip OA and knee OA. CONCLUSIONS: We found that a number of CpG sites and genes across the genome were differentially methylated in OA patients, a remarkable portion of which seem to be involved in potential etiologic mechanisms of OA. Genes involved in skeletal developmental pathways and embryonic organ morphogenesis may be a potential area for further OA studies.

Overexpression of MMP13 in human osteoarthritic cartilage is associated with the SMAD-independent TGF-ß signalling pathway.

INTRODUCTION: In vitro and animal model of osteoarthritis (OA) studies suggest that TGF-ß signalling is involved in OA, but human data is limited. We undertook this study to elucidate the role of TGF-ß signalling pathway in OA by comparing the expression levels of TGFB1 and BMP2 as ligands, SMAD3 as an intracellular mediator, and MMP13 as a targeted gene between human osteoarthritic and healthy cartilage. METHODS: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary OA or hip fractures as controls. RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression. Mann-Whitney test was utilized to compare the expression levels of TGFB1, BMP2, SMAD3 and MMP13 in human cartilage between OA cases and controls. Spearman's rank correlation coefficient (rho) was calculated to examine the correlation between the expression levels of the four genes studied and non-parametric regression was used to adjust for covariates. RESULTS: A total of 32 OA cases (25 hip OA and 7 knee OA) and 21 healthy controls were included. The expression of TGFB1, SMAD3, and MMP13 were on average 70%, 46%, and 355% higher, respectively, whereas the expression of BMP2 was 88% lower, in OA-affected cartilage than that of controls (all p < 0.03), but no difference was observed between hip and knee OA (all p > 0.4). The expression of TGFB1 was correlated with the expression of SMAD3 (rho = 0.50, p = 0.003) and MMP13 (rho = 0.46, p = 0.007) in OA-affected cartilage and the significance became stronger after adjustment for age, sex, and BMI. The expression of BMP2 was negatively correlated with both TGFB1 (rho = -0.50, p = 0.02) and MMP13 (rho = -0.48, p = 0.02) in healthy cartilage, but the significance was altered after adjustment for the covariates. There was no correlation between the expression of SMAD3 and MMP13. CONCLUSIONS: Our results demonstrate that MMP13 expression is associated with an increased expression of TGFB1 in OA-affected cartilage, possibly through SMAD-independent TGF-ß pathway. Furthermore, TGF-ß/SMAD3 is overactivated in OA cartilage; yet, the consequence of this overactivation remains to be established.

Relationship between blood plasma and synovial fluid metabolite concentrations in patients with osteoarthritis.

OBJECTIVE: To investigate the relationship between plasma and synovial fluid (SF) metabolite concentrations in patients with osteoarthritis (OA). METHODS: Blood plasma and SF samples were collected from patients with primary knee OA undergoing total knee arthroplasty. Metabolic profiling was performed by electrospray ionization tandem mass spectrometry using the AbsoluteIDQ kit. The profiling yielded 168 metabolite concentrations. Correlation analysis between SF and plasma metabolite concentrations was done on absolute concentrations as well as metabolite concentration ratios using Spearman's rank correlation (ρ) method. RESULTS: A total of 69 patients with knee OA were included, 30 men and 39 women, with an average age of 66 ± 8 years. For the absolute metabolite concentrations, the average ρ was 0.23 ± 0.13. Only 8 out of 168 metabolite concentrations had a ρ ≥ 0.45, with a p value ≤ 2.98 × 10(-4), statistically significant after correcting multiple testing with the Bonferroni method. For the metabolite ratios (n = 28,056), the average ρ was 0.29 ± 0.20. There were 4018 metabolite ratios with a ρ ≥ 0.52 and a p value ≤ 1.78 × 10(-6), significant after correcting multiple testing. Sex-separate analyses found no difference in ρ between men and women. Similarly, there was no difference in ρ between people younger and older than 65 years. CONCLUSION: Correlation between blood plasma and SF metabolite concentrations are modest. Metabolite ratios, which are considered proxies for enzymatic reaction rates and have higher correlations, should be considered when using blood plasma as a surrogate of SF in OA biomarker identification.

Classification of osteoarthritis phenotypes by metabolomics analysis.

OBJECTIVES: To identify metabolic markers that can classify patients with osteoarthritis (OA) into subgroups. DESIGN: A case-only study design was utilised. PARTICIPANTS: Patients were recruited from those who underwent total knee or hip replacement surgery due to primary OA between November 2011 and December 2013 in St. Clare's Mercy Hospital and Health Science Centre General Hospital in St. John's, capital of Newfoundland and Labrador (NL), Canada. 38 men and 42 women were included in the study. The mean age was 65.2±8.7 years. OUTCOME MEASURES: Synovial fluid samples were collected at the time of their joint surgeries. Metabolic profiling was performed on the synovial fluid samples by the targeted metabolomics approach, and various analytic methods were utilised to identify metabolic markers for classifying subgroups of patients with OA. Potential confounders such as age, sex, body mass index (BMI) and comorbidities were considered in the analysis. RESULTS: Two distinct patient groups, A and B, were clearly identified in the 80 patients with OA. Patients in group A had a significantly higher concentration on 37 of 39 acylcarnitines, but the free carnitine was significantly lower in their synovial fluids than in those of patients in group B. The latter group was further subdivided into two subgroups, that is, B1 and B2. The corresponding metabolites that contributed to the grouping were 86 metabolites including 75 glycerophospholipids (6 lysophosphatidylcholines, 69 phosphatidylcholines), 9 sphingolipids, 1 biogenic amine and 1 acylcarnitine. The grouping was not associated with any known confounders including age, sex, BMI and comorbidities. The possible biological processes involved in these clusters are carnitine, lipid and collagen metabolism, respectively. CONCLUSIONS: The study demonstrated that OA consists of metabolically distinct subgroups. Identification of these distinct subgroups will help to unravel the pathogenesis and develop targeted therapies for OA.

SMAD3 is associated with the total burden of radiographic osteoarthritis: the Chingford study.

BACKGROUND: A newly-described syndrome called Aneurysm-Osteoarthritis Syndrome (AOS) was recently reported. AOS presents with early onset osteoarthritis (OA) in multiple joints, together with aneurysms in major arteries, and is caused by rare mutations in SMAD3. Because of the similarity of AOS to idiopathic generalized OA (GOA), we hypothesized that SMAD3 is also associated with GOA and tested the hypothesis in a population-based cohort. METHODS: Study participants were derived from the Chingford study. Kellgren-Lawrence (KL) grades and the individual features of osteophytes and joint space narrowing (JSN) were scored from radiographs of hands, knees, hips, and lumbar spines. The total KL score, osteophyte score, and JSN score were calculated and used as indicators of the total burden of radiographic OA. Forty-one common SNPs within SMAD3 were genotyped using the Illumina HumanHap610Q array. Linear regression modelling was used to test the association between the total KL score, osteophyte score, and JSN score and each of the 41 SNPs, with adjustment for patient age and BMI. Permutation testing was used to control the false positive rate. RESULTS: A total of 609 individuals were included in the analysis. All were Caucasian females with a mean age of 60.9±5.8. We found that rs3825977, with a minor allele (T) frequency of 20%, in the last intron of SMAD3, was significantly associated with total KL score (ß = 0.14, Ppermutation = 0.002). This association was stronger for the total JSN score (ß = 0.19, Ppermutation = 0.002) than for total osteophyte score (ß = 0.11, Ppermutation = 0.02). The T allele is associated with a 1.47-fold increased odds for people with 5 or more joints to be affected by radiographic OA (Ppermutation = 0.046). CONCLUSION: We found that SMAD3 is significantly associated with the total burden of radiographic OA. Further studies are required to reveal the mechanism of the association.

Probiotic administration attenuates myocardial hypertrophy and heart failure after myocardial infarction in the rat.

BACKGROUND: Probiotics are extensively used to promote gastrointestinal health, and emerging evidence suggests that their beneficial properties can extend beyond the local environment of the gut. Here, we determined whether oral probiotic administration can alter the progression of postinfarction heart failure. METHODS AND RESULTS: Rats were subjected to 6 weeks of sustained coronary artery occlusion and administered the probiotic Lactobacillus rhamnosus GR-1 or placebo in the drinking water ad libitum. Culture and 16s rRNA sequencing showed no evidence of GR-1 colonization or a significant shift in the composition of the cecal microbiome. However, animals administered GR-1 exhibited a significant attenuation of left ventricular hypertrophy based on tissue weight assessment and gene expression of atrial natriuretic peptide. Moreover, these animals demonstrated improved hemodynamic parameters reflecting both improved systolic and diastolic left ventricular function. Serial echocardiography revealed significantly improved left ventricular parameters throughout the 6-week follow-up period including a marked preservation of left ventricular ejection fraction and fractional shortening. Beneficial effects of GR-1 were still evident in those animals in which GR-1 was withdrawn at 4 weeks, suggesting persistence of the GR-1 effects after cessation of therapy. Investigation of mechanisms showed a significant increase in the leptin:adiponectin plasma concentration ratio in rats subjected to coronary ligation, which was abrogated by GR-1. Metabonomic analysis showed differences between sham control and coronary artery ligated hearts particularly with respect to preservation of myocardial taurine levels. CONCLUSIONS: The study suggests that probiotics offer promise as a potential therapy for the attenuation of heart failure.

Long-term outcomes for Kienböck's disease.

BACKGROUND: The precise etiology of Kienböck's disease is unclear. Controversy exists regarding the appropriate treatment modality. The present study sought to investigate and compare surgical and nonsurgical treatment outcomes of patients suffering from Kienböck's disease in the province of Newfoundland and Labrador (NL), Canada. METHODS: The present study was a retrospective analysis of 66 patients. The primary outcome was the Disabilities of the Arm, Shoulder, and Hand (DASH) score. Student's t test was used to assess differences in outcomes between treatment groups. One-way ANOVA was used to assess differences in primary outcome in time since first assessed in an effort to examine progression over time. Pearson correlation was used to assess for correlation between primary outcome and age at diagnosis. RESULTS: The average age was 38.6 ± 11.4 (18-70) years; Four patients were excluded due to inaccessible imaging. Of the remaining patients, 44 were treated conservatively, while 18 were treated surgically. The DASH scores for the surgical group were 23.7 ± 24.5 (0.9-82.8) and nonsurgical group were 20.0 ± 20.1 (1.7-81). As expected, the surgical group was mainly comprised of late-stage Kienböck's. When both groups were compared, there was no significant difference in the DASH scores. There were no difference in DASH scores within groups according to time since first diagnosed (<5 years; between 5 and 10 years; and >10 years). A positive correlation was found between age at diagnosis and DASH score (r = 0.42, p = 0.007), despite treatment modality. This finding remained significant after accounting for confounding factors (p = 0.029). CONCLUSION: The DASH score for the surgical group was 23.7 ± 24.5 (0.9-82.8) and nonsurgical group was 20.0 ± 20.1 (1.7-81). No significant difference in DASH scores was found between surgically and nonsurgically treated patients. A positive association was found between the age at diagnosis of Kienböck's and DASH score, which suggests that patients diagnosed and treated later in life tend not to do as well.

Changes in the transcriptome of abdominal subcutaneous adipose tissue in response to short-term overfeeding in lean and obese men.

BACKGROUND: Obesity is caused by the excessive accumulation of adipose tissue as a result of a chronic energy surplus. Little is known regarding the molecular mechanisms involved in the response to an energy surplus in human adipose tissue at the genomic level. OBJECTIVE: The objective was to investigate changes in the transcriptome of abdominal subcutaneous adipose tissue after a positive energy challenge induced by overfeeding in both lean and obese subjects to identify novel obesity candidate genes. DESIGN: A total of 26 men were recruited and classified on the basis of percentage body fat (measured by dual-energy X-ray absorptiometry) as lean (<20%) or obese (>25%) to participate in the baseline comparison. Sixteen men participated in the overfeeding study (8 lean and 8 obese). Adipose tissue biopsy samples were collected from all subjects at the subumbilical region. Global gene expression profiles were determined at baseline and after a 7-d hypercaloric diet at 40% above normal energy requirements by using whole human genome DNA microarrays. RESULTS: Overfeeding induced differential expression in 45 genes. Six genes displayed a significant interaction effect between adiposity status and overfeeding treatment, including transferrin (TF), stearoyl-CoA desaturase (SCD), transaldolase 1 (TALDO1), cathepsin C (CTSC), insulin receptor substrate 2 (IRS2), and pyruvate dehydrogenase kinase, isozyme 4 (PDK4). Overfeeding resulted in changes in expression of these genes in lean subjects, whereas no significant changes were evident in obese subjects. CONCLUSIONS: Differential expression of these 6 genes may represent a protective mechanism at the molecular level in lean subjects in response to an energy surplus. These genes represent valuable candidates for downstream studies related to obesity.

Lack of association of ghrelin precursor gene variants and percentage body fat or serum lipid profiles.

Ghrelin has been recognized for its involvement in food intake, control of energy homeostasis, and lipid metabolism. However, the roles of genetic variations in the ghrelin precursor gene (GHRL) on body compositions and serum lipids are not clear in humans. Our study investigated five single-nucleotide polymorphisms (SNPs) within GHRL to determine their relationship with body fat percentage (BF), trunk fat percentage (TF), lower body (legs) fat percentage (LF), and serum lipids in 1,464 subjects, which were recruited from the genetically homogeneous population of Newfoundland and Labrador (NL), Canada. Serum glucose, insulin, total cholesterol, high-density lipoprotein-cholesterol, low-density lipoprotein-cholesterol, and triglycerides were determined. Five SNPs are rs35684 (A/G: a transition substitution in exon 1), rs4684677 (A/T: a missense mutation), rs2075356 (C/T: intron), rs26802 (G/T: intron), and rs26311 (A/G: near the 3' untranslated region) of GHRL were genotyped using TaqMan validated or functionally tested SNP genotyping assays. Our study found no significant evidence of an allele or genotype association between any of the variant sites and body compositions or serum lipids. Furthermore, haplotype frequencies were not found to be significantly different between lean and obese subjects. In summary, the results of our study do not support a significant role for genetic variations in GHRL in the differences of body fat and serum lipid profiles in the NL population.

Altered calcium homeostasis is correlated with abnormalities of fasting serum glucose, insulin resistance, and beta-cell function in the Newfoundland population.

Alteration of extracellular calcium concentration may be involved in glucose metabolism in a number of pathways. The present study was designed to investigate the relationship between total serum calcium and 1) fasting serum glucose, 2) insulin, 3) insulin resistance, and 4) beta-cell function in 1,182 healthy subjects from the province of Newfoundland and Labrador, Canada. All variables were log10 transformed, and confounding factors including age, trunk fat percentage, serum phosphorus, magnesium, 25-OH vitamin D, and parathyroid hormone were adjusted before analyses. Significant positive correlations between glucose and insulin resistance with calcium were found in both sexes, whereas an inverse correlation between beta-cell function and calcium was found only in women. Similar results were found in medication-free women and men, as well as in pre- and postmenopausal women. Subjects with low calcium levels had the lowest concentration of glucose and the least insulin resistance, whereas subjects with high calcium levels had the highest concentration of glucose and insulin resistance in women but not in men. This relationship remained after calcium was adjusted for 25-OH vitamin D and parathyroid hormone. Our results suggest that alteration of serum calcium homeostasis is significantly correlated with the abnormality of glucose level, insulin resistance, and beta-cell function.

Comparison of multifrequency bioelectrical impedance analysis with dual-energy X-ray absorptiometry for assessment of percentage body fat in a large, healthy population.

BACKGROUND: Bioelectrical impedance analysis (BIA) is widely used in clinics and research to measure body composition. However, the results of BIA validation with reference methods are contradictory, and few data are available on the influence of adiposity on the measurement of body composition by BIA. OBJECTIVE: The goal was to determine the effects of sex and adiposity on the difference in percentage body fat (%BF) predicted by BIA compared with dual-energy X-ray absorptiometry (DXA). DESIGN: A total of 591 healthy subjects were recruited in Newfoundland and Labrador. %BF was predicted by using BIA and was compared with that measured by DXA. Methods agreement was assessed by Pearson's correlation and Bland and Altman analysis. Differences in %BF among groups based on sex and adiposity were analyzed by using one-factor analysis of variance with Bonferroni correction. RESULTS: Correlations between BIA and DXA were 0.88 for the whole population, 0.78 for men, and 0.85 for women. The mean %BF determined by BIA (32.89 +/- 8.00%) was significantly lower than that measured by DXA (34.72 +/- 8.66%). The cutoffs were sex specific. BIA overestimated %BF by 3.03% and 4.40% when %BF was <15% in men and <25% in women, respectively, and underestimated %BF by 4.32% and 2.71% when %BF was >25% in men and >33% in women, respectively. CONCLUSIONS: BIA is a good alternative for estimating %BF when subjects are within a normal body fat range. BIA tends to overestimate %BF in lean subjects and underestimate %BF in obese subjects.