Confounding factors of the expression of mTBI biomarkers, S100B, GFAP and UCH-L1 in an aging population.

OBJECTIVES: To evaluate some confounding factors that influence the concentrations of S100 calcium binding protein B (S100B), glial fibrillary acidic protein (GFAP), and ubiquitin carboxyl-terminal hydrolase L-1 (UCH-L1) in older individuals. Indeed, recent guidelines have proposed the combined use of S100B and the "GFAP-UCH-L1" mTBI test to rule out mild traumatic brain injuries (mTBI). As older adults are the most at risk of mTBI, it is particularly important to understand the confounding factors of those mTBI rule-out biomarkers in aging population. METHODS: The protein S100B and the "GFAP and UCH-L1" mTBI test were measured using Liaison XL (Diasorin) and Alinity I (Abbott), respectively, in 330 and 341 individuals with non-suspected mTBI from the SarcoPhAge cohort. RESULTS: S100B, GFAP and UCH-L1 were all significantly correlated with renal function whereas alcohol consumption, Geriatric Depression Score (GDS), smoking habits and anticoagulant intake were not associated with any of these three biomarkers. Body mass index (BMI) and age were associated with GFAP and UCH-L1 expression while sex and mini-mental state examination (MMSE) were only associated with GFAP. According to the manufacturer's cut-offs for mTBI rule-out, only 5.5 % of participants were positive for S100B whereas 66.9 % were positive for the "GFAP-UCH-L1" mTBI test. All positive "GFAP-UCH-L1" mTBI tests were GFAP+/UCH-L1-. Among individuals with cystatin C>1.55 mg/L, 25 % were positive for S100B while 90 % were positive for the mTBI test. CONCLUSIONS: Our data show that confounding factors have different impacts on the positivity rate of the "GFAP-UCH-L1" mTBI test compared to S100B.

Unveiling the Detrimental Effect of Glipizide on Structure and Function of Catalase: Spectroscopic, Thermodynamics and Simulation Studies.

Free radicals, products of oxidative processes, induce cellular damage linked to diseases like Parkinson's and diabetes due to increased reactive oxygen species (ROS) levels. Catalase, crucial for scavenging ROS, emerges as a therapeutic agent against ailments including atherosclerosis and tumor progression. Its primary function involves breaking down hydrogen peroxide into water and oxygen. Research on catalase-drug interactions reveals structural changes under specific conditions, affecting its activity and cellular antioxidant balance, highlighting its pivotal role in defending against oxidative stress-related diseases. Hence, targeting catalase is considered an effective strategy for controlling ROS-induced cellular damage. This study investigates the interaction between bovine liver catalase and glipizide using spectroscopic and computational methods. It also explores glipizide's effect on catalase activity. More than 20% inhibition of catalase enzymatic activity was recorded in the presence of 50 µM glipizide. To investigate the inhibition of catalase activity by glipizide, we performed a series of binding studies. Glipizide was found to form a complex with catalase with moderate affinity and binding constant in the range of 3.822 to 5.063 × 104 M-1. The binding was spontaneous and entropically favourable. The α-helical content of catalase increased from 24.04 to 29.53% upon glipizide complexation. Glipizide binding does not alter the local environment surrounding the tyrosine residues while a notable decrease in polarity around the tryptophan residues of catalase was recorded. Glipizide interacted with numerous active site residues of catalase including His361, Tyr357, Ala332, Asn147, Arg71, and Thr360. Molecular simulations revealed that the catalase-glipizide complex remained relatively stable in an aqueous environment. The binding of glipizide had a negligible effect on the secondary structure of catalase, and hydrogen bonds persisted consistently throughout the trajectory. These results could aid in the development of glipizide as a potent catalase inhibitor, potentially reducing the impact of reactive oxygen species (ROS) in the human body.

Binding of a Drug (Colchicine) to L-Asparaginase Enzyme Using Multispectroscopic, Thermodynamics, and Simulation Studies: Possible Implication in Acute Lymphoblastic Leukemia Treatment.

The research aims to elucidate how drug interactions affect the activity of L-asparaginase (L-ASNase), an essential enzyme in cancer treatment, especially for acute lymphoblastic leukemia (ALL). Understanding these interactions is crucial for optimizing treatment effectiveness and reducing adverse effects. This study explores the intricate molecular interactions and structural dynamics of L-ASNase upon binding with colchicine. Fluorescence quenching experiments were conducted at various temperatures (298, 303, and 310 K), revealing notable interactions between L-ASNase and colchicine. These interactions were characterized by a reduction in fluorescence intensity and a blue shift in emission maxima. Additional analyses, including the determination of Stern-Volmer quenching constants (KSV), bimolecular quenching rate constants (kq), and thermodynamic parameters, indicated a static quenching mechanism with moderate binding affinities (Ka: 1.40-2.71 × 104 M-1) across different temperatures. Thermodynamic study suggested positive enthalpy and entropy changes (ΔH° = -10.26 kcal mol-1; ΔS° = -14.19 cal mol-1 K-1), suggesting a spontaneous reaction with negative ΔG° values (-5.86 to -6.03 kcal mol-1). FRET measurements supported optimal distances (r and Ro) for FRET occurrence, reinforcing the static quenching mechanism. Molecular docking further supported these findings, revealing a 1:1 stoichiometric binding ratio for L-ASNase:colchicine and elucidating specific binding orientations and interactions critical for complex stability. Subsequent molecular dynamics simulations spanning 100 ns underscored the stability of the L-ASNase-colchicine complex, with minimal deviations observed in key structural parameters such as RMSD, RMSF, Rg, and SASA. Additionally, spectroscopic analyses, including circular dichroism (CD), synchronous fluorescence, and 3D fluorescence provided insights into the conformational changes and alterations in the microenvironment of aromatic amino acid residues in L-ASNase upon colchicine binding. Moreover, L-ASNase activity was slightly reduced by 25% in the presence of colchicine. This comprehensive investigation sheds light on the molecular intricacies of the L-ASNase-colchicine complex, advancing our understanding of drug-target interactions and offering potential avenues for therapeutic applications.

Non-enzymatic glycation and aggregation of camel immunoglobulins induce breast cancer cell proliferation.

Glycation of biomolecules results in the formation of advanced glycation end products (AGEs). Immunoglobulin G (IgG) has been implicated in the progression of various diseases, including diabetes and cancer. This study purified three IgG subclasses (IgG1, IgG2, and IgG3) from Camelus dromedarius colostrum using ammonium sulfate fractionation and chromatographic procedures. SDS-PAGE was performed to confirm the purity and molecular weight of the IgG subclasses. Several biochemical and biophysical techniques were employed to study the effect of glycation on camel IgG using methylglyoxal (MGO), a dicarbonyl sugar. Early glycation measurement showed an increase in the fructosamine content by ~four-fold in IgG2, ~two-fold in IgG3, and a slight rise in IgG1. AGEs were observed in all classes of IgGs with maximum hyperchromicity (96.6%) in IgG2. Furthermore, glycation-induced oxidation of IgGs led to an increase in carbonyl content and loss of -SH groups. Among subclass, IgG2 showed the highest (39.7%) increase in carbonyl content accompanied by 82.5% decrease in -SH groups. Far UV-CD analysis illustrated perturbation of ß-sheet structure during glycation reaction with MGO. Moreover, glycation of IgG proceeds to various conformational states like aggregation and increased hydrophobicity. In addition, the cytotoxicity assay (MTT) illustrated the proliferation of breast cancer cells (MCF-7) with IgG2 treatment.

Agitation does not induce fibrillation in reduced hen egg-white lysozyme at physiological temperature and pH.

Several proteins and peptides tend to form an amyloid fibril, causing a range of unrelated diseases, from neurodegenerative to certain types of cancer. In the native state, these proteins are folded and soluble. However, these proteins acquired ß-sheet amyloid fibril due to unfolding and aggregation. The conversion mechanism from well-folded soluble into amorphous or amyloid fibril is not well understood yet. Here, we induced unfolding and aggregation of hen egg-white lysozyme (HEWL) by reducing agent dithiothreitol and applied mechanical sheering force by constant shaking (1000 rpm) on the thermostat for 7 days. Our turbidity results showed that reduced HEWL rapidly formed aggregates, and a plateau was attained in nearly 5 h of incubation in both shaking and non-shaking conditions. The turbidity was lower in the shaking condition than in the non-shaking condition. The thioflavin T binding and transmission electron micrographs showed that reduced HEWL formed amorphous aggregates in both conditions. Far-UV circular dichroism results showed that reduced HEWL lost nearly all alpha-helical structure, and ß-sheet secondary structure was not formed in both conditions. All the spectroscopic and microscopic results showed that reduced HEWL formed amorphous aggregates under both conditions.

Effect of Antihypertensive Drug (Chlorothiazide) on Fibrillation of Lysozyme: A Combined Spectroscopy, Microscopy, and Computational Study.

Amyloid fibrils abnormally accumulate together in the human body under certain conditions, which can result in lethal conditions. Thus, blocking this aggregation may prevent or treat this disease. Chlorothiazide (CTZ) is a diuretic and is used to treat hypertension. Several previous studies suggest that diuretics prevent amyloid-related diseases and reduce amyloid aggregation. Thus, in this study we examine the effects of CTZ on hen egg white lysozyme (HEWL) aggregation using spectroscopic, docking, and microscopic approaches. Our results showed that under protein misfolding conditions of 55 °C, pH 2.0, and 600 rpm agitation, HEWL aggregated as evidenced by the increased turbidity and Rayleigh light scattering (RLS). Furthermore, thioflavin-T, as well as trans electron microscope (TEM) analysis confirmed the formation of amyloid structures. An anti-aggregation effect of CTZ is observed on HEWL aggregations. Circular dichroism (CD), TEM, and Thioflavin-T fluorescence show that both CTZ concentrations reduce the formation of amyloid fibrils as compared to fibrillated. The turbidity, RLS, and ANS fluorescence increase with CTZ increasing. This increase is attributed to the formation of a soluble aggregation. As evidenced by CD analysis, there was no significant difference in α-helix content and ß-sheet content between at 10 µM CTZ and 100 µM. A TEM analysis of HEWL coincubated with CTZ at different concentrations validated all the above-mentioned results. The TEM results show that CTZ induces morphological changes in the typical structure of amyloid fibrils. The steady-state quenching study demonstrated that CTZ and HEWL bind spontaneously via hydrophobic interactions. HEWL-CTZ also interacts dynamically with changes in the environment surrounding tryptophan. Computational results revealed the binding of CTZ to ILE98, GLN57, ASP52, TRP108, TRP63, TRP63, ILE58, and ALA107 residues in HEWL via hydrophobic interactions and hydrogen bonds with a binding energy of -6.58 kcal mol-1. We suggest that at 10 µM and 100 µM, CTZ binds to the aggregation-prone region (APR) of HEWL and stabilizes it, thus preventing aggregation. Based on these findings, we can conclude that CTZ has antiamyloidogenic activity and can prevent fibril aggregation.

Binding Studies of Caffeic and p-Coumaric Acid with α-Amylase: Multispectroscopic and Computational Approaches Deciphering the Effect on Advanced Glycation End Products (AGEs).

Alpha-amylase (α-amylase) is a key player in the management of diabetes and its related complications. This study was intended to have an insight into the binding of caffeic acid and coumaric acid with α-amylase and analyze the effect of these compounds on the formation of advanced glycation end-products (AGEs). Fluorescence quenching studies suggested that both the compounds showed an appreciable binding affinity towards α-amylase. The evaluation of thermodynamic parameters (ΔH and ΔS) suggested that the α-amylase-caffeic/coumaric acid complex formation is driven by van der Waals force and hydrogen bonding, and thus complexation process is seemingly specific. Moreover, glycation and oxidation studies were also performed to explore the multitarget to manage diabetes complications. Caffeic and coumaric acid both inhibited fructosamine content and AGE fluorescence, suggesting their role in the inhibition of early and advanced glycation end-products (AGEs). However, the glycation inhibitory potential of caffeic acid was more in comparison to p-coumaric acid. This high antiglycative potential can be attributed to its additional -OH group and high antioxidant activity. There was a significant recovery of 84.5% in free thiol groups in the presence of caffeic acid, while coumaric attenuated the slow recovery of 29.4% of thiol groups. In vitro studies were further entrenched by in silico studies. Molecular docking studies revealed that caffeic acid formed six hydrogen bonds (Trp 59, Gln 63, Arg 195, Arg 195, Asp 197 and Asp 197) while coumaric acid formed four H-bonds with Trp 59, Gln 63, Arg 195 and Asp 300. Our studies highlighted the role of hydrogen bonding, and the ligands such as caffeic or coumaric acid could be exploited to design antidiabetic drugs.

Associations of Bone Mineral Density with RANKL and Osteoprotegerin in Arab Postmenopausal Women: A Cross-Sectional Study.

Background and objective: There is limited information as to the association of several key bone markers with bone mineral density (BMD) in understudied ethnic groups. This study investigated the relationship between circulating levels of osteoprotegerin (OPG) and receptor activator of nuclear factor kappa-Β ligand (RANKL) with BMD in Arab postmenopausal women. Materials and methods: In this cross-sectional study, a total of 617 Saudi postmenopausal women from the Osteoporosis Registry of the Chair for Biomarkers of Chronic Diseases were included. Anthropometric data, BMD, and biochemical data were retrieved from the registry. Participants were stratified into three groups based on T-score; n = 169 with osteoporosis, n = 282 with osteopenia, and n = 166 normal. Analysis of bone markers including RANKL, OPG, osteocalcin, and N-terminal telopeptide (NTx) was completed using commercially available bioassays. Results: The results suggested that OPG was significantly and positively correlated with age in the osteoporosis group (r = 0.29, p < 0.05), while it was inversely correlated with BMD femoral neck left (r = −0.56, p < 0.001) and BMD femoral neck right (r = −0.37, p < 0.05) in the same group. Moreover, RANKL showed a significant inverse correlation with NTx in the osteopenia group (r = −0.37, p < 0.05). Furthermore, the RANKL/OPG ratio had a positive and significant correlation with BMI (r = 0.34, p < 0.05), BMD femoral neck left (r = 0.36, p < 0.05) and BMD femoral neck right (r = 0.35, p < 0.05) in the osteopenia group. By contrast, it showed a significant inverse correlation with waist to hip ratio in the osteoporosis group (r = −0.38, p < 0.05). Multiple regression analysis showed that OPG contributes to BMD variations in the osteopenia group (p = 0.03). Conclusions: In conclusion, changes in circulating levels of RANKL and OPG might be a protective mechanism contrary to the increased bone loss in postmenopausal women.

The association between iron and vitamin D status in Arab adolescents.

OBJECTIVE: Both vitamin D and Fe micronutrient deficiencies are common in Saudi Arabia but the association between them is unclear. The present study aimed to determine whether Fe indices are associated with vitamin D status and other metabolic markers in Arab adolescents. DESIGN: Single-centre, cross-sectional study gathering anthropometrics, glucose and lipid profile. Serum 25-hydroxyvitamin D (25(OH)D), Fe, total iron-binding capacity (TIBC), transferrin saturation (%) and other parameters were measured. SETTING: Vitamin D School Project Database, King Saud University (2014-2016). PARTICIPANTS: Arab adolescents aged 10-17 years randomly selected from the Vitamin D School Project Database (170 Saudi students; 100 girls, seventy boys). RESULTS: Among Fe indices, only TIBC was found to be significantly and inversely associated with 25(OH)D (r = -0·20; P < 0·01) and only in girls (r = -0·20; P < 0·05). Among cardiometabolic parameters, serum Fe was associated with TAG in boys (r = 0·36; P < 0·01) and inversely associated with HDL-cholesterol in girls (r = -0·29; P < 0·05). Age was the most significant predictor of serum Fe for all participants, accounting for 5 % (R2 = 0·05; P = 0·004) of variance perceived. Serum 25(OH)D and age, on the other hand, were the most significant predictors for TIBC, accounting for 10·1 % (R2 = 0·10; P < 0·001) of variance perceived. CONCLUSIONS: Among healthy Arab adolescents, the association between vitamin D and Fe indices, particularly TIBC, is modest, inverse and sex-dependent. Larger studies with inclusion of markers such as hepcidin and ferritin, vitamin D metabolites and endogenous sex hormones may provide a clearer view of this complex association.

Strong parent-child correlation in circulating vitamin B12 levels and its association with inflammatory markers in Saudi families.

Vitamin B12 deficiency leads to adverse effects on human health, but limited information is available as to whether abnormal vitamin B12 levels are associated between parents and offspring. The present study aimed to assess the association between circulating levels of vitamin B12 in Saudi parents and their children as well as its association with pro-inflammatory markers. A total of 104 Saudi families: 49 fathers, 63 mothers, 94 sons and 79 daughters were selected for the study. Fasting blood samples and anthropometrics were collected. Biochemical parameters, various pro-inflammatory markers and vitamin B12 were measured. Results showed a significant positive correlation between B12 levels in most parent-offspring pairs: mother-daughter (N = 46 pairs, r = 0.72, p < 0.0001); father-daughter (N = 39, r = 0.62, p < 0.0001) and mother-son (N = 51, r = 0.42, p < 0.01). This association was absent in father-son pairs (N = 48, r = 0.26, p = 0.09). Also, B12 was inversely associated with tumor necrosis factor-α and plasminogen activator inhibitor-1 in parents (r = -0.32; p < 0.01 and r = -0.31; p < 0.01 respectively) and children (r = -0.14; p < 0.01 and r = -0.19; p < 0.01 respectively). A significant inverse correlation was found between vitamin B12 and leptin in mothers (r = -0.31, p < 0.05). Our study suggests a strong familial component between B12 levels indicating a possible genetic influence on individual B12 status. Our study also suggests an inverse correlation between circulating levels of vitamin B12 and pro-inflammatory markers. The present study highlights the importance of extending screening in families of patients with abnormal B12 levels and expanding treatment, if necessary, to maximize clinical benefits.

Circulating spexin levels are influenced by the presence or absence of gestational diabetes.

The study aimed to determine whether circulating spexin (SPX) is modified during the course of pregnancy and whether it is affected by the presence of glucose intolerance, i.e., Gestational Diabetes Mellitus (GDM). This prospective study included 102 pregnant women (63 non-GDM and 39 GDM; mean age 29.4 ±â€¯5.1 years; mean BMI 28.0 ±â€¯6.1 kg/m2). Anthropometrics, glycemic and lipid profiles, as well measurements of circulating adipocytokines and SPX were measured at baseline and after 3 and 6 months. In GDM patients, SPX levels increased significantly after 6-months, in parallel with a borderline significant increase in glucose (p = 0.07). In non-GDM patients, however, median SPX level decreased from baseline to 6-months (p < 0.01), and this change was not associated with changes in glucose levels. Change in glucose from baseline to 6-months was positively associated with change in SPX in GDM patients only (R = 0.37; p < 0.05). SPX levels are positively influenced by glucose intolerance in pregnant women with GDM, while they decrease in control women without GDM.

Coexistence of Pre-sarcopenia and Metabolic Syndrome in Arab Men.

OBJECTIVE: The present single-center observational study determined the prevalence and coexistence of sarcopenia, pre-sarcopenia, and metabolic syndrome (MetS) among apparently healthy Arab men and whether having both conditions present a unique cardiometabolic profile that is distinct than having the conditions separately. METHODS: A total of 471 out of 530 Arab men aged 20-77 years old were included after screening for the presence of pre-sarcopenia (ALM/ht2 < 7.26 kg/m2), sarcopenia (presence of both low muscle mass and low function), and MetS. MetS screening was done using the definition by the NCEP-ATP III. Based on the screening results, the participants were classified as control (normal) group (N = 328), MetS only (N = 73), pre-sarcopenia only (N = 64), and MetS + pre-sarcopenia (N = 6). RESULTS: Pre-sarcopenia without MetS was observed in 64 participants (13.6%), while MetS without pre-sarcopenia was observed in 73 participants (15.5%). MetS + pre-sarcopenia was observed only in 6 participants (1.3%). None of the participants had sarcopenia. Age- and BMI-adjusted comparisons showed that those with MetS + pre-sarcopenia had the highest diastolic blood pressure and triglyceride levels as compared to all groups (p values < 0.001). MetS + pre-sarcopenia group also had the highest levels of glucose and the lowest lean arms-legs/BMI ratio than control and pre-sarcopenia groups (p values < 0.001 and 0.005, respectively). CONCLUSION: The prevalence of pre-sarcopenia + MetS is low among young adult Arab men, but shows a unique cardiometabolic profile that is worse than those having only one of the conditions. Further investigations should be done among Arab women and the elderly.

Susceptibility to type 2 diabetes may be modulated by haplotypes in G6PC2, a target of positive selection.

BACKGROUND: The endoplasmic reticulum enzyme glucose-6-phosphatase catalyzes the common terminal reaction in the gluconeogenic/glycogenolytic pathways and plays a central role in glucose homeostasis. In most mammals, different G6PC subunits are encoded by three paralogous genes (G6PC, G6PC2, and G6PC3). Mutations in G6PC and G6PC3 are responsible for human mendelian diseases, whereas variants in G6PC2 are associated with fasting glucose (FG) levels. RESULTS: We analyzed the evolutionary history of G6Pase genes. Results indicated that the three paralogs originated during early vertebrate evolution and that negative selection was the major force shaping diversity at these genes in mammals. Nonetheless, site-wise estimation of evolutionary rates at corresponding sites revealed weak correlations, suggesting that mammalian G6Pases have evolved different structural features over time. We also detected pervasive positive selection at mammalian G6PC2. Most selected residues localize in the C-terminal protein region, where several human variants associated with FG levels also map. This region was re-sequenced in ~560 subjects from Saudi Arabia, 185 of whom suffering from type 2 diabetes (T2D). The frequency of rare missense and nonsense variants was not significantly different in T2D and controls. Association analysis with two common missense variants (V219L and S342C) revealed a weak but significant association for both SNPs when analyses were conditioned on rs560887, previously identified in a GWAS for FG. Two haplotypes were significantly associated with T2D with an opposite effect direction. CONCLUSIONS: We detected pervasive positive selection at mammalian G6PC2 genes and we suggest that distinct haplotypes at the G6PC2 locus modulate susceptibility to T2D.

Effects of a multi-strain probiotic supplement for 12 weeks in circulating endotoxin levels and cardiometabolic profiles of medication naïve T2DM patients: a randomized clinical trial.

BACKGROUND: The present randomized clinical trial characterized the beneficial effects of a multi-strain probiotics supplementation on improving circulating endotoxin levels (primary endpoint) and other cardiometabolic biomarkers (secondary endpoint) in patients with T2DM. METHODS: A total of 78 adult Saudi T2DM patients (naïve and without co-morbidities) participated in this clinical trial and were randomized to receive twice daily placebo or probiotics [(2.5 × 109 cfu/g) containing the following bacterial strains: Bifidobacterium bifidum W23, Bifidobacterium lactis W52, Lactobacillus acidophilus W37, Lactobacillus brevis W63, Lactobacillus casei W56, Lactobacillus salivarius W24, Lactococcus lactis W19 and Lactococcus lactis W58 (Ecologic®Barrier)] in a double-blind manner for 12 weeks. Anthropometrics and cardiometabolic profiles were obtained at baseline and after 12/13 weeks of treatment. RESULTS: After 12/13 weeks of intervention and using intention-to-treat analysis, no difference was noted in endotoxin levels between groups [Placebo - 9.5% vs. Probiotics - 52.2%; (CI - 0.05 to 0.36; p = 0.15)]. Compared with the placebo group however, participants in the probiotics groups had a significant but modest improvement in WHR [Placebo 0.0% vs. Probiotics 1.11%; (CI - 0.12 to - 0.01; p = 0.02)] as well as a clinically significant improvement in HOMA-IR [Placebo - 12.2% vs. Probiotics - 60.4%; (CI - 0.34 to - 0.01; p = 0.04)]. CONCLUSION: Using a multi-strain probiotic supplement daily for 12/13 weeks significantly improved HOMA-IR and modestly reduced abdominal adiposity among medication naïve T2DM patients. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT01765517 , Registered January 10, 2013.

Splice variants of the extracellular region of RON receptor tyrosine kinase in lung cancer cell lines identified by PCR and sequencing.

BACKGROUND: Altered expression of receptor tyrosine kinases (RTKs) is a major driver of growth and metastasis of cancers. Recepteur d'origine nantais (RON) receptor is a single-pass transmembrane RTK aberrantly expressed in a number of cancers. Efforts to block deregulated RON signaling in tumors using small molecule kinase inhibitors or antibodies are complicated by the presence of unknown number/types of isoforms of RON, which, despite having similar sequences, are localized differently and mediate varied functions. The objective of this study was to identify splice variants of RON transcripts between exons 1 and 10 that code for the extracellular region. METHODS: Direct cDNA sequencing was performed for the transcript between exons 1-10 of RON by Sanger sequencing in various lung cancer cell lines. RESULTS: PCR amplification and bi-directional sequencing of cDNA for section between exons 1 and 10 from lung cancer cell lines revealed the presence of several splice variants of RON transcripts; the variants were formed by skipping of exons 2, 2-3, 5-6, 6 and 8-9. Each of these transcript variants were found in one or more cell lines. While the variants formed by skipping of exons 2, 2-3 and 5-6 resulted in loss of 63, 106 and 109 amino acids, respectively, and didn't cause reading-frameshift, the transcripts formed by skipping of exons 6 and 8-9 caused reading-frameshift. Splice variant lacking exons 8-9 was found in 13 out of 23 cell lines tested. CONCLUSION: Lung cancer cell lines contain several splice variants of RON which involve skipping of exons coding for extracellular region. Some of the splicing changes result in reading-frameshift and the N-terminally truncated isoforms are expected to be secreted out. The ubiquitous nature of alternative splicing events in RON suggests the need for isoform specific approaches to functional analysis and therapeutic targeting of RON.

Sex-specific vitamin D effects on blood coagulation among overweight adults.

BACKGROUND: Overweight adults are at increased risk for cardiovascular disease and vitamin D deficiency, whereas an important feature to vitamin D physiology is its sex dependence. The aim of this study was to examine whether vitamin D status improvement exerts a sexually dimorphic effect on serum proteins associated with cardiovascular risk among overweight adults. MATERIALS AND METHODS: Unprocessed serum from age- and BMI-matched men (n = 26) and premenopausal women (n = 24) with vitamin D deficiency and after they achieved sufficiency through a 12-month nutritional intervention was analysed using our previously published depletion-free quantitative proteomics method. Key findings were verified with ELISA. Differentially expressed proteins were subjected to in silico bioinformatics assessment using principal component analysis, hierarchical clustering and Metacore™ pathway analysis. All mass spectrometry proteomic data are available via ProteomeXchange (identifier: PXD003663). RESULTS: A total of 282 proteins were differentially expressed after the intervention between men and women (P-value ≤ 0·05), in which the blood coagulation pathway was significantly enriched. In agreement with the proteomics findings, ELISA measurements showed vitamin K-dependent protein C, von Willebrand factor, fibrinogen gamma chain and multimerin-1 proteins, of relevance to blood coagulation, to be differentially affected (P-value ≤ 0·05) between sexes after vitamin D status correction. CONCLUSIONS: This study identified novel protein-level molecular indicators on the sexually dimorphic effect of vitamin D status correction associated with blood coagulation among overweight adults. These sex-mediated vitamin D effects should be factored in the design and interpretation of vitamin D observational and interventional studies testing cardiometabolic outcomes.

SNPs in FNDC5 (irisin) are associated with obesity and modulation of glucose and lipid metabolism in Saudi subjects.

BACKGROUND: Irisin is a recently identified myokine that plays an important role in preventing obesity and insulin resistance. We investigated whether the common FNDC5 (irisin precursor) gene variants influence susceptibility to obesity and type 2 diabetes (T2D) and verified the impact of FNDC5 gene variants on serum irisin levels, glucose and lipid metabolism in a Saudi population. METHODS: Genomic DNA from 814 (394 T2DM and 414 controls) subjects were genotyped for the five common SNPs (rs3480A/G, rs1746661G/T, rs1298190A/G, rs726344A/G and rs1570569G/T) of the FNDC5 gene using the TaqMan genotyping assay. Biochemical parameters and hematic concentrations of irisin and insulin as well as anthropometric indices were collected. RESULTS: Serum irisin levels were higher in T2DM patients compared to controls (p < 0.0001). Analyses of FNDC5 SNPs showed that: 1) The rs3480 GG associates with decreased risk of obesity (p = 0.005; odds ratio: 0.48) and lower body mass index (BMI) values (p = 0.03). In addition, GGAAG was identified as the protective haplotype against risk of obesity (p = 0.001; odds ratio: 0.23). 2) The rs1746661 G allele associates with higher triglyceride (TG) levels (p = 0.019). 3) The rs157069 TT genotype associates with higher fasting insulin (p = 0.029) and HOMA-IR (p = 0.002) as well as with lower circulating irisin levels (p = 0.016). CONCLUSIONS: SNPs in FNDC5 gene correlates with obesity and glucose-lipid metabolism possibly because they modulate the serum levels of irisin.

Sensitivity of various adiposity indices in identifying cardiometabolic diseases in Arab adults.

BACKGROUND: Obesity is a recognized risk factor for various cardiometabolic diseases and several indices are used clinically to assess overall cardiometabolic risk. This study aims to determine the sensitivity of six anthropometric indices [Body mass index (BMI), waist, waist-to-hip ratio (WHR), waist-to-height ratio (WHtR), body adiposity index (BAI) and visceral adiposity index (VAI)] in determining diabetes mellitus type 2, coronary heart disease, dyslipidemia, hypertension and metabolic syndrome (MetS) in Saudi adults recruited from two independent cohorts (2008-2009 and 2013-2014). METHODS: A total of 6,821 Saudi adults [2008-2009, N = 3,971 (1,698 males and 2,273 females); 2013-2014, N = 2,850 (926 males and 1,924 females)] aged 18-70 years old were included in this descriptive, cross-sectional study. Anthropometrics were obtained and fasting blood samples analyzed for glucose and lipids. BMI, WHR, WHtR, BAI and VAI were computed mathematically. RESULTS: VAI was the most sensitive index in determining DMT2 (AUC 0.72; p < 0.001) in the 2008-2009 cohort and MetS (AUC = 0.84; p < 0.001) in the 2013-2014 cohort. WHR was most discriminating for CHD in both cohorts (AUC 0.70 and 0.84 for 2008-2009 and 2013-2014, p values <0.001, respectively). WHtR was most sensitive but rather modest in determining hypertension (AUC 0.66; p < 0.001), while waist circumference was most sensitive for dyslipidemia (AUC 0.72; p < 0.001) in the 2008-2009 cohort and MetS (AUC 0.85; p < 0.001) in the 2013-2014 cohort. BAI was the least sensitive adiposity index. CONCLUSION: Sensitivity of adiposity indices regarding cardiometabolic diseases highlight the importance of body fat distribution in determining overall cardiometabolic risk, with indices involving abdominal obesity being more clinically significant than BMI and BAI. The sensitivity of these adiposity indices should be noted in assessing a particular cardiometabolic disease.

Habitual physical activity is associated with circulating irisin in healthy controls but not in subjects with diabetes mellitus type 2.

BACKGROUND: Irisin, a novel myokine, has been shown to increase following vigorous exercise, with studies suggesting that it mediates some of the beneficial effects of exercise. Irisin might play a role in 'browning' of white adipocytes, thus increasing energy expenditure. The role of irisin in exercise and energy expenditure in subjects with diabetes mellitus type 2 (DMT2) remains largely unknown. We aimed to investigate the association between circulating irisin and habitual physical activity in subjects with and without DMT2. MATERIAL AND METHODS: In this cross-sectional study, 164 Saudi adults: 81 non-DMT2 controls [age: (mean ± SD) 51.6 ± 10.9; BMI: 29.6 ± 4.3 kg/m(2) ] and 83 DMT2 subjects [age: 54.3 ± 10.3 year; BMI: 29.4 ± 4.7 kg/m(2) ] were studied. Anthropometric and fasting serum biochemical data were collected. Circulating irisin was measured using an enzyme-linked immunosorbent assay (ELISA). Frequency intensity time (FIT) index was used to assess the level of habitual physical activity. RESULTS: We observed significantly higher levels of irisin in DMT2 subjects than in controls (P < 0.001). FIT index was positively associated (r = 0.20, P = 0.03) with circulating irisin in controls only. Additionally, irisin levels were significantly higher in tertile 3 (0.75 ± 0.07 µg/mL) than tertile 1 (0.49 ± 0.06 µg/mL) of the FIT index in healthy controls, whilst no such relation with physical activity was observed in DMT2 subjects. CONCLUSION: This cross-sectional study has shown a weak association of irisin with physical activity levels in healthy controls but not in DMT2 subjects, suggesting the possibility of discordant regulation in the condition of DMT2.

A nonsense polymorphism (R392X) in TLR5 protects from obesity but predisposes to diabetes.

The TLR5 gene encodes an innate immunity receptor. Mice lacking Tlr5 (T5KO) develop insulin resistance and increased adiposity. Owing to the segregation of a dominant nonsense polymorphism (R392X, rs5744168), a portion of humans lack TLR5 function. We investigated whether the nonsense polymorphism influences obesity and susceptibility to type 2 diabetes (T2D). R392X was genotyped in two cohorts from Saudi Arabia, a region where obesity and type 2 diabetes (T2D) are highly prevalent. The nonsense allele was found to protect from obesity (p(combined) = 0.0062; odds ratio, 0.51) and to associate with lower body mass index (BMI) (p(combined) = 0.0061); this allele also correlated with a reduced production of proinflammatory cytokines. A significant interaction was noted between rs5744168 and sex in affecting BMI (p(interaction) = 0.006), and stratification by gender revealed that the association is driven by females (p(combined) = 0.0016 and 0.0006 for obesity and BMI, respectively). The nonsense polymorphism also associated with BMI in nonobese women. After correction for BMI, the 392X allele was found to represent a risk factor for T2D with a sex-specific effect (p(interaction) = 0.023) mediated by females (p = 0.021; odds ratio, 2.60). Fasting plasma glucose levels in nondiabetic individuals were also higher in women carrying the nonsense allele (p = 0.012). Thus, in contrast to T5KO mice, loss of human TLR5 function protects from weight gain, but in analogy to the animal model, the nonsense allele predisposes to T2D. These effects are apparently sex-specific. Data in this study reinforce the hypothesis that metabolic diseases, including T2D, are associated with immune dysregulation.