A major portion of available fatty acids for adipocyte uptake is derived from lipoprotein lipase (LPL)-mediated hydrolysis of circulating lipoprotein particles. In vivo studies aimed at identifying the precise role of adipocyte-derived LPL in fat storage function of adipose tissue have been unable to provide conclusive evidence due to compensatory mechanisms that activate endogenous fatty acid synthesis. To address this gap in knowledge, we have measured the effect of reducing adipocyte LPL expression on intracellular lipid accumulation using a well-established cultured model of adipocyte differentiation.siRNA specific for mouse LPL was transfected into 3T3-L1 adipocytes. Expression of LPL was measured by quantitative real-time PCR and cell surface-associated LPL enzymatic activity was measured by colorimetric detection following substrate (p-nitrophenyl butyrate) hydrolysis. Apolipoprotein CII and CIII expression ratios were also measured by qRT-PCR. Intracellular lipid accumulation was quantified by Nile Red staining.During differentiation of 3T3-L1 pre-adipocytes, LPL mRNA expression increases 6-fold resulting in a 2-fold increase in cell surface-associated LPL enzymatic activity. Parallel to this increase in LPL expression, we found that intracellular lipids increased ~10-fold demonstrating a direct correlation between adipocyte-derived LPL expression and lipid storage. We next reduced LPL expression in adipocytes using siRNA transfections to directly quantify the contributions of adipocyte-derived LPL to lipid storage, This treatment reduced LPL mRNA expression and cell surface-associated LPL enzymatic activity to ~50% of non-treated controls while intracellular lipid levels were reduced by 80%. Exogenous addition of purified LPL (to restore extracellular lipolytic activity) or palmitate (as a source of free fatty acids) to siRNA-treated cells restored intracellular lipid levels to those measured for non-treated controls. We also found that adipocytes express apolipoprotein CII and CIII and, in addition, the apoCII/apoCIII ratio remains largely unchanged in cells with reduced LPL expression.We provide evidence that adipocyte-derived LPL is required for efficient fatty acid uptake and storage, and that adipocytes express their own source of apoCII and apoCIII for regulating extracellular LPL activity. These findings demonstrate that adipocytes are capable of producing the necessary enzymatic components and co-factors for efficient lipid storage independent of vascular sources. The adipocyte plays a crucial role in metabolic regulation, serving as a storage depot for fatty acids and as an endocrine cell to manage energy utilization and feeding behavior ,2. The mThe availability of VLDL-derived fatty acids for energy needs and adipocyte storage is dependent upon hydrolytic activity of lipoprotein lipase (LPL) -6, and tLipoprotein particles that are transported out of the vasculature represent a significant source of fatty acids for adipocyte storage. The release of fatty acids from these particles is likely to be dependent on adipocyte-derived LPL activity. One of our recent studies provides evidence that HSPG found on the surface of adipocytes are essential for lipid accumulation in adipocytes . AlthougAn essential element necessary for this mechanism to be effective is adipocyte-derived LPL activity -27. Stud2 and passaged twice weekly. To differentiate 3T3-L1 cells into adipocytes, cells were incubated with 250 nM dexamethasone, 450 μM 3-isobutyl-1-methylxanthine, and 167 nM insulin for 2 days, followed by 167 nM insulin for an additional 3 days.3T3-L1 cells were obtained from American Type Culture Collection  and grown in Dulbecco's modified Eagle's medium (DMEM)  supplemented with 10% (v/v) fetal calf serum , 1 mM sodium pyruvate, 0.1 mM non-essential amino acids, 2 mM L-glutamine, 100 μg/ml streptomycin sulfate, and 100 units/ml penicillin. Cells were cultured at 37°C with 10% CO® pre-designed small interfering RNA (siRNA) directed toward mouse lipoprotein lipase (LPL) was synthesized by Ambion Inc. . The target sequence for the siLPL is on exon 2, with a sense sequence of GCAAAUUUGCCCUAAGGACtt and an antisense sequence of GUCCUUAGGGCAAAUUUGCtt. Cells were transfected with siRNA (100 nM) using DharmaFECT™ 3 transfection reagent according to the manufacturer's specific protocol for 3T3-L1 cells . Total RNA was purified from cells using RNeasy  and converted to cDNA using TaqMan Reverse Transcriptase . LPL, leptin, apoCII, apoCIII, and β-actin expression levels were measured by quantitative Real Time PCR analysis (qRT-PCR) of cDNA samples. Primers specific for LPL [GenBank:NM_008509] were designed to amplify 231 base pairs flanking intron 4. Primer sequences for LPL were: upstream, CTGCTGGCGTAGCAGGAAGT; downstream, GCTGGAAAGTGCCTCCATTG. Primers specific for leptin [GenBank:NM_008493] were designed to amplify 213 base pairs flanking intron 2. Primer sequences for leptin were: upstream, TGACACCAAAACCCTCATCA; downstream, TCATTGGCTATCTGCAGCAC. Primers specific for apoCII [GenBank: NM_009695] were designed to amplify 147 base pairs flanking intron 2. Primer sequences for apoCII were: upstream, GCAGGGCTCCCTCTTAAGTT; downstream, AAAATGCCTGCGTAAGTGCT. Primers specific for apoCIII [GenBank: NM_023114] were designed to amplify 99 base pairs flanking intron 3. Primer sequences for apoCIII were: upstream, GGCTGGATGGACAATCACTT; downstream, TGGTTGGTCCTCAGGGTTAG. Primers specific for β-actin [GenBank:NM_007393] were designed to amplify 287 base pairs flanking intron 3. Primer sequences for β-actin were: upstream, CCTGAACCCTAAGGCCAACC; downstream, CAGCTGTGGTGGTGAAGCTG. qRT-PCR was performed using ABsolute QPCR SYBR Green Mix  with the following cycling parameters: 1 cycle, 95°C, 15 min; 40 cycles, 95°C, 15 sec, 60°C, 1 min. β-actin mRNA levels were measured in parallel with identical cycling conditions and used to normalize values obtained for LPL, leptin, apoCII, and apoCIII expression. Relative quantitation was performed using the comparative CT method. For this analysis, the amount of target message  was normalized to the internal reference (β-actin) and compared to the calibrator .Purified Silencer® .For straight RT-PCR reactions of leptin, apoCII, apoCIII and β-actin, isolated mRNA was reverse transcribed as described above and cDNA amplification was performed using TaKaRa Ex Taq™  with the following cycling parameters: 1 cycle, 95°C, 2 min; 40 cycles, 95°C, 30 sec, 60°C, 30 sec, 72 °C 1 min. Amplified products were separated on 3% agarose gels and stained with Gel Star-1cm-1.Cells, grown in 6-well plates, were rinsed twice with phosphate buffered saline, pH 7 (PBS), and incubated with 200 μg/ml heparin  prepared in PBS for 15 min at 25°C. Following incubation, the heparin solution was centrifuged  to remove cell debris and mixed with an equal volume of 2 mM p-nitrophenyl butyrate (PNPB). Absorbance at 405 nm was recorded following a 10 min incubation using a Genesys UV Spectrophotometer. LPL activity is reported as the amount of p-nitrophenol product formed over the 10 min incubation. The molar extinction coefficient of p-nitrophenol at 405 nm is 11,000 MPalmitate , purified bovine LPL, and fatty acid-free albumin were purchased from Sigma. Palmitate-albumin complexes were prepared as follows. Palmitate was dissolved in 3% albumin (wt/v) to a final concentration of 10 mM in 1X Hank's Balanced Salt Solution. The mixture was slowly heated to 50°C and stirred overnight. The mixture was then cooled to 37°C, stirred for an additional 3 hours and sterile filtered through a 0.2 μm syringe filter. Protein was quantified using the BCA™ Protein Assay Kit .excitation 488 nm; λemission 585 nm). Nile Red fluorescence was quantitated using a Shimadzu RF-1501 spectrofluorophotometer .Cells were rinsed twice with PBS, pH 7, and trypsinized with phenol red-free 0.05% Trypsin-EDTA  for 5 min at 37°C. Cells were fixed in suspension with 0.5% paraformaldehyde (v/v) in PBS for 10 min at 25°C. A stock solution of 1 mg/ml Nile Red  was prepared in acetone and added to the cell preparation to a final concentration of 5 μg/ml. Cells were incubated at 25°C for 30 min in the dark. Flow cytometry was performed using a FACScan flow cytometer  LPL mRNA expression changes due to siRNA treatment, 2) the resulting affect on cell surface LPL activity levels, and 3) performed a quantitative evaluation of intracellular lipid accumulation Fig. . SimilarSince siRNA-mediated inhibition of LPL in adipocytes significantly reduced intracellular lipid storage, we reasoned that the exogenous addition of purified LPL (to restore extracellular lipase activity lost due to siRNA treatment) or palmitate (to supply cells with a source of available free fatty acid thus abrogating the need for LPL hydrolysis activity) would be expected to reverse the phenotype seen in siRNA treated cells. To establish control values, adipocytes were treated with or without siRNA for LPL and intracellular lipid accumulation was quantified by Nile Red staining as was done in Figure ApoCII and apoCIII are known to be, respectively, a strong activator and strong inhibitor of LPL ,33. WithSince apoCII and apoCIII have opposite effects on LPL activity, it is generally accepted that the state of LPL activity depends on the ratio of apoCII/apoCIII expression. A high ratio of apoCII/apoCIII promotes LPL activity, whereas a low apoCII/apoCIII ratio inhibits LPL function. Since this ratio can significantly affect extracellular LPL activity and in turn fatty acid uptake and storage by adipocytes, we determined apoCII and apoCIII expression levels in adipocytes treated with or without siRNA for LPL. Although expression of both regulators was largely unaffected by LPL-siRNA treatment, we did find a small but reproducible effect on their expression as a result of diminished LPL expression Fig. . ApoCII The generation of fatty acids from dietary sources and their uptake by cells are essential processes for efficient energy metabolism and storage. A general role for LPL in these processes has been confirmed from LPL gene ablation studies in mice which results in a lethal phenotype . NaturalIt is now well established that LPL requires apoCII as a co-factor for efficient triacylglycerol hydrolysis ,40. ThisThe author(s) declare that they have no competing interests.AMG carried out the experiments in this study and participated in the experimental design. RAO provided the original conceptual framework for the study, assisted with the experimental design and finalized the manuscript for submission. All authors read and approved the final version.


This study investigated the role of a negative FAST in the diagnostic and therapeutic algorithm of multiply injured patients with liver or splenic lesions.A retrospective analysis of 226 multiply injured patients with liver or splenic lesions treated at Bern University Hospital, Switzerland.FAST failed to detect free fluid or organ lesions in 45 of 226 patients with spleen or liver injuries (sensitivity 80.1%). Overall specificity was 99.5%. The positive and negative predictive values were 99.4% and 83.3%. The overall likelihood ratios for a positive and negative FAST were 160.2 and 0.2. Grade III-V organ lesions were detected more frequently than grade I and II lesions. Without the additional diagnostic accuracy of a CT scan, the mean ISS of the FAST-false-negative patients would be significantly underestimated and 7 previously unsuspected intra-abdominal injuries would have been missed.FAST is an expedient tool for the primary assessment of polytraumatized patients to rule out high grade intra-abdominal injuries. However, the low overall diagnostic sensitivity of FAST may lead to underestimated injury patterns and delayed complications may occur. Hence, in hemodynamically stable patients with abdominal trauma, an early CT scan should be considered and one must be aware of the potential shortcomings of a "negative FAST". The acr (ATLS®) . But, th (ATLS®) -6. Furth (ATLS®) ,8. This  (ATLS®) .®, an abdominal CT scan is indicated in hemodynamic normal trauma patients with impaired sensorium , and equivocal abdominal findings [The computed tomography (CT) scan of the abdomen is currently considered the gold standard for detecting intra- and retroperitoneal lesions in trauma patients -13. Accofindings .However, trauma centers are equipped with dedicated CT scanners to allow fast access to emergency patients, especially those with multiple injuries . ScanninThe aim of the present study was to investigate the role of FAST in the diagnostic algorithm in multiply injured patients in a modern ED with immediate access to a CT scanner. Therefore, we analysed the results of FAST in 226 multiply injured patients with liver or splenic lesions in relationship to the grade of organ injury. Additionally, we characterized the FAST-false-negative patients and determined the clinical consequences of a false-negative FAST.The study was conducted at the Bern University Hospital, Switzerland between January 2001 and July 2006. An average of 286  multiple injured patients are treated in our level I trauma centre during this time each year .® EUB-6500). In hemodynamically stable patients with the history of a blunt or penetrating abdominal trauma, impaired sensorium, and unclear abdominal clinical findings, a contrast enhanced helical abdominal CT scan (Siemens® Somatom Sensation 16) is conducted.Immediately after primary assessment of the patient by the attending surgeon, a senior resident in radiology performs bedside the FAST to detect free fluid  a FAST examination on admission, and (2) a spleen or liver lesion documented by (3) an abdominal intravenous contrast enhanced helical CT scan were included in this study. A total of 226 patients fulfilled these three criteria.In the first step, the original radiological reports of the FAST and CT scans, and inpatient records were systematically reviewed. Data concerning the mechanism of injury and accompanying extra- and intra abdominal injuries were collected.The injury severity score (ISS), according to Baker et al., was calculated and the Abbreviated-Injury-Scale (AIS) grading for chest and neurological trauma was used ,19. The Free intra-abdominal fluid or liver and/or spleen lesions detected by FAST were defined as FAST-positive. Free intra-abdominal fluid or organ lesions detected by CT scan, but not by FAST, were defined as FAST-false-negative.The sensitivity and specificity of FAST was then calculated. We further determined the diagnostic accuracy of FAST in relationship to the severity of the organ lesions as depicted by contrast enhanced helical CT scan according to Mirvis et al ,23.p < 0.05 was considered significant.Statistical calculations were performed using SigmaStat 1.0 . Means  were compared using the Mann-Whitney test, and proportions using the Chi-square and Fischer exact test. A value of A total of 164 male and 62 female multiply injured patients with liver or spleen injuries were included in this study. The mean age of the study cohort was 38 years (SD ± 15.0 years). The mean ISS was 17.8 (SD ± 6.9). A splenic lesion was found in 98 patients (44%), a hepatic lesion in 87 patients (38%), and the combination of splenic and hepatic lesions was found in 41 patients (18%).p = n.s.).In 45 of 226 patients with CT-confirmed spleen or liver injuries, the initial FAST failed to detect free fluid or organ lesions (sensitivity 80.1%). FAST showed free fluid without confirmation in the CT examination in one patient with a grade II liver contusion and a subcapsular hematoma (specificity 99.5%). The overall positive and negative predictive values were 99.4% and 83.3%, respectively. The overall likelihood ratios for a positive and negative FAST were 160.2 and 0.2, respectively. Of 41 patients with a combination of hepatic and splenic lesions, FAST was false negative in 7 cases (sensitivity 82.9%). The diagnostic accuracy of FAST was identical for splenic and hepatic injuries . In 21 of 53 patients with grade I hepatic or splenic lesions, FAST could not detect free fluid or any organ lesion (sensitivity 60.4%). In grade II lesions, the sensitivity was 78.6%, and in grade III lesions it was 88.6%. In 32 patients with grade IV and V spleen or liver injuries, FAST could always detect either free fluid or directly demonstrate the organ lesion (sensitivity 100%).Table Of the 7 FAST-false-negative patients with a combination of splenic and hepatic lesions, two had a grade III liver injury in combination with a grade I spleen injury. The five other patients had combinations of grade I and II hepatic and splenic lesions.p = n.s.). There was no difference in the gender ratio between these two groups (Chi-square test: p = n.s.).The mean age of the FAST-false-negative patients was 42 years (SD ± 19.1 years) and 37 years (SD ± 19.0 years) in the FAST-positive group (Mann-Whitney test: Table p = n.s.). The over-all mean ISS of the FAST-false-negative patients was 17.6 (SD ± 10.0). Without the additional diagnostic benefit of a CT scan, the mean ISS of these patients would have been 13.0 (SD ± 10.1), which would significantly underestimate the actual severity of their injuries (Mann-Whitney test: p = 0.0095).The ISS of the FAST-false-negative and FAST-positive patients in relation to the injured organ (spleen or liver) is shown in Table Additional and previously unsuspected intra-abdominal injuries detected by CT scans in the FAST-false-negative patients included: 2 renal contusions (grade II), 1 grade III renal laceration, 2 hemorrhages of the suprarenal gland, 1 colonic perforation, and 1 retroperitoneal hematoma. Of note, surgical intervention was only needed in the patient with colonic perforation. The other incidental findings could be treated conservatively.Further extra-abdominal severe injuries in the 45 FAST-false-negative patients included: 25 (56%) severe thoracic injuries (AIS grade 3 and 4), 8 (18%) severe acute brain injuries , and 5 (11%) unstable pelvic fractures.This clinical study implies that the FAST examination at the primary assessment fails to detect free fluid or organ lesions in 1 of every 5 patients with confirmed spleen or liver injury. On closer examination, it also shows that higher grade lesions were significantly more likely to be identified by FAST than lower grade lesions. This disparity in sensitivity is nicely shown in Table With an exceptional likelihood ratio of a positive result 160.2) FAST is an excellent test when positive. However, the inadequate likelihood ratio of a negative test (0.2) emphasizes the risk to miss intra-abdominal injuries. According to the literature, 11–34% of patients with even high grade spleen and liver injury show no evidence of hemoperitoneum and therefore may appear FAST negative . Morbid 60.2 FASTThe mechanism of injury alone doesn't seem to be a predictive factor for a positive abdominal CT scan too ,28. HoweOf note, 2 patients with grade II splenic lesions and negative initial FAST required a haemostatic splenorrhaphy and a splenectomy due to delayed haemorrhage . From the clinical point of view, it is crucial to thoroughly detect all abdominal injuries early. Even low grade lesions can be the source of relevant  bleeding ,30.Currently, the initial CT diagnostic workup is considered the gold-standard for the systematic evaluation of the polytraumatized patient -13. The FAST is an excellent test when positive. However, the inadequate likelihood ratio of a negative test (0.2) emphasizes the risk to miss intra-abdominal injuries, which has led to a significant underestimation of the injury pattern. High grade lesions can be detected reliably by FAST, but only a CT scan can determine the extent and pattern of parenchymal and vascular injury, and therefore allow for individually tailored and often non-operative therapeutic management. Hence, in hemodynamically stable patients with abdominal trauma, an early CT scan should be considered, and one must be aware of the potential shortcomings of a so-called "negative FAST".The major limitations of this study are its retrospective design and potential for selection bias. The diagnostic capability of ultrasonography depends on the skill and experience of the examiners. Furthermore, with only a few patients with grade IV and V splenic and liver injuries included in this series, our conclusions are cautious.The authors declare that they have no competing interests.BS coordinated the statistical analysis, interpreted the results and drafted the manuscript. JK collected the radiological data and performed the statistical analysis. DI participated in the interpretation of data and draft of the manuscript. MS collected the clinical data and performed the statistical analysis. RK and ML revised the manuscript critically for radiological and emergency medicine contents. DC gave final approval of the version to be published. AK conceived, designed and coordinated the study. HZ gave final approval of the version to be published.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2342/9/3/prepub


Staphylococcus aureus, Mycobacterium tuberculosis and Escherichia coli O157:H7 EDL993) were assessed via comparative genomics methods for theirsuitability as antibacterial targets according to a number of criteria, including theessentiality of the protein, its level of sequence conservation, and its distribution inpathogens, bacteria and eukaryotes . Each protein was scored andranked based on weighted variants of these criteria in order to prioritize proteins aspotential novel broad-spectrum targets for antibacterial drugs. A number of proteinsproved to score highly in all three species and were robust to variations in the scoringsystem used. Sensitivity analysis indicated the quantitative contribution of each metricto the overall score. After further analysis of these targets, tRNA methyltransferase(trmD) and translation initiation factor IF-1 (infA) emerged as potential and novelantimicrobial targets very worthy of further investigation. The scoring strategy usedmight be of value in other areas of post-genomic drug discovery.Single and multiple resistance to antibacterial drugs currently in use is spreading,since they act against only a very small number of molecular targets; finding noveltargets for anti-infectives is therefore of great importance. All protein sequences fromthree pathogens (


In Escherichia coli, two genetically distinct forms of SSADHs had been described that are essential for preventing accumulation of toxic levels of succinic semialdehyde (SSA) in cells.In mammals succinic semialdehyde dehydrogenase (SSADH) plays an essential role in the metabolism of the inhibitory neurotransmitter γ-aminobutyric acid (GABA) to succinic acid (SA). Deficiency of SSADH in humans results in elevated levels of GABA and γ-Hydroxybutyric acid (GHB), which leads to psychomotor retardation, muscular hypotonia, non-progressive ataxia and seizures. E coli gabD gene by X-ray crystallographic studies and compare these data with the structure of human SSADH. In the E. coli SSADH structure, electron density for the complete NADP+ cofactor in the binding sites is clearly evident; these data in particular revealing how the nicotinamide ring of the cofactor is positioned in each active site.Here we structurally characterise SSADH encoded by the E. coli SSADH permits this enzyme to use NADP+, whereas in contrast the human enzyme utilises NAD+. Furthermore, the structure of E. coli SSADH gives additional insight into human mutations that result in disease.Our structural data suggest that a deletion of three amino acids in  The products of the gab operon (which comprises gabT (γ-aminobutyrate transferase), gabD (SSADH), gabP (GABA permease) and gabC (a regulatory gene) sad gene encodes for a NAD+ dependent SSADH (EC 1.2.1.16 and shares 32% identity with gabD) and is an orphan genesad gene is induced by exposure to exogenous SSA and functions primarily to prevent its accumulation in the cell. Furthermore, the sad gene product may also enable growth on putrescine as the nitrogen source In Burkholderia pseudomallei, without a substrate or cofactor (PDB ID: 3ifg and 3ifh), was deposited into the PDB by the Seattle Structural Genomic Centre for Infectious Disease.Recently, the structure of human SSADH has been published gabD gene product (NADP+-dependant) SSADH from E. coliE. coli SSADH is also redox regulated, furthermore it reveals that the bacterial SSADH is structurally suited for NADP+, rather than NAD+ (as utilised by its human counterpart).Despite these recent studies, the structural information of SSADH and its interactions with cofactor remains scarce. Here we report a 2.3 Å X-ray crystal structure of the E. coli SSADH was purified as a tetrameric molecule  which is in agreement with the previous description in the literature 1H NMR, as shown in mK of the purified enzyme is 16.94±2.2 µM and maxV is 40.92±1.3 µM. The enzyme activity measured in the presence of NADP+ is approximately 20-fold higher than that measured in the presence of NAD+ (data not shown) as described previously Recombinant E. coli SSADH reveals four monomers  in the asymmetric unit  and 13 α helices (1–13). The secondary structure assignments used in this report are as shown in + is bound to the active site giving rise to a binary complex, whereas the structure of the N-terminal His-tag was not resolved.The SSADH monomer  adopts a+ via two tandem Rossmann folds in a (βα)4β formation. This is a variation of the classic Rossmann fold 2β motif (residues 148–208) forms the first β strand of the second (βα)2β motif (residues 205–254). The oligomerisation domain comprises an elongated 3-stranded β-sheet (the B-sheet), which interacts with two other monomers in the final tetrameric assembly.The catalytic domain consists of a central 7 stranded β-sheet (the D-sheet) flanked by 2 helices on one side and 3 helices on the other. The catalytic loop (residues 282–290) is located adjacent to the cofactor binding site. The cofactor binding domain interacts with NADP2 buried in the dimer interface  is formed by domain swapping; such that strand s3B of the oligomerisation domain in monomer A forms β-sheet hydrogen bonding with strand s7D of the catalytic domain in monomer B to make a 10 stranded β-sheet . The scrnterface .2 buried in the interface and 10 H-bonds  and superposes with a root-mean-square deviation of 0.79 Å over 472 Cα  and inactive  state has recently been determined  E. coli . These dn of DTT . InteresE. coli SSADH reveals two major regions of structural variation: the first involves the cofactor binding site  resembles the canonical ALDH fold Comparison of human and +  and the active site residues  of human SSADH structures (PDB ID: 2w8o and 2w8q)+ results in formation of the thioacyl enzyme intermediate and NAD(P)H. Lastly, the conserved E254 residue acts as a general base to deprotonate a water molecule prior to its attack on the thioacyl enzyme intermediate resulting in formation of SA and regeneration of the C288 residue. The general base, E254, in all monomers can be modelled in two alternative conformations according to the electron density. It is suggested that the two conformations of E254 is likely to be associated with different stages of the catalytic process, with one conformation “a” tion “a”  being foE. coli SSADH structure, electron density for the cofactor in the binding sites is clearly evident . In human SSADH, 261RKN263  to catalyse the deacylation process.Amongst the well defined cofactor crystal structures, three different conformations of NAD(P)E. coli SSADH numbering in parentheses). The majority (25 mutations) give rise to truncations, deletions, insertions as well as splice site mutants . All these residues superpose well between the human and E. coli structures . The DNA sequence of the PCR product was shown to have a single amino acid change to that of the gabD gene sequence in the database, which is thought to represent a naturally occurring variant in E. coli, the SSADH cDNA was excised from the recombinant pCR® -Blunt vector using the restriction enzymes EcoRI and HindIII, then ligated into pRSETc/His_TEV plasmid as previously described E. coli BL21(DE3) pLysS cells and the transformant was stored at −80°C. For expression of SSADH recombinant protein, transformed E. coli BL21(DE3) cells were propagated in 2YT growth medium in the presence of 100 µg/mL ampicillin and 36 µg/ml chloramphenicol at 37°C to A600 = 0.6 followed by induction at 16°C with 0.5 mM IPTG for 18 hours.The cDNA encoding SSADH was isolated from Harvested cells were treated with lysozyme (1 mg/ml) at 4°C for 30 min and lysed in lysis buffer containing 300 mM NaCl, 10 mM imidazole, 5 mM β-mercaptoethanol, 0.01% triton X-100, 50 mM Tris (pH 8.0) by sonication on ice. Clarified cell lysate was loaded onto a Nickel Chelating Sepharose Fast Flow column , the His-tagged SSADH protein was eluted in 150 mM NaCl, 250 mM imidazole, 5 mM β-mercaptoethanol, 50 mM Tris (pH 8.0). Purified His-tagged SSADH were further purified using a S200 16/60 size exclusion column  pre equilibrated with 100 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol, 30 mM Tris (pH 7.5) for all experiments described in this paper.+ and SSA 0–400 mM at 30°C. The rate at which NADP+ was converted to NADPH was monitored fluorometrically (excitation: 355 nm and emission: 460 nm). SSADH oxidative inhibition (H2O2) assays were carried out by incubating various concentrations of H2O2 with SSADH (1 µg/ml) in a Na phosphate buffer (100 mM pH 8.4), containing 0.75 mM EDTA for 1 hour at room temperature, the reactions were terminated by adding 5.0 mM methionine. To reverse the effect of the H2O2, 10 mM DTT was added to H2O2 treated SSADH and incubated for a further 10 minutes. After the addition of 2.0 mM NADP+ and 0.15 mM SSA, the activity of H2O2 treated SSADH was measured spectrophotomically at 340 nm at 30°C.Enzyme kinetics were carried out using purified SSADH (2 µg/ml) in a Na phosphate buffer (100 mM pH 8.0), containing 1.1 mM NADP1H NMR spectroscopic analysis was carried out with a Bruker DPX 400 MHz spectrometer using purified SSADH in the presence of 125 µM SSA for 240 min as above.Pure SSADH was concentrated to 6 mg/mL and buffer exchanged into 20 mM NaCl, 10 mM β-mercaptoethanol, 5% glycerol, 1.0 mM NADP, 1.0 mM succinic semialdehyde, 30 mM Tris (pH 7.5) using a 10 KDa molecular weight cut off concentrator (Millipore).Crystals of SSADH were grown using hanging drop vapour diffusion by mixing 2.0 µL of protein (6 mg/mL) with 1.0 µL of reservoir solution containing 0.2 M ammonium tartrate, 26–31% polyethylene glycol 3350, 10 mM β-mercaptoethanol and 0.1 M Tris (pH 7.2–7.5). Crystals were dehydrated for 48 hours in 0.2 M ammonium tartrate, 32% polyethylene glycol 3350, 10 mM β-mercaptoethanol and 0.1 M Tris (pH 7.2–7.5) after three days. Crystals were soaked in reservoir solution supplemented with 5% 2-methyl-2,4-pentanediol for 5 minutes then soaked for a further 5 minutes in reservoir solution supplemented with 10% 2-methyl-2,4-pentanediol for cryoprotection before freezing in liquid nitrogen for data collection. Data were collected at the Australian Synchrotron High-throughput protein crystallography (PX1) beam.P4212 with unit cell dimensions of a = 151.88 Å, b = 151.88 Å, c = 165.77 Å, α = β = γ = 90.0°. These data are consistent with four molecules in the asymmetric unit. These data were merged and scaled using MOSFLM free with neither a sigma nor a low-resolution cut-off applied to the data. Summaries of the statistics are provided in SSADH crystals diffracted to 2.3 Å resolution and belong to the space group The structure of SSADH was solved using molecular replacement and the program PHASER A ‘mixed’ model consisting of conserved side chains with all other non-alanine/glycine residues truncated at Cγ atom was then created using the SCRWL server Rfree. Model building and structural validation was performed using COOT Rfree reached 28%. The presence of each water molecule was manually validated. The NADP+ moiety was modelled into the density using PHENIX ligandfit th percentile; Refinement and model building preceded using one molecule (chain A) in the asymmetric unit, with the other chains built using non-crystallographic symmetry operators. Maximum likelihood refinement using REFMACCoordinates and structure factors have been deposited in the Protein Data Bank with accession number 3jz4.http://hdl.handle.net/102.100.100/42) Diffraction image datasets and refinement logs are available online on TARDIS  substrate alone in phosphate buffer which contains succinic semialdehyde (X), 4,4-dihydroxybutanoic acid (Y) and succinic acid (Z). (b) Succinic acid alone with phosphate buffer, showing only the singlet peak of succinic acid (Z). For SSADH enzyme assay, incubation of succinic semialdehyde and SSADH in the presence of NADP+ at 0 min (c) and 240 min (d) showing all substrates  has been converted to succinic acid (Z), NADP+ is marked with W.(0.21 MB TIF)Click here for additional data file.Figure S22-sheet or a cylinder representing an Î±-helix. The secondary structure elements are coloured according the Alignment of E. coli SSADH with human SSADH. Conserved residues have been highlighted according to the following, polar (green), non-polar (yellow), acidic (red) and basic (blue). The secondary structure (E. coli SSADH above the sequence and human below the sequence) has been marked with either an arrow designating a Î(1.46 MB TIF)Click here for additional data file.Figure S3SSADH activity in an oxidised and reduced environment. The first column shows the untreated activity of SSADH. In the second column the E. coli SSADH enzyme was oxidised by incubating it with 200 Î¼M H2O, which has 11–13% of untreated SSADH activity. In the third column the addition of 10 mM DTT to previously oxidised enzyme, which rescued the inhibition to 80% that of the normal activity.(0.16 MB TIF)Click here for additional data file.Figure S4A–F. Human point mutations causing a dramatic loss of acticvity  mapped onto the E. coli SSADH structure. A cartoon representation of E. coli SSADH monomer A  showing the 17 point mutations (magenta spheres) that map to the mature human protein. A small region of monomer B catalytic domain (dark grey) and monomer C oligomerisation domain (light grey) have been included to illustrate the proximity of point mutations with regard to dimer and tetramer interfaces. Labelled, dashed red boxes highlight the area of E. coli SSADH that have been enlarged for analysis. A–F) Show the equivalent E. coli SSADH residues (magenta sticks) to the human point mutants and their interactions (black dashed lines) within their immediate surrounds with other residues (orange sticks). A–B) Mutations in this region would be anticipated to disrupt dimer and tetramerisation, while C) we would anticipate decreased stability in this region due to loss of stabilising interactions with the N255S mutation. D) A loss of NAD/P+ binding efficiency would be expected with the addition of a large negatively charged residue, G268E, into the positively charged adenosine-ribose binding pocket of SSADH. E) We anticipate an overall loss of structural integrity of this loop region from either of the mutations G409D (loss of +/+ backbone conformation) and P382L/Q (disruption of aromatic ring stacking interactions with F419). F) The introduction of a large charged residue (N335K) in a buried surface on the catalytic loop would be anticipated to greatly disrupt the structural integrity and catalytic ability of SSADH.(2.71 MB TIF)Click here for additional data file.Table S1Intermolecular contacts with respect to monomer A.(0.18 MB DOC)Click here for additional data file.Table S2Residues involved in significant NADP+ binding using Molprobity (0.07 MB DOC)Click here for additional data file.


Systematic identification of protein-drug interaction networks is crucial to correlate complex modes of drug action to clinical indications. We introduce a novel computational strategy to identify protein-ligand binding profiles on a genome-wide scale and apply it to elucidating the molecular mechanisms associated with the adverse drug effects of Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors are a new class of preventive therapies for the treatment of cardiovascular disease. However, clinical studies indicated that one CETP inhibitor, Torcetrapib, has deadly off-target effects as a result of hypertension, and hence it has been withdrawn from phase III clinical trials. We have identified a panel of off-targets for Torcetrapib and other CETP inhibitors from the human structural genome and map those targets to biological pathways via the literature. The predicted protein-ligand network is consistent with experimental results from multiple sources and reveals that the side-effect of CETP inhibitors is modulated through the combinatorial control of multiple interconnected pathways. Given that combinatorial control is a common phenomenon observed in many biological processes, our findings suggest that adverse drug effects might be minimized by fine-tuning multiple off-target interactions using single or multiple therapies. This work extends the scope of chemogenomics approaches and exemplifies the role that systems biology has in the future of drug discovery. Both the cost to launch a new drug and the attrition rate during the late stage of the drug discovery and development process are increasing. Torcetrapib is a case in point, having been withdrawn from phase III clinical trials after 15 years of development and an estimated cost of US $800 M. Torcetrapib represents a new class of therapies for the treatment of cardiovascular disease; however, clinical studies indicated that Torcetrapib has deadly side-effects as a result of hypertension. To understand the origins of these adverse drug reactions from Torcetrapib and other related drugs undergoing clinical trials, we introduce a systematic strategy to identify off-targets in the human structural proteome and investigate the roles of these off-targets in impacting human physiology and pathology using biochemical pathway analysis. Our findings suggest that potential side-effects of a new drug can be identified at an early stage of the development cycle and be minimized by fine-tuning multiple off-target interactions. The hope is that this can reduce both the cost of drug development and the mortality rates during clinical trials. These findings suggest that adverse drug reactions might be modulated by the fine-tuning of the off-target binding network and exemplify the role of systems biology in the future of drug discovery.In this paper, we apply this strategy to identify and analyze a panel of unknown off-targets for Cholesteryl Ester Transfer Protein (CETP) inhibitors. CETP inhibitors represent a new preventive therapy for cardiovascular disease through raising HDL cholesterol. However, clinical studies have revealed that one of the CETP inhibitors, Torcetrapib, has deadly off-target effects as a result of hypertension The ligand binding site of CETP (PDB id: 2OBD) is assumed to be a long tunnel interacting with two cholesteryl oleates (2OB) and two 1,2-dioleoyl-Sn-glycero-3-phosphocholines (PCW) molecules in the native state , howeverAlthough only approximately 15% of human proteins have known 3D structures deposited in the Protein Data Bank (PDB) Most of the predicted ligand binding sites of CD1B, LPTP, and FABP have a similar topology to that of CETP. The drug molecule binds to a cavity formed by anti-parallel beta-sheets and capped by other structural components such as a helix. The others, NR, EF, and HEME all have alpha-helical architectures that are completely different from the secondary structure surrounding the binding site of CETP. These differences illustrate the necessity of tools like SMAP that can find local structural similarities even when global similarity is non-existent. From a functional perspective, it is not surprising that lipid binding proteins act as off-targets for CETP inhibitors since they are required to bind similar cognate hydrophobic ligands such as PCW. It is noteworthy that glycolipid transfer protein, one of the lipid binding proteins, has significant structural similarity to nuclear hormone receptors. For example, the FATCAT http://www.ihop-net.org/UniPub/iHOP/in?dbrefs_1=NCBI_LOCUSLINK__ID|1071). Several top-ranked off-targets appear in the same sentences with each other more than 3 times in the literature. They include phospholipid transfer proteins, nuclear receptors, including PPAR, major histocompatibility complex class II that is similar to CD1B, apolipoprotein A-1, and angiotension I converting enzyme.We searched for possible functional correlations between CETP and the putative off-targets using the iHOP http://funsimmat.bioinf.mpi-inf.mpg.de/index.php). From 204 off-targets, 148 structures had annotated GO terms and 94 structures had detectable similarities with a Resnik score The functional similarity between CETP and the off-targets is further quantitatively measured using gene ontology (GO) relationships found with the FunSimMat web server To further support our off-target predictions we conducted docking studies on CETP and the identified off-targets, which also provides insights into the molecular mechanisms of off-target binding. It has been established that the binding affinity calculated from docking programs is not necessarily reliable 3, respectively. As shown in Importantly, the binding profiles for the three CETP inhibitors  are different from each other across the panel of off-targets. JTT-705 is the most promiscuous inhibitor. In contrast, Torcetrapib failed to dock into some of the off-targets, and Anacetrapib is suitable to be docked into the least number of off-targets. The difference between their off-target binding profiles can be partly explained by their different complexity By incorporating the predicted off-targets into biological pathways it is possible for us to correlate the predicted off-target interactions with the observed pleotropic effects of Torcetrapib, Anacetrapib and JTT-705. Among them, the negative effect of Torcetrapib on blood pressure in phase III clinical trials could be deduced. Also deducible was an explanation for the increased death from infection and cancer As shown in According to the normalized docking scores , TorcetrThe effects of CETP inhibitors on inflammation are shown in NF-κB regulates genes involved in cell proliferation and cell survival and hence is an interesting drug target in cancer treatment. Inhibition of NF-κB can potentially halts tumor progression and eliminate tumors oncogenes and participates in cancer development Recent experiments have shown that PPARα and PPARγ can induce extracellular signal-regulated kinase (Erk) and/or p38 phosphorylation and then activate the MAPK/Erk signaling pathway Effects of PPAR and RXR are also regulated by fatty acid binding proteins (FABP) Another type of highly ranked off-target, CD1, can also be directly related to the side- effect of infection through its function as an antigen-presenting protein in the immune system. T cells will recognize antigens presented by CD1 proteins and activate a cell-mediated immune response against microbial infections 2+ homeostasis and nitric oxide. It has also been observed that the lack of S100A1 (an EF protein) expression could lead to hypertension 2+ levels but also directly control the transcriptional activity of the tumor suppressor p53 through interactions with its regulatory sequences Other putative off-targets such as ubiquinol-cytochrome-c reductases, globin-like proteins, EF hand-like calcium binding proteins (EFs), and LPTP are also directly or indirectly associated with hypertension, inflammation, and/or cancer. Recent studies suggest that ubiquinol-cytochrome-c reductase expression is indirectly regulated by steroid hormones in response to hypertension In summary, most of the putative off-targets for CETP inhibitors are involved in interconnected lipid metabolism and signaling networks which activate or mediate various biological process such as hypertension, stress regulation In vitro, in vivo and clinical studies indicate that CETP inhibitors exhibit pleotropic effects in humans through the interaction with unknown off-targets. We have identified a panel of proteins that likely bind to CETP inhibitors leading to the observed clinical indications. The putative off-target interactions are consistent with existing experimental data and provide insights into the molecular mechanisms of the side-effect profile of CETP inhibitors. Drug promiscuity depends not only on the similarity of ligand binding pockets in the related proteins but also the complexity of the drug itself in silico simulation of the influence of small molecules on biological systems. In the interim it is noted that the analysis of incomplete networks is still invaluable in making new discoveries in biomedicine as exemplified by several recent studies The chemical systems biology approach developed here is limited by available protein structures that currently only cover approximately 50% of the human proteome, although the structural coverage of the human proteome will steadily increase with progress in structural genomics Besides SMAP used in this study, a number of web servers for ligand binding site search are available, for example, SiteEngine in vitro screening, in vivo screening and clinical trials.Despite the success of ligand binding search algorithms in protein function prediction and drug design Even with the current limited structural coverage of the human proteome, our predications are able to provide a testable hypothesis as to the suitability of a lead compound prior to conducting a clinical trial. Thus our findings have implications for drug discovery and development. In contrast to the conventional drug discovery process in which drug leads are optimized to reduce promiscuous binding, the possible combinatorial control of aldosterone regulation by CETP inhibitors suggests that adverse drug effects can be minimized through fine tuning of multiple off-target interactions. Although it is desirable for a drug to bind the primary target in a highly specific way, this is difficult to achieve considering the inherent similarity among protein binding pockets within and across gene families. Moreover, many biological process involve combinatorial control to provide redundancy and homeostasis N, which is the alignment length between two proteins:5,985 structures or models that cover approximately 57% of the human proteome were searched against CETP (PDB id: 2obd) ligand binding sites using the sequence order independent profile-profile alignment (SOIPPA) algorithm Using this statistical model, 276 off-targets are identified with p-values less than 1.0e-3.The putative 276 off-targets are subject to further investigation using more computationally intensive protein-ligand docking. After removing three structures with the same fold as CETP, JTT-705, the smallest CETP inhibitor, is docked to the remaining 273 structures using two commonly used fast docking programs, Surflex 2.1 An all-against-all global structural similarity analysis between the 204 putative off-targets was computed using CE http://sts-fw.bioengr.uic.edu/castp) with default settings.The volume of the binding pocket is computed using the CASTp server http://zinc.docking.org) Drug-like molecules are downloaded from ZINC .(0.09 MB DOC)Click here for additional data file.Figure S2Structural coverage of the human proteome vs. alignment length between the protein sequence and the structural template.(0.06 MB DOC)Click here for additional data file.Figure S3CE Z-score distributions of putative off-targets.(0.06 MB DOC)Click here for additional data file.Figure S4Structural clusters of helix-like proteins.(0.07 MB DOC)Click here for additional data file.Figure S5Global structure similarity between glycolipid transport protein (PDB: 1tfj) and nuclear hormone receptor ligand binding domain (PDB: 1yow).(0.17 MB DOC)Click here for additional data file.Figure S6Regression curves of eHiTs score for CETP and its off-targets dependent on the number of carbon atoms for a) 2obd, b) 1yow, c) 1y0s, d) 2p54, e) 1zeo, and f) 1ie8.(0.38 MB DOC)Click here for additional data file.Figure S7Correlation of eHiTS score between CETP and its off-targets binding with random ligands with different sizes. a) 1yow; b) 1y0s; c) 2p54; d) 1zeo; e) 1ie8; f) 1tfj.(2.04 MB DOC)Click here for additional data file.Figure S8Correlation of the off-target interaction network of CETP inhibitors with the clinical indication through interconnected biological pathways.(0.23 MB DOC)Click here for additional data file.Figure S9The different regulation effects of Torcetrapib, Anacetrapib and JTT-705 on hypertension, inflammation and cancer through combinational control of other identified off-targets.(0.10 MB DOC)Click here for additional data file.Table S1Putative off-targets of CETP inhibitors across the human structural genome identified from the off-target pipeline SMAP.(0.05 MB DOC)Click here for additional data file.Table S2GO based similarity between CETP and off-targets.(0.03 MB DOC)Click here for additional data file.Table S3Vector distances and Pearson correlations of carbon atom dependent average eHiTS docking scores between binding pockets of CETP and six classes of off-targets.(0.05 MB DOC)Click here for additional data file.


Triceratops and other ceratopsids (horned dinosaurs) are interpreted variously as display structures or as weapons against conspecifics and predators. Lesions  on Triceratops skulls have been used as anecdotal support of intraspecific combat similar to that in modern horned and antlered animals. If ceratopsids with different cranial morphologies used their horns in such combat, this should be reflected in the rates of lesion occurrence across the skull.The horns and frill of G-test of independence to compare incidence rates of lesions in Triceratops  and the related ceratopsid Centrosaurus , for the nasal, jugal, squamosal, and parietal bones of the skull. The two taxa differ significantly in the occurrence of lesions on the squamosal bone of the frill (P = 0.002), but not in other cranial bones (P>0.20).We used a Triceratops using its horns in combat and the frill being adapted as a protective structure for this taxon. Lower pathology rates in Centrosaurus may indicate visual rather than physical use of cranial ornamentation in this genus, or a form of combat focused on the body rather than the head.This pattern is consistent with  Triceratops battling with conspecifics or the predator Tyrannosaurus have become ingrained in both the scientific and the popular mind. Lesions (wounded or diseased areas) on the horns, frill, and face of Triceratops specimens have been cited as evidence in support of the defensive and offensive nature of the animal's cranial ornamentation Triceratops have been anecdotal, focusing on generating speculative scenarios to explain instances of hypothesized injury Images of the three-horned dinosaur Triceratops and Centrosaurus for bony lesions .A Triceratops and Centrosaurus, because all cranial elements should then show similar rates of incidence. We also rule out predatory attacks as the primary cause of the lesions, because similar large predators (tyrannosaurid theropods) were present in the habitats for both genera, and we would thus expect similar patterns of osteological abnormalities in both. Alternatively, it might be claimed that Triceratops had more frequent occurrence of lesions on the squamosal because this element forms a greater proportion of the frill's exposed area, and was thus more likely to be injured, than in Centrosaurus  caused the differing prevalence of lesions between us e.g., . We testnot be due to combat. We only claim that the overall pattern in all of the specimens is consistent with intraspecific combat in Triceratops.It is important to note that we do not claim to infer a precise cause for individual pathologies on certain specimens , the evolutionary predecessors of ceratopsids, possessed a thin, enlarged frill but lacked elongated brow or nasal horns. Thus, the primitive function of the frill (in addition to a role in jaw muscle attachment) was probably that of display rather than cervical protection Triceratops may have been an exaptation for cervical protection, in addition to a role in display. This suggests interesting possibilities for the factors that drove the evolution of cranial morphology in ceratopsids. Display probably was an important function for the horns and frills in all ceratopsids, but not the only one. Horned combat, and the consequences of injury from this combat, may have been another important selective factor. Recent discoveries strongly suggest that Centrosaurus evolved from an ancestor with a Triceratops-like horn configuration Centrosaurus) lost their long brow horns or changed combat styles as a way to reduce cranial injury. This interpretation also suggests that the frill may not have had a protective function within Centrosaurus , but instead functioned for species recognition and/or other forms of visual display. Centrosaurus and some other ceratopsids may have focused blows on an opponent's torso rather than the skull; this is suggested by the occurrence of fractured ribs in Centrosaurus, Pachyrhinosaurus, and ChasmosaurusTriceratops, as well as rates of cranial bony anomalies in additional taxa, may be informative in further evaluating this hypothesis. Clearly, horned dinosaurs used their cranial ornamentations for a variety of functions.Non-ceratopsid neoceratopsians  is quite similar to other chasmosaurine from these formations (Torosaurus and Diceratus). No distinction was made between Triceratops species, Triceratops horridus and Triceratops prorsus, because of general similarity in horn morphology as well as the difficulty in assessing species for incomplete skulls. A similar approach was used for Centrosaurus. Two species, Centrosaurus apertus and Centrosaurus brinkmani, which differ only in minor details of the horns and frill, were combined in the sample. Additional isolated centrosaurine specimens from the Dinosaur Park Formation of Alberta were also assumed to belong to Centrosaurus.Specimens of Cranial elements chosen for comparison included the nasal , jugal, squamosal, and parietal , and theCentrosaurus, some specimens preserved the midline bar of the frill and only one side. In this case, the elements were scored as belonging to the side that was predominantly preserved, in order to avoid inflating the sample size. Similarly, parietals consisting primarily of the midline bar were also scored only as a single element.Each examined element was coded as “pathological” or “normal,” and the side of the element (right or left) was also recorded. Because the parietal bone is fused into a single bilaterally symmetric element, right and left sides of the bone were distinguished relative to the midline. In G-test of independence was applied to each 2×2 matrix For each element, the data were assembled into a 2×2 matrix, with rows representing taxon and columns representing pathological state. For associated specimens preserving both right and left elements, each side was counted separately in the matrix. This is appropriate, because the pathologies counted here presumably represented discrete events in the life of the animal instead of systemic conditions Triceratops than in Centrosaurus , we conducted a second analysis looking at the frill as a single element. As before, left and right frills  were counted separately, and disarticulated or isolated frill elements were considered to represent a single “frill” in order to maximize sample size. This latter assumption was modified for specimens recovered from bonebeds, in order to reduce the possibility that a single individual  would be over counted. Here, the number of squamosals and parietals from each bone bed sample were compared, and the element with the greatest representation was chosen as the N of frills for that locality. For instance, a site with 5 parietals and 8 squamosals would be considered to have 8 individual frills .The squamosal, relative to the parietal, forms a greater proportion of the frill in rosaurus . In ordeTable S1ffracture callus; pperiosteal reactive boneSpecimens included in this study, by element. Each specimen number listed is for a single element. Where numbers are listed twice  or indicated with a parentheses (2), this indicates that two elements from the same individual were included in the sample. Abbreviations: AMNH, American Museum of Natural History, New York, New York; ASU, Appalachian State University, Boone, North Carolina; CCM, Carter County Museum, Ekalaka, Montana; CMN, Canadian Museum of Nature, Ottawa, Ontario; DMNH, Denver Museum of Nature and Science, Colorado; RAM, Raymond M. Alf Museum of Paleontology, Claremont, California; ROM, Royal Ontario Museum, Toronto, Ontario; SDSM, South Dakota School of Mines and Technology Museum of Geology, Rapid City; TLAM, Timber Lake Area Museum, South Dakota; TMP, Royal Tyrrell Museum of Palaeontology, Drumheller, Alberta; UCMP, University of California Museum of Paleontology, Berkeley; USNM, National Museum of Natural History, Washington, D.C.; YPM, Yale Peabody Museum of Natural History, New Haven, Connecticut. (0.02 MB DOC)Click here for additional data file.


Glycyrrhiza glabra) inhibits 11β-hydroxysteroid dehydrogenase type 1 that catalyzes the activation of glucocorticoids. Thus, oral administration of GA is postulated to ameliorate the MetS.The metabolic syndrome (MetS) is a cluster of metabolic abnormalities comprising visceral obesity, dyslipidaemia and insulin resistance (IR). With the onset of IR, the expression of lipoprotein lipase (LPL), a key regulator of lipoprotein metabolism, is reduced. Increased activation of glucocorticoid receptors results in MetS symptoms and is thus speculated to have a role in the pathophysiology of the MetS. Glycyrrhizic acid (GA), the bioactive constituent of licorice roots (p < 0.05) but non-significant increase in the abdominal muscle, kidney, liver, heart and the subcutaneous and visceral adipose tissues (p > 0.05) of the GA-treated rats compared to the control. Decrease in adipocyte size (p > 0.05) in both the visceral and subcutaneous adipose tissue depots accompanies such selective induction of LPL expression. Consistent improvement in serum lipid parameters was also observed, with decrease in serum free fatty acid, triacylglycerol, total cholesterol and LDL-cholesterol but elevated HDL-cholesterol (p > 0.05). Histological analysis using tissue lipid staining with Oil Red O showed significant decrease in lipid deposition in the abdominal muscle and quadriceps femoris (p < 0.05) but non-significant decrease in the heart, kidney and liver (p > 0.05).In this study, daily oral administration of 50 mg/kg of GA for one week led to significant increase in LPL expression in the quadriceps femoris (Results from this study may imply that GA could counteract the development of visceral obesity and improve dyslipidaemia via selective induction of tissue LPL expression and a positive shift in serum lipid parameters respectively, and retard the development of IR associated with tissue steatosis. Lipoprotein lipase (LPL) is the major enzyme responsible for the hydrolysis of circulating triacylglycerol (TAG) moiety of both classes of TAG-rich lipoproteins; the chylomicrons and very-low-density lipoprotein (VLDL), generating free fatty acids (FFA) that are either oxidized in the muscles or re-esterified in the adipose tissues, and glycerol that is returned to the liver. LPL plays a central role in overall lipoprotein metabolism, where (i) the successive interaction of VLDL with LPL generates the low-density lipoproteins (LDL) that are involved in forward cholesterol transport and (ii) the remnant lipoprotein particles so formed from LPL catalysis contributes to the maturation of high-density lipoprotein (HDL) precursors, the latter of which is then involved in reverse cholesterol transport ,2. PertuThe MetS refers to a constellation of metabolic abnormalities characterized by the co-existence of insulin resistance (IR), visceral obesity, hyperglycaemia, hypertension and dyslipidaemia. The syndrome has become a recognizable clinical cluster of risk factors that are predictive of the progression to cardiovascular disease and type 2 diabetes mellitus (T2DM) . Both viDyslipidaemia, the hallmark of the MetS which is manifested in the more severe form in T2DM, is characterized by (i) increased flux of FFA, (ii) elevated TAG level (hypertriglyceridaemia), (iii) reduced HDL level and (iv) a predominance of small, dense LDL. Elevated plasma FFA is viewed as the primary defect leading to the development of dyslipidaemia ,7 and IRGlycyrrhiza glabra and its pharmacologically active metabolite glycyrrhetic acid (GE) act as potent, non-selective inhibitors of both isoforms of 11β-HSD  and normalized to the β-actin (BAC) gene with the forward and reverse primers 5'-GTATGGGTCAGAAGGACTCC-3' and 5'-GTTCAATGGGGTACTTCAGG-3' and the probe 5'-(TET) CCTCTCTTGCTCTGGGC (BHQ1)-3' specific for Rattus norvegicus BAC mRNA [GenBank: BC063166]. The comparison of LPL expression between control and GA-treated rats were performed using the Comparative Ct (ΔΔCt) Method, with BAC as reference, GA-treated group as target and control group as calibrator. Agarose gel electrophoresis was carried out on amplicons generated from qRT-PCR reaction to ensure primer specificity.The expression of LPL was determined by qRT-PCR using the LPL forward and reverse primers 5'-CAGCAAGGCATACAGGTG-3' and 5'-CGAGTCTTCAGGTACATCTTAC-3' and the probe 5'-(6-FAM) TTCTCTTGGCTCTGACC (BHQ1)-3' that are specific for et al. [et al. [Frozen tissues were cut into small cubes of approximately 5 × 5 × 5 mm on and embedded using the Optimal Cutting Temperature (OCT) Compound . Cryosectioning was performed at a temperature of -25°C where the embedded tissues were sectioned into 5 μm slices and adhered onto glass slides. Staining with Oil Red O (ORO) was performed in accordance to Koopman et al.  and capt [et al. . Images Eight contiguous views per tissue section were captured and analyzed for the lipid content. The level of lipid deposition of each tissue section was calculated as the average of these eight values.2) per field view per tissue section at 100× magnification.Adipose tissues that were fixed in 10% neutral-buffered formalin as aforementioned were processed by a Leica TP 1020 Automatic Tissue Processor and embedded in paraffin. 5 μm thick tissue sections were then stained with haematoxylin and eosin (H&E) followed by the measurement of the size of 100 adipocytes (μm©) MCS Beta 2006 while that of all other parameters was performed using the Statistical Package for the Social Sciences (SPSS) Version 16.0. Data distribution was analyzed using the Kolmogorov-Smirnov test. Parametric data were then analyzed with independent t-test and are presented as mean ± standard error while non-parametric with Mann-Whitney U-test and are reported as median (minimum – maximum). In all analyses, a p-value ≤ 0.05 was considered significant.Statistical analysis of LPL expression was performed using the Relative Expression Software Tool (REST11β-HSD: 11β-hydroxysteroid dehydrogenase; AM: abdominal muscle; CETP: cholesteryl ester transfer protein; FFA: free fatty acids; GA: glycyrrhizic acid; GE: glycyrrhetic acid; H&E: haematoxylin and eosin; HL: hepatic lipase; HDL: high-density lipoprotein; IR: insulin resistance; LDL: low-density lipoprotein; LPL: lipoprotein lipase; MetS: metabolic syndrome; ORO: Oil Red O; PPAR: peroxisome proliferator-activator receptor; PPRE: peroxisome proliferator response element; QF: quadriceps femoris; SAT: subcutaneous adipose tissue; T2DM: type 2 diabetes mellitus; TAG: triacylglycerol; VAT: visceral adipose tissue; VLDL: very-low-density lipoprotein.The authors declare that they have no competing interests.WYAL was involved in all bench work, data acquisition, analysis and interpretation and manuscript preparation. YYC had part in the optimization of the qRT-PCR conditions and SYL had part in histological work. SHT, KAK and SNASH participated in the coordination of the study and helped in drafting the manuscript. All authors read and approved the final manuscript.


Increased resting heart rate (RHR) may be associated with increased cardiovascular morbidity. Our aim was to explore the possibility that increased RHR is associated with the prevalence of the metabolic syndrome (MetS) in a sample of apparently healthy individuals and those with cardiovascular risk factors.We performed a cross-sectional analysis in a large sample of apparently healthy individuals who attended a general health screening program and agreed to participate in our survey. We analyzed a sample of 7706 individuals (5106 men and 2600 women) with 13.2% of men and 8.9% of the women fulfilling the criteria for the MetS. The participants were divided into quintiles of resting heart rate. Multiple adjusted odds ratio was calculated for having the MetS in each quintile compared to the first.The multi-adjusted odds for the presence of the MetS increased gradually from an arbitrarily defined figure of 1.0 in the lowest RHR quintile (<60 beats per minute (BPM) in men and <64 BPM in women) to 4.1 and 4.2 in men and women respectively in the highest one (≥80 BPM in men and ≥82 BPM in women).Raised resting heart rate is significantly associated with the presence of MetS in a group of apparently healthy individuals and those with an atherothrombotic risk. The strength of this association supports the potential presence of one or more shared pathophysiological mechanisms for both RHR and the MetS. There are multiple lines of emerging evidence which suggest that resting heart rate (RHR) is associated with the presence and/or the potential to develop cardiovascular disease -4. We haWe have currently analyzed data which has been collected during the last five years from the Tel Aviv Medical Center Inflammation Survey (TAMCIS), a registered data bank of the Israeli Ministry of Justice -13. ThisBaseline RHR was obtained manually at enrolment with one radial pulse measurement over a period of 60 seconds with the patient in a sitting position, after sitting in a quiet room for 5 minutes.The diagnosis of the metabolic syndrome was based on the National Cholesterol Education Program (NCEP) ATP III Criteria  with theBlood was drawn during the morning hours following a fasting period of at least 12 hours using a standard Vacutainer gel tube . Triglycerides, HDL-C and glucose concentration were measured using a Bayer Advia 1650 chemistry analyzer and respective Bayer kits . Blood pressure was obtained on two separate measurements following a five minute resting period.All data was summarized and displayed as mean (Standard deviation (SD)) for the continuous variables and as the number of patients (percentage) in each group for categorical variables.In order to characterize the population we divided the patients of each gender into quintiles of resting heart rate and analyzed all results accordingly. For all categorical variables the Chi-Square test was used for assessing the overall statistical significance between the quintiles, while the One-Way Analysis of Variance was used for all continuous variables as well as for calculating the P value for the linear trend between the quintiles.In order to better evaluate the magnitude of the association between each component of the MetS and the RHR, we calculated the estimated marginal means for the groups with and without each component, adjusting for age, former or never smoking status, alcohol consumption, exercise activity, oral temperature, family history of premature cardiovascular disease (CVD), use of aspirin as well as the presence of all four others components of the MetS, using analysis of covariance (ANCOVA) under a general linear model.Finally, in order to quantify the relative odds of having the MetS as a function of the quintiles of RHR, we arbitrarily defined the lowest quintile as 1.0 and calculated the adjusted odds ratio (OR) for having the MetS for each of the higher quintiles with adjustment for age, former or never smoking status, alcohol consumption, exercise activity, oral temperature, family history of CVD and the use of aspirin, using logistic regression. All above analyses were considered significant at p < 0.05 (two tailed). The SPSS statistical package was used to perform all statistical evaluation .We have currently performed a cross-sectional analysis regarding the presence or absence of the MetS in a sample of 5,106 men and 2,600 women with respective mean (SD) ages of 43 (11) and 44 (11) years. This cohort included a total of 674 males and 231 females defined as having the MetS. The baseline characteristics of both men and women in relation to quintiles of RHR are reported in Tables We have presently documented a relatively strong association between RHR and the presence of the MetS. Such a strong association might suggest that there is a shared pathophysiological pathway which is yet to be revealed. We believe therefore, that RHR is an extremely easy-to-perform and almost costless parameter which may improve the early detection of cardiovascular risk. Of special note might be the fact that even a small increment in RHR (for example from the first to the second quintile) had a clear influence on the odds of having MetS. Moreover, the prevalence of the MetS was three times higher in individuals in the fourth quintile and this was still within the "normal" RHR that is - to date - accepted as being between 60 and 80. Again, this might be another observation to support the notion that one should look at RHR as a continuous variable, putting into perspective the currently used normal limits.Sympathetic overactivity or parasympathetic underactivity might underly the aforementioned observation. In fact, sympathetic imbalance has been described for several of the components of the MetS including hypertension , waist cIn a previous study which solely involved men, we addressed the question of the association between RHR and the presence of microinflammatory changes . It is kResting heart rate measurements are known to be influenced by multiple factors. In the current study design, although there were multiple exclusions and statistical adjustments performed, we could not account for every factor. Thus we acknowledge several limitations to the present study. Firstly, the RHR was obtained by single measurements following five minutes of rest and not by repeated measurements. Since all of the analyses were based on this single measurement and since unconditioned individuals might require more time to return to their usual heart rate at rest, it is possible that our results were accordingly influenced. On the other hand, we believe that the large number of individuals analysed dilutes this possible effect and makes it unlikely that repeated measurements or longer resting prior to measurement would influence our results significantly. Secondly, we did not exclude patients with abnormal thyroid function, mainly because the survey does not include those measurements. We did however, exclude all individuals reporting thyroid disease or taking thyroid replacement therapy. Thirdly, we did not measure insulin levels and thus could not calculate the HOMA index. Lacking this objective laboratory parameter, we could only assume that there is a shared pathophysiological pathway and that future studies in this field might reveal what it is. Fourthly, the individuals were not evaluated routinely by echocardiography and thus we were not able to exclude individuals with a cardiomyopathy, including such a cardiomyopathy secondary to tachycardia. Due to the relatively healthy population evaluated and the large number of individuals we believe that the number of individuals with a significant cardiomyopathy included is negligible and would therefore not have influenced the results significantly. Finally, we did not exclude patients regularly performing sport activity, although we did adjust our results to their levels of physical activity. In addition, since our population is mainly referred from places of employment, there exists the possibility of "healthy worker" selection bias and thus generalization of the results might be limited.The question of "normalcy" for RHR remains. Our study does support, at least in relation to the presence of the MetS, the finding that the lower the RHR is, the lower the dysmetabolic risk. This information might be relevant if new "normal values" for RHR are to be determined in the future.The authors declare that they have no competing interests.OR and AS have participated in the design of the study, performed the statistical analyses and drafted the paper. SB and SI conceived the study, participated in its design and coordination and helped to draft and review the manuscript. MC, NS and OKB helped in the data organization and retrieval, English editing and final draft preparation. All of the authors have read and approved the final manuscript.


A decade has passed since metabolic syndrome (MetS) was documented to be highly prevalent in the kingdom of Saudi Arabia. No follow-up epidemiologic study was done. This study aims to fill this gap. In this cross-sectional, observational study, a total of 2850 randomly selected Saudi adults aged 18–55 years were recruited. Subjects' information was generated from a database of more than 10,000 Saudi citizens from the existing Biomarkers Screening in Riyadh Program (RIYADH Cohort), Saudi Arabia. Anthropometrics included body mass index (BMI), blood pressure, as well as waist and hip circumferences. Fasting blood glucose and lipid profile were determined using routine laboratory procedures. The definition of ATP-III (NHANES III) was used for the diagnosis of the full MetS. The overall prevalence of complete MetS was 35.3% [Confidence-Interval (CI) 33.5–37.01]. Age-adjusted prevalence according to the European standard population is 37.0%. Low HDL-cholesterol was the most prevalent of all MetS risk factors, affecting 88.6% (CI 87.5–89.7) and hypertriglyceridemia the second most prevalent, affecting 34% (CI 32.3–35.7) of the subjects. The prevalence of the full MetS decreased from previous estimates but remains high, while dyslipidemia remains extremely high, affecting almost 90% of middle-aged Arabs. Screening for dyslipidemia among Saudi adults is warranted, especially among those most at risk. Scientific inquiry into the molecular causes of these manifestations should be pursued as a first step in the discovery of etiologic therapies. Metabolic syndrome (MetS), which is the clinical term used for the co-occurrence of several cardiovascular risk factors, is quite prevalent in many developed nations. Al-Nozha and colleagues previously reported that MetS was almost 40% among adults in a kingdom-wide sample population taken from 1995–2000 In this cross-sectional community-based study, a total of 2,850 Saudi adults aged 18–55 years were included. Subjects were part of the capital-wide Biomarker Screening in Riyadh (BSR), a collaborative effort between the Biomarkers Research Program (BRP) of King Saud University (KSU) and the Ministry of Health in Riyadh, Kingdom of Saudi Arabia (RIYADH Cohort). Ethical approval was obtained from the College of Medicine Research Center Ethics Committee of KSU, Riyadh, KSA. In brief, BSR was launched to identify and employ novel biomarkers of chronic non-communicable diseases, including diabetes mellitus (DM), cardiovascular diseases (CVD), and obesity, among consenting Saudis. Subjects were recruited randomly from different Primary Health Care Centers (PHCC) across Riyadh. No expatriates were included in the conduct of this study. Each participating subject submitted a signed consent form, a general questionnaire containing demographic, past and present medical history, as well as diet information from the food frequency questionnaire. Subjects' information was taken from the existing database of more than 10,000 subjects (RIYADH Cohort).2).Consenting adults were invited to their respective PHCC after a 10 h overnight fasted state. Anthropometric measurements included height (cm), weight (cm), waist (cm) and hip (cm) circumferences and were taken and noted by trained nurses. Systolic and diastolic blood pressures were taken twice with a 15-minute interval. The average of both readings was recorded. Body mass index (BMI) was calculated as weight (kg) over height . This biochemical analyzer (Konelab) was calibrated routinely prior to the analysis of all serum samples using quality control samples provided . Glucose test method employed glucose oxidase and modified Trinder color reaction catalyzed by the enzyme peroxidase. Triglycerides test method employed hydrolysis, phosphorylation and oxidation leading to the formation of quinoeimine dye which is absorbed at 510 nm. HDL-cholesterol test method employed homogenous enzymatic colorimetric test by cholesterol oxidase coupled with poly(ethylene-glycol) to the amino groups. Measurement ranges were as follows: glucose (0.3–20.0 mmol/L); triglycerides (0.05–11.0 mmol/L), HDL-cholesterol (0.16–2.80 mmol/L). All samples fell within normal detection limits. Standard International Units (mmol/L) were used to record the results. For validation purposes, randomized samples were sent to a reference lab within the kingdom .The definition of MetS used was according to the National Cholesterol Education Program-Third Adult Treatment Panel (NCEP ATP III), three or more of the following criteria must be fulfilled: fasting blood glucose level ≥5.6 mmol/l; blood pressure ≥130/85 mm Hg; triglycerides ≥1.7 mmol/l; HDL cholesterol <1.03 mmol/l for men and <1.29 mmol/l for women; and waist circumference >102 cm for men and >88 cm for women Statistical analysis was done using the Statistical Package for Social Sciences (SPSS) version 11.5 . Continuous variables are shown as mean ± standard deviation. Age adjustment was done with a European standard population. Frequencies were presented as percentage . MetS prevalence was presented as overall prevalence, age-adjusted prevalence and prevalence according to age.p-values 0.004, 0.001<0.001 respectively) while females had significantly higher prevalence of abdominal obesity . The over-all prevalence of MetS was 35.3% (CI 33.5–37.01), while an age adjusted prevalence was almost 37.0%. Low HDL-cholesterol remains the most common MetS component with almost 9 out of 10 [88.6% (CI 87.5–89.7)] of subjects affected followed by hypertriglyceridemia with an over-all prevalence of 34% (CI 32.3–35.7) . Males hThere is some improvement in the prevalence of MetS in the kingdom, particularly in Riyadh region where the study was conducted. Prevalence of MetS in urban areas was previously recorded at 44.1% (1) compared to 37.0% in the present study. The prevalence of dyslipidemia as evidenced by low HDL-cholesterol and high triglyceride levels, however, remained alarmingly high, continuing to be the most common cardiovascular risk factor among Saudi adults. High prevalence of low HDL-cholesterol was also noted among the elderly in Spain While the prevalence of MetS in males is higher, it is not significantly different from females. Similar findings were found in a recent study by Lin and colleagues, where even if a higher prevalence of MetS among males was observed, the mortality risk was greater among postmenopausal women In summary, the prevalence of the full MetS among urban Saudi adults remains high, but has considerably decreased. Prevalence of low-HDL cholesterol on the other hand remains extremely prevalent and begs for tougher measures in early intervention and further scientific exploration.


We have recently determined the optimal cut-off of the homeostatic model assessment of insulin resistance for the diagnosis of insulin resistance (IR) and metabolic syndrome (MetS) in non-diabetic residents of Tehran, the capital of Iran. The aim of the present study is to establish the optimal cut-off at the national level in the Iranian population with and without diabetes.Data of the third National Surveillance of Risk Factors of Non-Communicable Diseases, available for 3,071 adult Iranian individuals aging 25-64 years were analyzed. MetS was defined according to the Adult Treatment Panel III (ATPIII) and International Diabetes Federation (IDF) criteria. HOMA-IR cut-offs from the 50th to the 95th percentile were calculated and sensitivity, specificity, and positive likelihood ratio for MetS diagnosis were determined. The receiver operating characteristic (ROC) curves of HOMA-IR for MetS diagnosis were depicted, and the optimal cut-offs were determined by two different methods: Youden index, and the shortest distance from the top left corner of the curve.The area under the curve (AUC) (95%CI) was 0.650 (0.631-0.670) for IDF-defined MetS and 0.683 (0.664-0.703) with the ATPIII definition. The optimal HOMA-IR cut-off for the diagnosis of IDF- and ATPIII-defined MetS in non-diabetic individuals was 1.775 . The optimal cut-offs in diabetic individuals were 3.875  and 4.325  for ATPIII- and IDF-defined MetS, respectively.We determined the optimal HOMA-IR cut-off points for the diagnosis of MetS in the Iranian population with and without diabetes. Insulin resistance, which represents a reduced physiological response of the peripheral tissues to the action of the normal levels of insulin, is amajor finding in several metabolic disorders, including type 2 diabetes and metabolic syndrome (MetS) . TherefoThe homeostasis model assessment of insulin resistance (HOMA-IR), which is developed for application in large epidemiologic investigations , is an aPopulation-based studies for defining cut-off values of insulin resistance for diagnosis of MetS are limited. In this study, we sought, for the first time, to evaluate the distribution and optimal cut-off value of HOMA-IR for identifying MetS in a Middle Eastern population with and without diabetes.The data obtained from the third National Surveillance of Risk Factors of Non-Communicable Diseases in Iran (SuRFNCD-2007)  were anaWeight and height of participants were determined in light clothing and without shoes. Portable calibrated electronic weighing scale and portable measuring inflexible bars were used. Waist circumference was measured using constant tension tape at the end of a normal expiration, with arms relaxed at the sides, at the midpoint between the lower part of the lowest rib and the highest point of the hip on the mid-axillary line. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared. Blood pressure was measured with a calibrated Omron M7 sphygmomanometer (HEM-780-E). The mean value of three measurements, made at intervals of 5 minutes, was used for analysis. Blood samples were collected following 12 h overnight fast. Fasting plasma glucose was measured by the enzymatic colorimetric method using glucose oxidize test . Serum total cholesterol, triglyceride, and high density lipoprotein-cholesterol (HDL-cholesterol) were determined by enzymatic methods . Low density lipoprotein-cholesterol (LDL-cholesterol) was calculated using the formula of Friedewald et al. . When se2 were defined as normal subjects .MetS was defined according to the Adult Treatment Panel III (ATPIII)  and Inte2 + (1 - specificity)2] were calculated ), and the point with shortest distance value form the point  [(1 - sensitivity)lculated . These alculated . We alsoPrimary analyses were performed without covariate adjustment to reflect standard use of blood test results in clinical practice. Subsidiary analyses of surrogate measures considered additional adjustment for age and sex. To control whether this would lead to over-fitting HOMA-IR in statistical models, analyses were repeated with fasting insulin as an alternative to HOMA-IR. For fasting insulin, we also considered additional adjustment for fasting glucose (to assess adjusted discrimination compared with discrimination using HOMA-IR). For each surrogate measure, we compared the ROC of the fuller model with that of the sparser model. P < 0.05 was considered statistically significant.Table S1; Additional file Age and sex distribution of HOMA-IR cut-offs from the 50th to the 95th percentile along with their corresponding sensitivity and specificity for the diagnosis of IDF-MetS in non-diabetic and diabetic individuals are shown in Tables S2 and S3  for ATPIII-defined MetS and 4.325 for IDF-defined MetS .As depicted in Figure ROC analyses showed that the diagnosis of MetS is made more accurately by using HOMA-IR than fasting insulin. Insulin resistance (using any surrogate) or fasting insulin predicts ATPIII-defined MetS more accurately than IDF-defined MetS. For example, the HOMA-IR AUC (95% CI) for IDF-defined MetS was 0.650 (0.631-0.670) compared with 0.683 (0.664-0.703) for ATPIII-defined MetS. One reason that fasting insulin underperformed HOMA-IR is that information about plasma glucose is contained within the latter measure. Additional adjustment for age and sex was performed in subsidiary analyses. This did not influence the accuracy of MetS diagnosis by both definitions, i.e. crude versus age- and sex-adjusted ROCs (for both HOMA-IR and fasting insulin models) had statistically equal performance. Adjustment for fasting glucose decreased the accuracy of MetS diagnosis by both HOMA-IR and fasting insulin (data not shown).We demonstrated that the risk of MetS increased with rising HOMA-IR percentiles. The optimal cut-off point of HOMA-IR for the diagnosis of MetS in our population was estimated to be 1.775 in non-diabetics and around 4 in diabetic patients. In line with previous population-based studies ,31, we fHOMA-IR, developed in 1985 by Matthews and co-workers , was useFor inter-population comparisons, it is necessary to know normal values of HOMA-IR for each population. Although HOMA-IR has been widely used, there is hardly any consensus on the cut-off points for classification of insulin resistance. Some authors have tried to find HOMA-IR cut-offs in subjects who had increased tendencies toward insulin resistance or MetS, but their findings were not consistent -18. TablDifferent cut-off points might be selected to optimize sensitivity versus specificity depending on the purpose. A screening test requires high sensitivity and moderate specificity, whereas a diagnostic test requires a much higher specificity. Although insulin resistance may be at the core of the cluster of metabolic abnormalities that characterizes MetS, our data suggest that MetS, defined by conventional criteria, is not always synonymous with insulin resistance ,38. The The prevalence of MetS in our sample was 33.6% and 34.8% for ATPIII and IDF definitions, respectively. Our results regarding MetS prevalence, insulin levels, and HOMA-IR values suggest that women have a higher propensity to insulin resistance. The available reports on the prevalence of MetS show variable results 23%-40%), depending on ethnicity and the criteria used %, depend, the higIn conclusion, we showed that risk for MetS increases with increasing HOMA-IR percentiles. The optimal cut-off point of HOMA-IR for MetS diagnosis is 1.775 in non-diabetics and approximately 4 in diabetic individuals. Further prospective studies are warranted to elucidate the performance of these cut-offs in predicting incident diabetes or cerebrovascular disease in our country. A fairly large proportion of our participants were excluded because of missing lab results. Although excluded participants were randomly scattered across age, sex, BMI, and residential area categories of SuRFNCD-2007, and their exclusion is thus unlikely to have caused a significant problem in our analysis, this can be considered as a limitation of our study and is to be addressed in future work.The authors declare that they have no competing interests.AE participated in the design of the study and interpreted the results. HA and OK participated in statistical analysis, interpreted the results, and wrote the manuscript. AZ helped with statistical analysis and writing the manuscript. MN and AR interpreted the results and wrote the manuscript. MH and FA participated in the design of the study and conducting it. All authors read and approved the final manuscript.Supplementary Tables 1-4. Table S1 - Clinical and laboratory characteristics of participants, Table S2 - Age and sex distribution of HOMA-IR values in non-diabetic subjects . Table S3 - Age and sex distribution of HOMA-IR values in diabetic subjects (n = 366). Table S4 - Summary of reports (sorted by sample size) on HOMA-IR cut-off in different populations.Click here for file


COMMUNICATION – DIALOGUE – CO-OPERATION – SOLVING PROBLEMSThese are the words of nowadays Europe. Such approaches should be used in broad spectrum of life as well as in toxicology. It is therefore admirable that EUROTOX commissars, mainly past-president Prof. Liesivuori, invited all heads of European toxicological societies to discuss current issues of toxicology in Europe during IUTOX meeting held in Barcelona in July 2010. Renowned scientists and regulatory officers from Government, Industry and Academia made appearance and talked about contribution of toxicological research to society, importance of communication and education. The strategic meeting was successful. Prof. Liesivuori expressed gratitude to all delegates for the great suggestions and key findings resulting from the brainstorming sessions conducted during the meeting. Majority of delegates agreed that education in toxicology and related fields is the top priority for future. Communication towards society, in the sense of explaining possible risks, should be an important concern as well.I can only hope for success and wish for better and safer future		Taking a good example from EUROTOX, on the verge of summer the Slovak Toxicology Society SETOX organizes meeting of the toxicological societies from the Central Europe, i.e. Slovak, Czech, Polish, Austrian and Hungarian societies of toxicology. First positive step was done by Visegrad Fund and Federation of European Toxicologists & European Societies of Toxicology, which supported this meeting. It is essential for successful co-operation to establish strong connections, which can be further crucial for meaningful and highly profitable information to society.