Targeted whole exome sequencing and Drosophila modelling to unveil the molecular basis of primary ovarian insufficiency.

STUDY QUESTION: Can a targeted whole exome sequencing (WES) on a cohort of women showing a primary ovarian insufficiency (POI) phenotype at a young age, combined with a study of copy number variations, identify variants in candidate genes confirming their deleterious effect on ovarian function? SUMMARY ANSWER: This integrated approach has proved effective in identifying novel candidate genes unveiling mechanisms involved in POI pathogenesis. WHAT IS KNOWN ALREADY: POI, a condition occurring in 1% of women under 40 years of age, affects women's fertility leading to a premature loss of ovarian reserve. The genetic causes of POI are highly heterogeneous and several determinants contributing to its prominent oligogenic inheritance pattern still need to be elucidated. STUDY DESIGN, SIZE, DURATION: WES screening for pathogenic variants of 41 Italian women with non-syndromic primary and early secondary amenorrhoea occurring before age 25 was replicated on another 60 POI patients, including 35 French and 25 American women, to reveal statistically significant shared variants. PARTICIPANTS/MATERIALS, SETTING, METHODS: The Italian POI patients' DNA were processed by targeted WES including 542 RefSeq genes expressed or functioning during distinct reproductive or ovarian processes (e.g. DNA repair, meiosis, oocyte maturation, folliculogenesis and menopause). Extremely rare variants were filtered and selected by means of a Fisher Exact test using several publicly available datasets. A case-control Burden test was applied to highlight the most significant genes using two ad-hoc control female cohorts. To support the obtained data, the identified genes were screened on a novel cohort of 60 Caucasian POI patients and the same case-control analysis was carried out. Comparative analysis of the human identified genes was performed on mouse and Drosophila melanogaster by analysing the orthologous genes in their ovarian phenotype, and two of the selected genes were fruit fly modelled to explore their role in fertility. MAIN RESULTS AND THE ROLE OF CHANCE: The filtering steps applied to search for extremely rare pathogenic variants in the Italian cohort revealed 64 validated single-nucleotide variants/Indels in 59 genes in 30 out of 41 screened women. Burden test analysis highlighted 13 ovarian genes as being the most enriched and significant. To validate these findings, filtering steps and Burden analysis on the second cohort of Caucasian patients yielded 11 significantly enriched genes. Among them, AFP, DMRT3, MOV10, FYN and MYC were significant in both patient cohorts and hence were considered strong candidates for POI. Mouse and Drosophila comparative analysis evaluated a conserved role through the evolution of several candidates, and functional studies using a Drosophila model, when applicable, supported the conserved role of the MOV10 armitage and DMRT3 dmrt93B orthologues in female fertility. LARGE SCALE DATA: The datasets for the Italian cohort generated during the current study are publicly available at ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/): accession numbers SCV001364312 to SCV001364375. LIMITATIONS, REASONS FOR CAUTION: This is a targeted WES analysis hunting variants in candidate genes previously identified by different genomic approaches. For most of the investigated sporadic cases, we could not track the parental inheritance, due to unavailability of the parents' DNA samples; in addition, we might have overlooked additional rare variants in novel candidate POI genes extracted from the exome data. On the contrary, we might have considered some inherited variants whose clinical significance is uncertain and might not be causative for the patients' phenotype. Additionally, as regards the Drosophila model, it will be extremely important in the future to have more mutants or RNAi strains available for each candidate gene in order to validate their role in POI pathogenesis. WIDER IMPLICATIONS OF THE FINDINGS: The genomic, statistical, comparative and functional approaches integrated in our study convincingly support the extremely heterogeneous oligogenic nature of POI, and confirm the maintenance across the evolution of some key genes safeguarding fertility and successful reproduction. Two principal classes of genes were identified: (i) genes primarily involved in meiosis, namely in synaptonemal complex formation, asymmetric division and oocyte maturation and (ii) genes safeguarding cell maintenance (piRNA and DNA repair pathways). STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C621_2016 and 08C924_2019) provided to IRCCS Istituto Auxologico Italiano, and by 'Piano Sostegno alla Ricerca' (PSR2020_FINELLI_LINEA_B) provided by the University of Milan; M.P.B. was supported by Telethon-Italy (grant number GG14181). There are no conflicts of interest.

High-resolution array-CGH analysis on 46,XX patients affected by early onset primary ovarian insufficiency discloses new genes involved in ovarian function.

STUDY QUESTION: Can high resolution array-CGH analysis on a cohort of women showing a primary ovarian insufficiency (POI) phenotype in young age identify copy number variants (CNVs) with a deleterious effect on ovarian function? SUMMARY ANSWER: This approach has proved effective to clarify the role of CNVs in POI pathogenesis and to better unveil both novel candidate genes and pathogenic mechanisms. WHAT IS KNOWN ALREADY: POI describes the progression toward the cessation of ovarian function before the age of 40 years. Genetic causes are highly heterogeneous and despite several genes being associated with ovarian failure, most of genetic basis of POI still needs to be elucidated. STUDY DESIGN, SIZE, DURATION: The current study included 67 46,XX patients with early onset POI (<19 years) and 134 control females recruited between 2012 and 2016 at the Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano. PARTICIPANTS/MATERIALS, SETTING, METHODS: High resolution array-CGH analysis was carried out on POI patients' DNA. Results of patients and female controls were analyzed to search for rare CNVs. All variants were validated and subjected to a gene content analysis and disease gene prioritization based on the present literature to find out new ovary candidate genes. Case-control study with statistical analysis was carried out to validate our approach and evaluate any ovary CNVs/gene enrichment. Characterization of particular CNVs with molecular and functional studies was performed to assess their pathogenic involvement in POI. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 37 ovary-related CNVs involving 44 genes with a role in ovary in 32 patients. All except one of the selected CNVs were not observed in the control group. Possible involvement of the CNVs in POI pathogenesis was further corroborated by a case-control analysis that showed a significant enrichment of ovary-related CNVs/genes in patients (P = 0.0132; P = 0.0126). Disease gene prioritization identified both previously reported POI genes (e.g. BMP15, DIAPH2, CPEB1, BNC1) and new candidates supported by transcript and functional studies, such as TP63 with a role in oocyte genomic integrity and VLDLR which is involved in steroidogenesis. LARGE SCALE DATA: ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/); accession numbers SCV000787656 to SCV000787743. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis for almost all of the CNVs identified. Inheritance studies of CNVs in some non-familial sporadic cases was not performed as the parents' DNA samples were not available. Addionally, RT-qPCR analyses were carried out in few cases as RNA samples were not always available and the genes were not expressed in blood. WIDER IMPLICATIONS OF THE FINDINGS: Our array-CGH screening turned out to be efficient in identifying different CNVs possibly implicated in disease onset, thus supporting the extremely wide genetic heterogeneity of POI. Since almost 50% of cases are negative rare ovary-related CNVs, array-CGH together with next generation sequencing might represent the most suitable approach to obtain a comprehensive genetic characterization of POI patients. STUDY FUNDING/COMPETING INTEREST(S): Supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C203_2012) and 'Ricerca Finalizzata' (GR-2011-02351636, BIOEFFECT) to IRCCS Istituto Auxologico Italiano.

Electron Density Analysis of Metal Clusters with Semi-Interstitial Main Group Atoms. Chemical Bonding in [Co6X(CO)16]- Species.

In this work, we propose a careful and thorough analysis of the chemical bond nature in high nuclearity metal carbonyl clusters having semi-interstitial main group atoms. We investigated the species [Co6X(CO)16]- (X = As, P), known for a rather interesting conformational flexibility of the cluster (leading to open or closed cages) and a corresponding polymorphism in the solid state (observed at least for X = As). The factors that trigger the molecular isomerism and the nature of X-Co and Co-Co interactions emerge from theoretical calculations and high resolution X-ray diffraction. Both energy and charge density atomic partitioning (QTAIM, EDA, IQA) are employed for this analysis, with the aim of revealing the stabilizing/destabilizing factors of the interaction between the cage and the semi-interstitial atoms in the various conformations.

Advances in Poly(4-aminodiphenylaniline) Nanofibers Preparation by Electrospinning Technique.

Polyaniline (PANI) nanofibers are drawing a great deal of interest from academia and industry due to their multiple applications, especially in biomedical field. PANI nanofibers were successfully electrospun for the first time by MacDiarmid and co-workers at the beginning of the millennium and since then many efforts have been addressed to improve their quality. However, traditional PANI prepared from aniline monomer shows some drawbacks, such as presence of toxic (i.e., benzidine) and inorganic (salts and metals) co-products, that complicate polymer post-treatment, and low solubility in common organic solvents, making hard its processing by electrospinning technique. Some industrial sectors, such as medical and biomedical, need to employ materials free from toxic and polluting species. In this regard, the oxidative polymerization of N-(4-aminophenyl)aniline, aniline dimer, to produce poly(4-aminodiphenylaniline), P4ADA, a kind of PANI, represents an innovative alternative to the traditional synthesis because the obtained polymer results free from carcinogenic and/or polluting co-products, and, moreover, more soluble than traditional PANI. This latter feature can be exploited to obtain P4ADA nanofibers by electrospinning technique. In this paper we report the advances obtained in the P4ADA nanofibers electrospinnig. A comparison among polyethylene oxide (PEO), polymethyl methacrylate (PMMA) and polystyrene (PS), as the second polymer to facilitate the electrospinning process, is shown. In order to increase the conductivity of P4ADA nanofibers, two strategies were adopted and compared: selective insulating binder removal from electrospun nanofibers by a rinsing tratment, afterwards optimizing the minimum amount of binder necessary for the electrospinning process. Moreover, the effect of PEO/P4ADA weight ratio on the fibers morphology and conductivity was highlighted.

Similar patterns of myocardial metabolism and perfusion in patients with type 2 diabetes and heart disease of ischaemic and non-ischaemic origin.

AIMS/HYPOTHESIS: Type 2 diabetes and insulin resistance are often associated with the co-occurrence of coronary atherosclerosis and cardiac dysfunction. The aim of this study was to define the independent relationships between left ventricular dysfunction or ischaemia and patterns of myocardial perfusion and metabolism in type 2 diabetes. METHODS: Twenty-four type 2 diabetic patients--12 with coronary artery disease (CAD) and preserved left ventricular function and 12 with non-ischaemic heart failure (HF)--were enrolled in a cross-sectional study. Positron emission tomography (PET) was used to assess myocardial blood flow (MBF) at rest, after pharmacological stress and under euglycaemic hyperinsulinaemia. Insulin-mediated myocardial glucose disposal was determined with 2-deoxy-2-[(18)F]fluoroglucose PET. RESULTS: There was no difference in myocardial glucose uptake (MGU) between the healthy myocardium of CAD patients and the dysfunctional myocardium of HF patients. MGU was strongly influenced by levels of systemic insulin resistance in both groups (CAD, r = 0.85, p = 0.005; HF, r = 0.77, p = 0.01). In HF patients, there was an inverse association between MGU and the coronary flow reserve (r = -0.434, p = 0.0115). A similar relationship was observed in non-ischaemic segments of CAD patients. Hyperinsulinaemia increased MBF to a similar extent in the non-ischaemic myocardial of CAD and HF patients. CONCLUSIONS/INTERPRETATION: In type 2 diabetes, similar metabolic and perfusion patterns can be detected in the non-ischaemic regions of CAD patients with normal cardiac function and in the dysfunctional non-ischaemic myocardium of HF patients. This suggests that insulin resistance, rather than diagnosis of ischaemia or left ventricular dysfunction, affects the metabolism and perfusion features of patients with type 2 diabetes.

Impact of increased visceral and cardiac fat on cardiometabolic risk and disease.

OBJECTIVE: Previous studies have highlighted the associations between abdominal, cardiac or total fat accumulation and cardiovascular disease. The aim of this study was to investigate the impact of different ectopic fat depots on measurements of metabolic dysfunction and cardiovascular disease risk. METHODS: Using magnetic resonance imaging in 113 subjects, we measured abdominal (visceral and subcutaneous) and cardiac (epicardial and extra-pericardial) fat depots and examined their association with overall (BMI) and abdominal obesity (waist circumference), dyslipidaemia (triglycerides, total and HDL cholesterol), glucose tolerance (by an oral glucose tolerance test) and insulin sensitivity, blood pressure and 10-year coronary heart disease risk by Framingham score. RESULTS: Fat accumulation was proportional to the degree of obesity, with body fat ranging from 14 to 33 kg, visceral fat from 0.8 to 1.8 kg and cardiac fat from 134 to 236 g. Most cardiac fat (70% on average) was extra-pericardial, with a wide variability for both cardiac depots (epicardial: 172-2008 mm(2); extra-pericardial: 100-5056 mm(2)). Only visceral and extra-pericardial fat, but not epicardial or subcutaneous fat, could discriminate between subjects with three or more factors of the metabolic syndrome or medium-to-high coronary heart disease risk score. Controlling for gender and BMI by multivariable analysis, the best marker of reduced insulin sensitivity was visceral fat (partial r = -0.35); extra-pericardial fat was the closest associate of increased blood pressure (partial r = 0.26) and both extra-pericardial and visceral fat clustered with hypertriglyceridaemia (partial r = 0.29 and 0.24; both P < 0.02). CONCLUSION: Increased epicardial fat per se does not necessarily translate into presence or prediction of disease. In contrast, increased deposition of visceral abdominal and extra-pericardial mediastinal fat are both associated with an enhanced cardiovascular disease risk profile.

Multicystic peritoneal mesothelioma treated by surgical cytoreduction and hyperthermic intra-peritoneal chemotherapy (HIPEC).

BACKGROUND: Multicystic peritoneal mesothelioma (MPM) is an extremely uncommon lesion with uncertain malignant potential. Multiple recurrences after surgical interventions and transition to aggressive malignancies have been reported. Here, we review our experience with cytoreduction and hyperthermic intraperitoneal chemotherapy (HIPEC) in the management of MPM. PATIENTS AND METHODS: Five women with MPM underwent 6 procedures of cytoreduction and close-abdomen HIPEC with cisplatin and doxorubicin. Three patients had recurrent disease after 1, 2 and 4 previous debulkings, respectively. RESULTS: Optimal cytoreduction (residual tumor nodules < or =2.5 mm) was performed in all the procedures. One grade 4 postoperative complication (NCI/CTCAE v.3.0) and no operative mortality occurred. Median follow-up was 31 months (range 3-102). MPM recurred in two patients: one is presently disease-free after a second cytoreduction with HIPEC and the other is alive with minimal stable disease. CONCLUSION: Definitive eradication by means of cytoreduction and HIPEC seems a safe and effective therapeutic option for MPM.

Crura augmentation with Bio-A® mesh for laparoscopic repair of hiatal hernia: single-institution experience with 100 consecutive patients.

BACKGROUND: The potential utility of both non-absorbable and absorbable meshes to reinforce the esophageal hiatus and prevent recurrent hernia has been investigated in observational studies and a few randomized clinical trials. Use of absorbable mesh has been associated with lesser side-effects, but the long-term safety and effectiveness are still debated. This rather scanty clinical evidence is due to heterogeneity and bias regarding the type of mesh and operation used, the modalities of follow-up, and the reporting of objective results. OBJECTIVES: The aim of the study was to assess safety, quality of life, and recurrence-free probability after laparoscopic repair of hiatal hernia reinforced with a synthetic absorbable mesh. METHODS: Observational, retrospective, single-center cohort study. All patients with hiatal hernia who underwent laparoscopic crura repair using a biosynthetic mesh (Gore Bio A® tissue reinforcement, Flagstaff, AZ) were included. Pre- and post-operative symptoms were assessed with the GERD-HRQL questionnaire. Objective follow-up consisted of upper gastrointestinal endoscopy and barium swallow study. RESULTS: From September 2011 to March 2016, a total of 100 patients underwent hiatal hernia repair using a Bio-A® mesh. All surgical procedures were completed laparoscopically. Postoperative morbidity rate was 10%. All patients had a minimum follow-up of 6 months, and the median follow-up was 30 (IQR = 22) months. No mesh-related complications occurred. The incidence of recurrent hernia ≥2 cm was 9%, and eight of the nine patients had a preoperative type III hernia. The median GERD-HRQL score was significantly reduced after operation (p < 0.001). The recurrence-free probability at 1 and 5 years was, respectively, 0.99 (CI 0.97-1.00) and 0.84 (CI 0.74-0.97), and no reoperation was required. No association was found between age, BMI, hernia size, previously failed surgical repairs and hernia recurrence. CONCLUSIONS: The use of a synthetic absorbable mesh to reinforce the esophageal hiatus is safe and appears to be effective and durable over a medium-term follow-up.

Insulin resistance and hyperinsulinemia: No independent relation to left ventricular mass in humans.

BACKGROUND: Hyperinsulinemia and insulin resistance may contribute to the development of cardiac hypertrophy. In humans, however, the evidence is inconclusive. METHODS AND RESULTS: We studied 50 nondiabetic subjects covering a wide range of age (20 to 65 years), body mass index (BMI, 19 to 40 kg x m(-2)), and mean blood pressure (72 to 132 mm Hg). Plasma insulin concentrations and secretory rates were measured at baseline and during an oral glucose tolerance test; insulin sensitivity was measured by the insulin clamp technique. Left ventricular mass (LVM) (by 2D M-mode echocardiography) was distributed normally and was higher in obese (BMI >/=27 kg x m(-2), n=16) or hypertensive patients (blood pressure >140/90 mm Hg, n=21) (50+/-8 and 55+/-10 g x m(-2.7), respectively) than in 13 nonobese, normotensive subjects (40+/-8 g x m(-2.7), P:=0.0004). In a multivariate model adjusting for sex, age, BMI, and blood pressure, neither insulin concentrations (fasting or postglucose) nor insulin sensitivity or secretory rates were significant correlates of LVM. Systolic blood pressure (P:=0.003) and BMI (P:=0.01) were the only independent correlates of LVM. From the regression, the impact of hypertension (as a systolic pressure of 180 versus 140 mm Hg=+20%) was twice as large as that of obesity (as a BMI of 35 versus 25 kg x m(-2)=+11%), the two factors being additive. CONCLUSIONS: When adequate account is taken of body mass and blood pressure, insulin, as concentration, secretion, or action, is not an independent determinant of LVM in nondiabetic subjects.

Effect of physiological hyperinsulinemia on gluconeogenesis in nondiabetic subjects and in type 2 diabetic patients.

Gluconeogenesis (GNG) is enhanced in type 2 diabetes. In experimental animals, insulin at high doses decreases the incorporation of labeled GNG precursors into plasma glucose. Whether physiological hyperinsulinemia has any effect on total GNG in humans has not been determined. We combined the insulin clamp with the (2)H(2)O technique to measure total GNG in 33 subjects with type 2 diabetes (BMI 29.0 +/- 0.6 kg/m(2), fasting plasma glucose 8.1 +/- 0.3 mmol/l) and in 9 nondiabetic BMI-matched subjects after 16 h of fasting and after euglycemic hyperinsulinemia. A primed-constant infusion of 6,6-(2)H-glucose was used to monitor endogenous glucose output (EGO); insulin (40 mU. min(-1). m(-2)) was then infused while clamping plasma glucose for 2 h (at 5.8 +/- 0.1 and 4.9 +/- 0.2 mmol/l for diabetic and control subjects, respectively). In the fasting state, EGO averaged 15.2 +/- 0.4 micromol. min(-1). kg(-1)(ffm) (62% from GNG) in diabetic subjects and 12.2 +/- 0.7 micromol. min(-1). kg(-1)(ffm) (55% from GNG) in control subjects (P < 0.05 or less for both fluxes). Glycogenolysis (EGO - GNG) was similar in the two groups (P = NS). During the last 40 min of the clamp, both EGO and GNG were significantly (P < 0.01 or less, compared with fasting) inhibited (EGO 7.1 +/- 0.9 and 3.6 +/- 0.5 and GNG 7.9 +/- 0.5 and 4.5 +/- 1.0 respectively) but remained significantly (P < 0.05) higher in diabetic subjects, whereas glycogenolysis was suppressed completely and equally in both groups. During hyperinsulinemia, GNG micromol. min(-1). kg(-1)(ffm) in diabetic and control subjects, was reciprocally related to plasma glucose clearance. In conclusion, physiological hyperinsulinemia suppresses GNG by approximately 20%, while completely blocking glycogenolysis. Resistance of GNG (to insulin suppression) and resistance of glucose uptake (to insulin stimulation) are coupled phenomena. In type 2 diabetes, the excess GNG of the fasting state is carried over to the insulinized state, thereby contributing to glucose overproduction under both conditions.

Troglitazone reduces LDL oxidation and lowers plasma E-selectin concentration in NIDDM patients.

Troglitazone, an oral antidiabetic agent with antioxidant properties, has previously been shown to increase the resistance of LDL to oxidation in vitro and in vivo in healthy volunteers. In a randomized, placebo-controlled, parallel-group study in 29 patients with NIDDM, we tested the effect of troglitazone (200 mg once daily) on the resistance of LDL to oxidation and on circulating levels of preformed lipid hydroperoxides and the adhesion molecule E-selectin. Resistance of LDL to oxidation was assessed by measuring 1) fluorescence development induced by copper treatment (lag phase), and 2) amount of thiobarbituric acid-reactive substances (TBARS) generated by incubation with umbilical vein endothelial cells. At 8 weeks, the lag phase was increased by 23% (P < 0.01 by analysis of covariance [ANCOVA]) in the patients receiving troglitazone (n = 18) compared with the group receiving placebo (n = 11). At the same time, TBARS were 3.63 +/- 0.10 nmol/l (vs. 5.32 +/- 0.10 nmol/l in the placebo group, P = 0.009), LDL hydroperoxide concentration was reduced from 1.48 +/- 0.03 to 1.19 +/- 0.03 ng/mg (no change in the placebo group, P < 0.01), and plasma E-selectin levels decreased from 56.5 +/- 2.33 to 43.7 +/- 1.77 microg/l (no change in the placebo group, P < 0.01). In NIDDM, troglitazone may slow down the development of atherosclerosis by modifying LDL-related atherogenic events.

Effect of vitamin C on forearm blood flow and glucose metabolism in essential hypertension.

In 9 patients with essential hypertension, we tested whether a high-dose (12 mg. min(-1)) vitamin C infusion into the brachial artery, by improving endothelium-dependent vasodilatation, would also attenuate the insulin resistance of deep forearm tissues. We measured the effect of vitamin C on acetylcholine (Ach)-induced vasodilatation and on forearm glucose uptake during systemic hyperinsulinemia; in all studies, the contralateral forearm served as the control. Intrabrachial Ach infusion produced a stable increase in forearm blood flow, from 2.6+/-0.3 to 10.6+/-2.1 mL. min(-1). dL(-1); when vitamin C was added, a further rise in forearm blood flow (to 13.4 mL. min(-1). dL(-1); P<0.03 vs Ach alone) was observed. In response to insulin, blood flow in both the infused and control forearms did not significantly change from baseline values (+10+/-16% and +2+/-11%, respectively). In contrast, when vitamin C was added, blood flow in the infused forearm increased significantly (to 3.7+/-0.7 mL. min(-1). dL(-1); P<0.02 vs 2.8+/-0.6 mL. min(-1). dL(-1) in the control forearm). Insulin stimulated whole-body glucose disposal to 20+/-2 micromol. min(-1). kg(-1), compatible with the presence of marked insulin resistance. Forearm glucose uptake was similarly stimulated after 80 minutes of insulin infusion (to 2.11+/-0.42 and 2.06+/-0.43 micromol. min(-1). dL(-1), infused and control, respectively). When intrabrachial vitamin C was added, no difference in glucose uptake was observed between the 2 forearms (infused, 2.37+/-0.44 micromol. min(-1). dL(-1)and control, 2.36+/-0. 53 micromol. min(-1). dL(-1)). Forearm O(2) uptake at baseline was also similar in the 2 forearms (infused, 9.7+/-0.7 micromol. min(-1). dL(-1) and control, 9.6+/-1.1 micromol. min(-1). dL(-1)) and was not changed by either insulin or vitamin C. We conclude that in the deep forearm tissues of patients with essential hypertension and insulin resistance, an acute improvement in endothelial function, obtained with pharmacological doses of vitamin C, restores insulin-mediated vasodilatation but does not improve insulin-mediated glucose uptake. Thus, the endothelial dysfunction of essential hypertension is unlikely to be responsible for their metabolic insulin resistance.

High-speed gas chromatography with direct resistively-heated column (ultra fast module-GC)-separation measure (S) and other chromatographic parameters under different analysis conditions for samples of different complexities and volatilities.

The influence of GC speed on the separation capability of a chromatographic system is reported measuring a series of parameters including separation measure (S), peak capacity (n), peak width (w), analysis time, t(b) (determined on the last eluting compound) and separation measure/analysis time ratio (S/t(b)) determined by analyzing a bergamot essential oil sample and a standard mixture of pesticides. Conventional GC, fast GC (with 10 m (FGC10) and 5 m (FGC5) narrow-bore columns), and direct resistively-heated ultra fast module-GC (UFM-GC) were the GC speed approaches used. The influence of different heating rates with a constant flow for FGC5, FGC 10, and UFM-GC and with variable flows for UFM-GC on S, n, w, S/t(b), and t(b) was also studied. The results of this study show that: (a) separation capability of the chromatographic system (i.e. S and n) and analysis time depend on the GC approaches. Within each GC approach, S and n and analysis time depend on the heating rates, although to a different extent, and S and n decrease much less than the gain in analysis time, in particular when fast heating rates are applied; (b) in UFM-GC, the loss of separation capability with heating rate can also be partially compensated by the choice of an appropriate flow rate that, within each heating rate, may contribute to increase S while reducing t(b); (c) within a specific GC approach, the chromatographic system (column and stationary phase) and conditions (heating and flow rates) must be such to achieve a suitable S-value when two analytes must be separated with a given resolution in a minimum analysis time.

Effects of troglitazone on insulin action and cardiovascular risk factors in patients with non-insulin-dependent diabetes.

OBJECTIVE: Insulin resistance is a potential target for pharmacologic intervention in non-insulin-dependent diabetes. Troglitazone is being evaluated as an insulin enhancer in insulin resistant states. RESEARCH DESIGN AND METHODS: We randomized 40 patients with non-insulin-dependent diabetes to diet plus placebo (n = 15) or diet plus troglitazone (n = 25; 200 mg/day) treatment for 8 weeks. Fasting endogenous glucose production (EGP, by the stable isotope technique) and whole-body insulin sensitivity (by the insulin suppression test) were measured at baseline and on days 3, 7, 14, 28, and 56 of treatment. RESULTS: By day 56, fasting plasma glucose had risen from 12.0 +/- 0.9 to 12.8 +/- 1.2 mmol/L in the placebo group and had fallen from 12.4 +/- 0.6 to 11.3 +/- 0.6 mmol/L in the troglitazone group (p = 0.03). This was the result of small improvements in whole-body insulin sensitivity (steady-state plasma glucose during the insulin suppression test: from 11.09 +/- 1.1 to 10.3 +/- 0.8 mmol/L versus 13.8 +/- 1.0 to 10.0 +/- 0.9 mmol/L, placebo versus troglitazone; p = 0.01) and EGP (from 103% +/- 3% versus 96% +/- 2% of baseline, placebo versus troglitazone; p = 0.09). The time course of insulin action showed an early (first week of treatment) decrease in EGP in the troglitazone group that was maintained throughout, whereas steady-state plasma glucose levels began to diverge toward the end of treatment. The effects of insulin on plasma free fatty acid and potassium concentrations were not different between placebo and troglitazone. The cardiovascular risk profile (heart rate; serum triglycerides; total, low-density lipoprotein, and high-density lipoprotein cholesterol; proinsulin; uric acid; plasminogen activator inhibitor-1 antigen and activity; 24-hour blood pressure monitoring and urinary albumin excretion) was unaltered by troglitazone treatment. CONCLUSIONS: Troglitazone as monotherapy for typical non-insulin-dependent diabetes had a modest anti-hyperglycemic effect and, at the dose used in this study, had no effect on cardiovascular risk factors.

Effect of insulin on renal sodium and uric acid handling in essential hypertension.

In normal subjects, insulin decreases the urinary excretion of sodium, potassium, and uric acid. We tested whether these renal effects of insulin are altered in insulin resistant hypertension. In 37 patients with essential hypertension, we measured the changes in urinary excretion of sodium, potassium, and uric acid in response to physiological euglycemic hyperinsulinemia (by using the insulin clamp technique at an insulin infusion rate of 6 pmol/min/kg). Glucose disposal rate averaged 26.6 +/- 1.5 mumol/min/kg, i.e., 20% lower than in normotensive controls (33.1 +/- 2.1 mumol/min/kg, P = .015) In the basal state, fasting plasma uric acid concentrations were higher in men than women (P < .001), were positively related to body mass index (r = 0.38, P = .02), waist/hip ratio (r = 0.35, P < .05), and serum triglyceride levels (r = 0.59, P = .0001), and negatively related to HDL cholesterol concentrations (r = -0.59, P = .0001) and glucose disposal rate (r = 0.42, P < .01). Uric acid clearance, on the other hand, was inversely related to body mass index (r = 0.41, P = .01), plasma uric acid (r = 0.65, P < .0001) and triglyceride concentrations (r = 0.39, P < .02), and directly related to HDL cholesterol levels (r = 0.52, P < .001). During insulin infusion, blood pressure, plasma uric acid and sodium concentration, and creatinine clearance did not change. In contrast, hyperinsulinemia caused a significant decrease in the urinary excretion of uric acid (2.67 +/- 0.12 to 1.86 +/- .14 mumol/min/1.73 m2, P = .0001), sodium (184 +/- 12 to 137 +/- 14 mumol/min/1.73 m2, P = .0001), and potassium (81 +/- 7 to 48 +/- 4 mumol/ min/1.73 m2, P = .0001). Both in absolute terms (clearance and fractional excretion rates) and percentagewise, these changes were similar to those found in normotensive subjects. Insulin-induced changes in urate excretion were coupled (r = 0.55, P < .0001) to the respective changes in sodium excretion. In hypertensive patients, higher uric acid levels and lower renal urate clearance rates cluster with insulin resistance and dyslipidemia. Despite insulin resistance of glucose metabolism, acute physiological hyperinsulinemia causes normal antinatriuresis, antikaliuresis, and antiuricosuria in these patients.

In vivo effect of insulin on intracellular calcium concentrations: relation to insulin resistance.

Elevated intracellular calcium concentrations ([Ca2+]i) have been described in essential hypertension and other insulin-resistant states. Our aim was to explore the relationship between insulin resistance and abnormal Ca2+ metabolism. In 50 nondiabetic subjects, half of whom had untreated essential hypertension, we simultaneously measured the in vivo effect of insulin on glucose metabolism (by the insulin clamp technique) and on platelet [Ca2+]i (by the Fura-2 method). In each subject, [Ca2+]i measurements (both in Ca(2+)-free medium and, sequentially, following in vitro Ca2+ loading) were obtained in the fasting state and after 2 hours of euglycemic hyperinsulinemia. In the fasting state, no association was found between any measure of [Ca2+]i and gender, age, body mass index (BMI), blood pressure, or insulin sensitivity. In contrast, following in vivo insulin, platelet [Ca2+]i increased significantly (from 23 +/- 1 to 28 +/- 1 nmol/L in Ca(2+)-free medium, P < .01) in the whole group, and an insulin-induced increase in [Ca2+]i was associated with insulin resistance (r = .35, P = .01) but not with hypertension (r = .2, P = .17) and with impaired glucose storage (as determined by indirect calorimetry, r = .39, P = .01) but not with glucose oxidation. Thus, the 12 most insulin-resistant subjects were characterized by a cluster of abnormalities (mild overweight, higher blood pressure and prevalence of hypertension, higher serum triglycerides and insulin response to oral glucose, and reduced glucose storage) that included an insulin-induced increase in [Ca2+]i (9 +/- 2 nmol/L, P < .001 v basal). We conclude that insulin resistance, rather than hypertension, is associated with an abnormal in vivo effect of insulin on platelet [Ca2+]i.

Characterization of the toxicity of distamycin derivatives on cancer cell lines and rat heart.

The cytotoxicity and cardiotoxicity of benzoyl mustard (FCE 24517) and epoxamido (FCE 24561) synthetic derivatives of distamycin A were reported in the present study. The 50% inhibiting concentration (IC50) of colony formation of FCE 24517 on human SNB-19 glioblastoma, A2780 ovarian cancer and DU 145 prostate cancer was at least three times lower than that of FCE 24561; on the same cell lines the IC50 of DXR was up to 14 and 240 times higher than that of FCE 24561 and FCE 24517, respectively. Isolated rat hearts perfused with concentrations of both derivatives equivalent to their respective IC50 values did not show any significant change in ECG parameters, contractility and coronary flow. Compared to control hearts, FCE 24517 10(-6) M induced a significant increase in PR interval, reduction in + dF/dtmax, heart rate and coronary flow, while FCE 24561 10(-6) M produced a modest but significant increase in S alpha T segment and decrease in + dF/dtmax. Rats treated with FCE 24561 3, 6 or 12 mg/kg, intravenously (i.v.), once weekly for 3 weeks had a modest increase in S alpha T segment and QRS complex duration, while a slight alteration of S alpha T segment and QRS complex duration were observed in rats given FCE 24517 1 or 2 mg/kg i.v. once weekly for 3 weeks. No cardiac histologic alterations were found in hearts from rats receiving FCE 24517 or FCE 24561. For comparison, the cardiotoxicity of doxorubicin (DXR) was evaluated in the same experimental models; perfusion of hearts with DXR 10(-6) M induced severe alterations in all parameters of the isolated hearts; the administration of DXR 3 mg/kg i.v. once a week for 3 weeks was associated with a widening of the S alpha T segment and QRS complex and cardiac histologic picture was markedly altered. In conclusion, distamycin A derivatives display elevated cytotoxicity while no substantial cardiotoxicity was observed.

Conventional inner diameter short capillary columns: an approach to speeding up gas chromatographic analysis of medium complexity samples.

Short capillary columns (5 m) with 0.25 mm inner diameter (I.D.) are applied to the GC analysis of medium complexity samples (up to 30 components) with the aim of shortening analysis time. This approach is complementary to fast GC with narrow-bore columns and is based on compensating the lower efficiency of short columns with conventional I.D.'s (0.25-0.32 mm) by using a stationary phase selectivity suitable to separate the components of the sample under investigation, so that the required resolution power is achieved but, at the same time, the analysis time is shortened. The qualitative and quantitative effectiveness of this approach is demonstrated through the analysis of: essential oils with different compositions (chamomile and rosemary), low-volatility triterpenes in a plant extract (Maytenus aquifolium and M. ilicfolium), thermolabile pyrethrins in a Pyrethrum extract, and a mixture of pesticides applied to protect medicinal plant crops. In all examples, GC analysis was five to ten times faster than with conventional columns.

An uncommon cause of massive intraperitoneal bleeding: leiomyoblastoma of the stomach.

A large exogastric leiomyoblastoma in a 48-year-old male revealed by asymptomatic upper abdominal mass is reported. Abdominal ultrasound, computerized tomography scan and magnetic resonance showed a 20 cm cystic lesion apart from liver and pancreas of undetermined origin. During hospitalization, massive intraperitoneal bleeding due to rupture of the mass was observed. An emergency laparotomy was carried out, and excision of a large, ruptured, cystic mass involving the greater gastric curvature was performed. Microscopy revealed a gastric leiomyoblastoma. Even if infrequent, massive intraperitoneal bleeding is a very serious complication of gastric leiomyoblastoma. Considering the difficulty of an accurate preoperative diagnosis and the risk of intraperitoneal rupture, the authors suggest that similar abdominal masses should be managed by quick diagnostic investigations and early surgical procedures.

[Re]

A chair conformation comparable to that observed for six-membered rings composed of tetrahedral carbon atoms is found for the cluster anion [Re(6)(µ-H)(5)(CO)(24)](-) (see picture; black spheres: Re, white spheres: µ-H; CO ligands omitted for clarity) in spite of the octahedral coordination at the Re centers. This is the first example of a carbonyl cluster exhibiting a cyclohexane-like geometry of the metallic framework.