Development of an analytical platform for the affinity screening of natural extracts by SEC-MS towards PPARα and PPARγ receptors.

BACKGROUND: Peroxisome proliferator-activated receptors (PPARs) belong to the superfamily of nuclear receptors and represent the targets for the therapeutical treatment of type 2 diabetes, dyslipidemia and hyperglycemia associated with metabolic syndrome. Some medicinal plants have been traditionally used to treat this kind of metabolic diseases. Today only few drugs targeting PPARs have been approved and for this reason, the rapid identification of novel ligands and/or chemical scaffolds starting from natural extracts would benefit of a selective affinity ligand fishing assay. RESULTS: In this paper we describe the development of a new ligand fishing assay based on size exclusion chromatography (SEC) coupled to LC-MS for the analysis of complex samples such as botanical extracts. The known PPARα and PPARγ ligands, WY-14643 and rosiglitazone respectively, were used for system development and evaluation. The system has found application on an Allium lusitanicum methanolic extract, containing saponins, a class of chemical compounds which have attracted interest as PPARs ligands because of their hypolipidemic and insulin-like properties. SIGNIFICANCE: A new SEC-AS-MS method has been developed for the affinity screening of PPARα and PPARγ ligands. The system proved to be highly specific and will be used to improve the throughput for the identification of new selective metabolites from natural souces targeting PPARα and PPARγ.

Resveratrol and its metabolites bind to PPARs.

Resveratrol, a modulator of several signaling proteins, can exert off-target effects involving the peroxisome proliferator-activated receptor (PPAR) transcription factors. However, evidence for the direct interaction between this polyphenol and PPARs is lacking. Here, we addressed the hypothesis that resveratrol and its metabolites control aspects of PPAR transcriptional activity through direct interaction with PPARs. Bioaffinity chromatographic studies with the immobilized ligand-binding domains (LBDs) of PPARγ and PPARα and isothermal titration calorimetry allowed the binding affinities of resveratrol, resveratrol 3-O-glucuronide, resveratrol 4-O-glucuronide, and resveratrol 3-O-sulfate to both PPAR-LBDs to be determined. Interaction of resveratrol, resveratrol 3-O-glucuronide, and resveratrol 4-O-glucuronide with PPARγ-LBD occurred with binding affinities of 1.4, 1.1, and 0.8 µM, respectively, although only resveratrol bound to the PPARα-LBD with a binding affinity of 2.7 µM. Subsequently, X-ray crystallographic studies were carried out to characterize resveratrol binding to the PPARγ-LBD at the molecular level. The electron density map from the crystal structure of the complex between PPARγ-LBD and resveratrol revealed the presence of one molecule of resveratrol bound to the LBD of PPARγ, with the ligand occupying a position close to that of other known PPARγ ligands. Transactivation assays were also performed in HepG2 cells, with the results showing that resveratrol was not a PPAR agonist but instead was able to displace rosiglitazone from PPARγ and Wy-14643 from PPARα with IC50 values of (27.4±1.8) µM and (31.7±2.5) µM, respectively. We propose that resveratrol acts as a PPAR antagonist through its direct interaction with PPARγ and PPARα.

Anelastic phenomena associated to water loss and collagen degradation in human dentin.

This work describes the anelastic and dynamic Young modulus behaviour of human dentin from room temperature up to 673 K. Human molars, extracted from individuals (males 55-70 years old) as part of their dental treatment, were cut to obtain bar-shaped samples subsequently used for mechanical spectroscopy experiments. In addition, thermo-gravimetric analysis (TGA) has been performed to assess a possible weight loss occurring in the same temperature range of mechanical spectroscopy tests. A broad and asymmetric internal friction (Q(-1)) maximum at 500 K has been observed during the heating of the as-prepared samples. This maximum is absent during the following cooling down to room temperature. It is therefore due to the occurrence of an irreversible transformation in the sample. TGA shows a remarkable weight loss in the same temperature range. This effect has been related to loss of fluids and degradation of collagen. Another set of samples, previously kept for 36 h under a vacuum of 10(-2)Pa, were submitted at room temperature to test at increasing strain from 6×10(-6) to 7×10(-4). The results show transient and fully recoverable Q(-1) increase and dynamic modulus (E) decrease. The phenomenon has been ascribed to the breaking of weak H-bonds between polypeptide chains forming the triple-helix with consequent increase of the mean length of vibrating chain segments.

Open tubular columns containing the immobilized ligand binding domain of peroxisome proliferator-activated receptors α and γ for dual agonists characterization by frontal affinity chromatography with mass spectrometry detection.

The peroxisome proliferator-activated receptors (PPARs) belong to the nuclear receptor superfamily. In the last years novel PPARs ligands have been identified and these include PPARα/γ dual agonists. To rapidly identify novel PPARs dual ligands, a robust binding assay amenable to high-throughput screening toward PPAR isoforms would be desirable. In this work we describe a parallel assay based on the principles of frontal affinity chromatography coupled to mass spectrometry (FAC-MS) that can be used to characterize dual agonists. For this purpose the ligand binding domain of PPARα receptor was immobilized onto the surface of open tubular capillaries to create new PPAR-alpha-OT columns to be used in parallel with PPAR-gamma-OT columns. The two biochromatographic systems were used in both ranking and Kd experiments toward new ureidofibrate-like dual agonists for subtype selectivity ratio determination. In order to validate the system, the Kd values determined by frontal analysis chromatography were compared to the affinity constants obtained by ITC experiments. The results of this study strongly demonstrate the specific nature of the interaction of the ligands with the two immobilized receptor subtypes.

New 2-(aryloxy)-3-phenylpropanoic acids as peroxisome proliferator-activated receptor α/γ dual agonists able to upregulate mitochondrial carnitine shuttle system gene expression.

The preparation of a series of 2-(aryloxy)-3-phenylpropanoic acids, resulting from the introduction of different substituents into the biphenyl system of the previously reported peroxisome proliferator-activated receptor α/γ (PPARα/γ) dual agonist 1, allowed the identification of new ligands with higher potency on PPARα and fine-tuned moderate PPARγ activity. For the most promising stereoisomer (S)-16, X-ray and calorimetric studies in PPARγ revealed, at high ligand concentration, the presence of two molecules simultaneously bound to the receptor. On the basis of these results and docking experiments in both receptor subtypes, a molecular explanation was provided for its different behavior as a full and partial agonist of PPARα and PPARγ, respectively. The effects of (S)-16 on mitochondrial acylcarnitine carrier and carnitine-palmitoyl-transferase 1 gene expression, two key components of the carnitine shuttle system, were also investigated, allowing the hypothesis of a more beneficial pharmacological profile of this compound compared to the less potent PPARα agonist fibrates currently used in therapy.

Synthesis, characterization and biological evaluation of ureidofibrate-like derivatives endowed with peroxisome proliferator-activated receptor activity.

A series of ureidofibrate-like derivatives was prepared and assayed for their PPAR functional activity. A calorimetric approach was used to characterize PPARγ-ligand interactions, and docking experiments and X-ray studies were performed to explain the observed potency and efficacy. R-1 and S-1 were selected to evaluate several aspects of their biological activity. In an adipogenic assay, both enantiomers increased the expression of PPARγ target genes and promoted the differentiation of 3T3-L1 fibroblasts to adipocytes. In vivo administration of these compounds to insulin resistant C57Bl/6J mice fed a high fat diet reduced visceral fat content and body weight. Examination of different metabolic parameters showed that R-1 and S-1 are insulin sensitizers. Notably, they also enhanced the expression of hepatic PPARα target genes indicating that their in vivo effects stemmed from an activation of both PPARα and γ. Finally, the capability of R-1 and S-1 to inhibit cellular proliferation in colon cancer cell lines was also evaluated.

Frontal affinity chromatography with MS detection of the ligand binding domain of PPARγ receptor: ligand affinity screening and stereoselective ligand-macromolecule interaction.

In this study we report the development of new chromatographic tools for binding studies based on the gamma isoform ligand binding domain (LBD) of peroxisome proliferator-activated receptor (PPARγ) belonging to the nuclear receptor superfamily of ligand-activated transcription factors. PPARγ subtype plays important roles in the functions of adipocytes, muscles, and macrophages with a direct impact on type 2 diabetes, dyslipidemia, atherosclerosis, and cardiovascular disease. In order to set up a suitable immobilization chemistry, the LBD of PPARγ receptor was first covalently immobilized onto the surface of aminopropyl silica particles to create a PPARγ-Silica column for zonal elution experiments and then onto the surface of open tubular (OT) capillaries to create PPARγ-OT capillaries following different immobilization conditions. The capillaries were used in frontal affinity chromatography coupled to mass spectrometry (FAC-MS) experiments to determine the relative binding affinities of a series of chiral fibrates. The relative affinity orders obtained for these derivatives were consistent with the EC(50) values reported in literature. The optimized PPARγ-OT capillary was validated by determining the K(d) values of two selected compounds. Known the role of stereoselectivity in the binding of chiral fibrates, for the first time a detailed study was carried out by analysing two enantioselective couples on the LBD-PPARγ capillary by FAC and a characteristic two-stairs frontal profile was derived as the result of the two saturation events. All the obtained data indicate that the immobilized form of PPARγ-LBD retained the ability to specifically bind ligands.

A randomized trial of prolonged high dose of interferon plus ribavirin for hepatitis C patients nonresponders to interferon alone.

Retreatment of chronic hepatitis C patients nonresponders to interferon (IFN) alone with the standard dose of IFN [3 million units (MU) thrice weekly (TIW)] plus ribavirin for 24 weeks has yielded low sustained virological response (SVR), averaging 8%. The aim of the present, open-labelled, randomized study was to evaluate the efficacy of IFN induction therapy followed by prolonged high dose of IFN plus ribavirin in nonresponders. One hundred and fifty-one patients were randomized to receive 5 MU daily of IFN alfa-2b (group 1, n = 73) or 5 MU TIW of IFN alfa 2b (group 2, n = 78) for 4 weeks followed by IFN (5 MU TIW) plus ribavirin (1000/1200 mg/daily) for 48 weeks in both groups. In an intention-to-treat analysis, the sustained virological response (SVR) at 24-week follow-up was 33 and 23% for group 1 and 2, respectively (P = 0.17). The overall SVR was 52 and 18% in patients with genotype 2/3 and 1/4, respectively. Among genotype 1/4 patients the SVR was 29 and 11% for age younger or older than 40 years. Compared with genotype 2/3 patients, the risk (95% confidence interval) of nonresponse to retreatment was 3.0-fold (1.17-8.0) in younger genotype 1/4 patients and 8.4-fold (3.0-23.29) in older genotype 1/4 patients. In conclusion these results suggest that retreatment with a reinforced regimen should be focused in nonresponder genotype 2/3 patients and younger genotype 1/4 patients, who are most likely to benefit. Induction therapy does not improve SVR.

Efficacy of prolonged 5 million units of interferon in combination with ribavirin for relapser patients with chronic hepatitis C.

summary. Retreatment of relapser patients with chronic hepatitis C with the standard dose of interferon (IFN) of 3 million units (MU) thrice weekly (tiw) plus ribavirin for 24 weeks achieves a sustained response in 30 and 73% of patients with genotype 1 and 2 or 3, respectively. The aim of this study was to evaluate the efficacy and safety of IFN alpha-2b induction therapy, followed by prolonged treatment with a high dose of IFN alpha-2b plus ribavirin in relapser patients. A total of 119 patients were randomized to receive IFN alpha-2b 5 MU daily (Group A: 59 patients) or IFN alpha-2b 5 MU tiw (Group B: 60 patients) for 4 weeks followed by IFN (5 MU tiw) and ribavirin (1000-1200 mg/day) for 48 weeks in both groups. The primary end point was hepatitis C virus (HCV)-RNA clearance at week 24 after the end of treatment. A sustained virological response (SVR) was achieved in 68 and 60% of Group A and B patients, respectively (P = 0.37). Logistic regression analysis identified genotype 2 or 3 as the only independent factor associated with response, whereas induction regimen and baseline viraemia levels did not affect the response. The overall SVR was 53 and 72% in patients with genotype 1 or 4 and 2 or 3, respectively. In conclusion, induction IFN therapy does not enhance the SVR to a 48-week combination therapy. Our study suggests that relapsed patients with genotype 1 or 4 may achieve significant response rates of approximately 50%, if retreated with 5 MU tiw IFN plus ribavirin for 48 weeks.

The cost-effectiveness of health education in improving knowledge and awareness about intestinal parasites in rural Bangladesh.

The impact of regular health education in improving knowledge, attitude and practices in the control of intestinal parasites was examined in four rural areas of Bangladesh; two areas received health education and the other two areas were controls. By the end of the 18-month study households receiving health education showed highly significant improvements in knowledge, water and sanitation facilities and personal hygiene compared with households in the control areas. Improving knowledge by 1% cost between US dollars 0.75 and 0.82 per household, while a 1% improvement in personal hygiene cost between US dollars 1.10 and 1.32 per household and water and sanitation between US dollars 1.39 and 1.52 per household.

Structures of solid and liquid during melting and solidification of indium.

During melting and solidification of polycrystalline indium the structures of the solid and liquid phases were investigated by X-ray diffractometry (XRD) in 1g conditions. The experiments showed that melting is immediately preceded and accompanied by an abnormal increase of vacancy concentration in the solid that enhances the diffusion. At the melting point (T(M)) the lattice planes facing the first formed liquid appear to be [002] and [101]; that is, those planes allocating first and second neighbors around a given atom, with shell radii very close to the mean distance of nearest neighbors in liquid as obtained from the radial distribution function (RDF). On this basis some types of solid-liquid interface (sharp or diffuse) are discussed; the possibility of a sudden texture change in the solid is also considered. On the other hand, the evolution of RDF curve of liquid indium, cooled down through T(M), shows that there are correlations between the structure of the liquid and of the forming solid during solidification. To avoid convective motions in the liquid and to achieve the best experimental conditions, we discuss the possibility of repeating the measurements in microgravity (microg) using thin oxide films as containers. This technique was already successfully tested by one of the investigators in the experiment ES 311 A-B carried out during the mission SPACELAB-1.

Three case definitions of malaria and their effect on diagnosis, treatment and surveillance in Cox's Bazar district, Bangladesh.

In countries where malaria is endemic, routine blood slide examinations remain the major source of data for the public health surveillance system. This approach has become inadequate, however, as the public health emphasis has changed from surveillance of laboratory-confirmed malaria infections to the early detection and treatment of the disease. As a result, it has been advocated that the information collected about malaria be changed radically and should include the monitoring of morbidity and mortality, clinical practice and quality of care. To improve the early diagnosis and prompt treatment (EDPT) of malaria patients, three malaria case definitions (MCDs) were developed, with treatment and reporting guidelines, and used in all static health facilities of Cox's Bazar district, Bangladesh (population 1.5 million). The three MCDs were: uncomplicated malaria (UM); treatment failure malaria (TFM); and severe malaria (SM). The number of malaria deaths was also reported. This paper reviews the rationale and need for MCDs in malaria control programmes and presents an analysis of the integrated surveillance information collected during the three-year period, 1995-97. The combined analysis of slide-based and clinical data and their related indicators shows that blood slide analysis is no longer used to document fever episodes but to support EDPT, with priority given to SM and TFM patients. Data indicate a decrease in the overall positive predictive value of the three MCDs as malaria prevalence decreases. Hence the data quantify the extent to which the mainly clinical diagnosis of UM leads to over-diagnosis and over-treatment in changing epidemiological conditions. Also the new surveillance data show: a halving in the case fatality rate among SM cases (from 6% to 3.1%) attributable to improved quality of care, and a stable proportion of TFM cases (around 7%) against a defined population denominator. Changes implemented in the EDPT of malaria patients and in the surveillance system were based on existing staff capacity and routine reporting structures.

[Incidence of chromosomal anomalies and congenital malformations in children born from assisted fertilization]. / Incidenza delle anomalie cromosomiche e delle malformazioni congenite nei bambini nati da procreazione medicalmente assistita.

The introduction and widespread application of Assisted Reproductive Techniques (ART) have raised major concern about the offspring 's health. The incidence of congenital and chromosomal anomalies after standard In Vitro Fertilization and Embryo Transfer seems to be similar to that expected in the general population. The prevalence of congenital malformations does not seem to be higher in children conceived by ICSI. On the other hand, it seems that there is a slight risk for transmission of chromosomal aberration of paternal origin and a certain risk of de novo sex-chromosomal and structural aberrations after ICSI. We report the results of the follow-up of 938 children conceived in our public ART by standard IVF(649) and by ICSI(289) from 21-2-1987 to 30-6-1999. The incidence of the congenital malformations results of the 1.8% (17/938); the incidence of chromosomal anomalies results 0.5% (5/938). The incidence of congenital malformations and chromosomal anomalies results 1.5% (10/649) and 0.6% (4/649), respectively, for standard IVF and 2.4% (7/289) and 0.3% (1/289) for ICSI. Our data seems to be reassuring but the incidence of chromosomal anomalies in ICSI children needs further investigation.

A study of the cost effectiveness of selective health interventions for the control of intestinal parasites in rural Bangladesh.

The study examined the cost effectiveness of 4 different regimens in reducing the prevalence and intensity of infection of Ascaris lumbricoides, Trichuris trichiura, and hookworm over an 18-mo period in randomized community samples of children aged 2-8 yr living in rural Bangladesh. The household was the unit of randomization in each community. The 4 regimens were (1) only chemotherapy to all household members at the commencement of the study (i.e., at an interval of 18 mo), (2) same as group (1) and regular health education throughout the study period, (3) chemotherapy to all household members at the commencement of the study and subsequent chemotherapy to all children at intervals of 6 mo, and (4) same as group 3 with the addition of regular health education throughout the study period. Health education (through home and school visits and focus group discussions) was aimed at increasing awareness of worm transmission and the disabilities caused by intestinal helminths. Simple ways of improving personal hygiene and sanitation through hand washing, nail trimming, wearing of shoes, and use of a latrine and clean water supplies were encouraged. Because albendazole is a broad spectrum anthelmintic, the cost effectiveness of the 4 interventions were compared by the weighted percentage reduction in prevalence and the weighted percentage reduction in intensities of infection as measured by geometric mean egg loads of all 3 worms combined. The most cost-effective strategy was the single albendazole mass chemotherapy at an interval of 18 mo. The 2 regimens involving health education were the least cost effective.

The role of diagnostic hysteroscopy and endometrial biopsy in assisted reproductive technologies.

OBJECTIVE: To study the incidence of unsuspected endouterine abnormalities in patients for whom IVF-ET repeatedly fails. DESIGN: Prospective study. SETTING: Infertility Unit of the Department of Obstetrics and Gynecology, Arcispedale Santa Maria Nuova, Reggio Emilia, Italy. PATIENT(S): One hundred patients for whom two IVF-ET cycles failed in which > or =2 good-quality embryos were transferred. INTERVENTION(S): In-office diagnostic hysteroscopy and endometrial biopsy. MAIN OUTCOME MEASURE(S): Relation between IVF-ET failure and unsuspected endouterine abnormalities. RESULT(S): In 18 patients, hysteroscopy showed an important unsuspected endouterine abnormality. Fifteen of these patients did not become pregnant after IVF-ET, and 3 became pregnant but had a spontaneous abortion. Histologic examination of the endometrium revealed chronic endometritis in 1 patient and tuberculous endometritis in another. CONCLUSION(S): Previous studies have reported that the incidence of endouterine abnormalities is high in patients undergoing IVF-ET. Our data confirm the previous reports and lead us to conclude that diagnostic hysteroscopy should be performed on all patients before they undergo IVF-ET.

The malaria control program in Papua New Guinea: epidemiological surveillance and a look into the future.

The four major phases of the malaria control program in Papua New Guinea are briefly described. Routine indicators utilized during Phases II and III (the periods when residual indoor spraying was used) are presented and their limitations outlined. The new epidemiological indicators developed during Phase IV and now included in the National Health Plan (1991-1995) are presented. Their future use and implications for the malaria control program are analyzed and discussed.