Publications by authors named "S B MARY"

83 Publications

Gain-of-function variants identified in vigabatrin-hypersensitive epileptic encephalopathies.

Brain Commun 2020 1;2(2):fcaa162. Epub 2020 Oct 1.

Faculty of Medicine and Health, School of Pharmacy, Brain and Mind Centre, The University of Sydney, Sydney, New South Wales 2006, Australia.

Variants in the gene encoding the β3-subunit of the γ-aminobutyric acid type A ( receptor are associated with various developmental and epileptic encephalopathies. Typically, these variants cause a loss-of-function molecular phenotype whereby γ-aminobutyric acid has reduced inhibitory effectiveness leading to seizures. Drugs that potentiate inhibitory GABAergic activity, such as nitrazepam, phenobarbital or vigabatrin, are expected to compensate for this and thereby reduce seizure frequency. However, vigabatrin, a drug that inhibits γ-aminobutyric acid transaminase to increase tonic γ-aminobutyric acid currents, has mixed success in treating seizures in patients with variants: some patients experience seizure cessation, but there is hypersensitivity in some patients associated with hypotonia, sedation and respiratory suppression. A variant that responds well to vigabatrin involves a truncation variant (p.Arg194*) resulting in a clear loss-of-function. We hypothesized that patients with a hypersensitive response to vigabatrin may exhibit a different γ-aminobutyric acid A receptor phenotype. To test this hypothesis, we evaluated the phenotype of variants in (p.Glu77Lys and p.Thr287Ile) associated with patients who are clinically hypersensitive to vigabatrin. We introduced the p.Glu77Lys and p.Thr287Ile variants into a concatenated synaptic and extrasynaptic γ-aminobutyric acid A receptor construct, to resemble the γ-aminobutyric acid A receptor expression by a patient heterozygous for the variant. The mRNA of these constructs was injected into oocytes and activation properties of each receptor measured by two-electrode voltage clamp electrophysiology. Results showed an atypical gain-of-function molecular phenotype in the p.Glu77Lys and p.Thr287Ile variants characterized by increased potency of γ-aminobutyric acid A without change to the estimated maximum open channel probability, deactivation kinetics or absolute currents. Modelling of the activation properties of the receptors indicated that either variant caused increased chloride flux in response to low concentrations of γ-aminobutyric acid that mediate tonic currents. We therefore propose that the hypersensitivity reaction to vigabatrin is a result of variants that exacerbate GABAergic tonic currents and caution is required when prescribing vigabatrin. In contrast, drug strategies increasing tonic currents in loss-of-function variants are likely to be a safe and effective therapy. This study demonstrates that functional genomics can explain beneficial and adverse anti-epileptic drug effects, and propose that vigabatrin should be considered in patients with clear loss-of-function variants.
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http://dx.doi.org/10.1093/braincomms/fcaa162DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7869430PMC
October 2020

Reduced Lymphatic Reserve in Heart Failure With Preserved Ejection Fraction.

J Am Coll Cardiol 2020 12;76(24):2817-2829

Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, United Kingdom.

Background: Microvascular dysfunction plays an important role in the pathogenesis of heart failure with preserved ejection fraction (HFpEF). However, no mechanistic link between systemic microvasculature and congestion, a central feature of the syndrome, has yet been investigated.

Objectives: This study aimed to investigate capillary-interstitium fluid exchange in HFpEF, including lymphatic drainage and the potential osmotic forces exerted by any hypertonic tissue Na excess.

Methods: Patients with HFpEF and healthy control subjects of similar age and sex distributions (n = 16 per group) underwent: 1) a skin biopsy for vascular immunohistochemistry, gene expression, and chemical (water, Na, and K) analyses; and 2) venous occlusion plethysmography to assess peripheral microvascular filtration coefficient (measuring capillary fluid extravasation) and isovolumetric pressure (above which lymphatic drainage cannot compensate for fluid extravasation).

Results: Skin biopsies in patients with HFpEF showed rarefaction of small blood and lymphatic vessels (p = 0.003 and p = 0.012, respectively); residual skin lymphatics showed a larger diameter (p = 0.007) and lower expression of lymphatic differentiation and function markers (LYVE-1 [lymphatic vessel endothelial hyaluronan receptor 1]: p < 0.05; PROX-1 [prospero homeobox protein 1]: p < 0.001) compared with control subjects. In patients with HFpEF, microvascular filtration coefficient was lower (calf: 3.30 [interquartile range (IQR): 2.33 to 3.88] l × 100 ml of tissue × min × mm Hg vs. 4.66 [IQR: 3.70 to 6.15] μl × 100 ml of tissue × min × mm Hg; p < 0.01; forearm: 5.16 [IQR: 3.86 to 5.43] l × 100 ml of tissue × min × mm Hg vs. 5.66 [IQR: 4.69 to 8.38] μl × 100 ml of tissue × min × mm Hg; p > 0.05), in keeping with blood vascular rarefaction and the lack of any observed hypertonic skin Na excess, but the lymphatic drainage was impaired (isovolumetric pressure in patients with HFpEF vs. control subjects: calf 16 ± 4 mm Hg vs. 22 ± 4 mm Hg; p < 0.005; forearm 17 ± 4 mm Hg vs. 25 ± 5 mm Hg; p < 0.001).

Conclusions: Peripheral lymphatic vessels in patients with HFpEF exhibit structural and molecular alterations and cannot effectively compensate for fluid extravasation and interstitial accumulation by commensurate drainage. Reduced lymphatic reserve may represent a novel therapeutic target.
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http://dx.doi.org/10.1016/j.jacc.2020.10.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724570PMC
December 2020

Crystal structure of (1',2',3)-1'-benzoyl-2'-(4-meth-oxy-phen-yl)-1-methyl-2',5',6',10b'-tetra-hydro-1'-spiro-[indoline-3,3'-pyrrolo-[2,1-]isoquinolin]-2-one.

Acta Crystallogr E Crystallogr Commun 2020 Oct 4;76(Pt 10):1548-1550. Epub 2020 Sep 4.

Department of Physics, Kings Engineering College, Irungattukottai, Sriperumbudur, Chennai-602117, Tamilnadu, India.

In the title spiro compound, CHNO, the central pyrrolidine ring is fused with the tetra-hydro-iso-quinoline ring, both having distorted envelope conformations, with the flap atoms being C and N, respectively. The meth-oxy-phenyl group is attached to the pyrrolidine ring, and is disordered over two positions, with refined occupancies of 0.638 (6):0.362 (6) Å. The central pyrrolidine ring is inclined relative to the tetra-hydro-iso-quinoline group, such that the dihedral between the non-flap atoms of each ring system is 11.29 (7)°. The spiro-linkage creates a dihedral angle of 83.26 (5)° between the indolinone ring and the non-flap atoms of the pyrrolidine ring. In the crystal, mol-ecules are linked C-H⋯O hydrogen bonds. For the major disorder component, these form (11) chains that propagate parallel to the axis.
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http://dx.doi.org/10.1107/S2056989020010300DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7534224PMC
October 2020

High Sodium Intake, Glomerular Hyperfiltration and Protein Catabolism in Patients with Essential Hypertension.

Cardiovasc Res 2020 Jul 16. Epub 2020 Jul 16.

Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow UK.

Aims: A blood pressure-independent metabolic shift toward a catabolic state upon high sodium (Na+) diet, ultimately favouring body fluid preservation, has recently been described in pre-clinical controlled settings. We sought to investigate the real-life impact of high Na+ intake on measures of renal Na+/water handling and metabolic signatures, as surrogates for cardiovascular risk, in hypertensive patients.

Methods And Results: We analysed clinical and biochemical data from 766 consecutive patients with essential hypertension, collected at the time of screening for secondary causes. The systematic screening protocol included 24h urine collection on usual diet and avoidance of renin-angiotensin-aldosterone system-confounding medications. Urinary 24h-Na+ excretion, used to define classes of Na+ intake (Low ≤2.3g/d; Medium 2.3-5g/d; High >5g/d), was an independent predictor of glomerular filtration rate after correction for age, sex, blood pressure, BMI, aldosterone and potassium excretion (p = 0.001; Low: 94.1 [69.9-118.8] vs High: 127.5 [108.3-147.8] ml/min/1.73m2). Renal Na+ and water handling diverged, with higher fractional excretion of Na+ and lower fractional excretion of water in those with evidence of High Na+ intake (FENa: Low 0.39% [0.30-0.47] vs. High 0.81% [0.73-0.98], p < 0.001; FEwater: Low 1.13% [0.73-1.72] vs. High 0.89% [0.69-1.12], p = 0.015). Despite higher FENa, these patients showed higher absolute 24h Na+ reabsorption and higher associated tubular energy expenditure, estimated by tubular Na+/ATP stoichiometry, accordingly (ΔHigh-Low = 18 [12-24] kcal/d, p < 0.001). At non-targeted LC/MS plasma metabolomics in an unselected subcohort (n = 67), metabolites which were more abundant in High vs. Low Na+ intake (p < 0.05) mostly entailed intermediates or end products of protein catabolism/urea cycle.

Conclusions: When exposed to high Na+ intake, kidneys dissociate Na+ and water handling. In hypertensive patients, this comes at the cost of higher glomerular filtration rate, increased tubular energy expenditure and protein catabolism from endogenous (muscle) or excess exogenous (dietary) sources. Glomerular hyperfiltration and the metabolic shift may have broad implications on global cardiovascular risk independent of blood pressure.

Translational Perspective: We herein show that high Na intake can adversely impact not only blood pressure control, but also renal function and metabolic balance in hypertensive patients. At variance with experimental preclinical studies, the catabolism of proteins appears to include also exogenous sources. Interventional studies where caloric intake is controlled but not restricted may identify preferential metabolic handling in different subjects/populations and test the effect of specific dietary strategies. Similarly, the potential benefit of medications like sodium-glucose cotransporter (SGLT)-2 inhibitors, which are known to reduce both glomerular hyperfiltration and excess tubular Na+ reabsorption, in non-diabetic hypertensive patients deserves further investigation in relation to the described Na+-reno-metabolic mechanisms.
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http://dx.doi.org/10.1093/cvr/cvaa205DOI Listing
July 2020

Development of Nonpeptidic Inverse Agonists of the Ghrelin Receptor (GHSR) Based on the 1,2,4-Triazole Scaffold.

J Med Chem 2020 10 21;63(19):10796-10815. Epub 2020 Sep 21.

IBMM, Univ Montpellier, CNRS, ENSCM, Faculty of Pharmacy, 34000 Montpellier, France.

GHSR controls, among others, growth hormone and insulin secretion, adiposity, feeding, and glucose metabolism. Therefore, an inverse agonist ligand capable of selectively targeting GHSR and reducing its high constitutive activity appears to be a good candidate for the treatment of obesity-related metabolic diseases. In this context, we present a study that led to the development of several highly potent and selective inverse agonists of GHSR based on the 1,2,4-triazole scaffold. We demonstrate that, depending on the nature of the substituents on positions 3, 4, and 5, this scaffold leads to ligands that exert an intrinsic inverse agonist activity on GHSR-catalyzed G protein activation through the stabilization of a specific inactive receptor conformation. Thanks to an in vivo evaluation, we also show that one of the most promising ligands not only exerts an effect on insulin secretion in rat pancreatic islets but also affects the orexigenic effects of ghrelin in mice.
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http://dx.doi.org/10.1021/acs.jmedchem.9b02122DOI Listing
October 2020

Quantum chemical insight into molecular structure, NBO analysis of the hydrogen-bonded interactions, spectroscopic (FT-IR, FT-Raman), drug likeness and molecular docking of the novel anti COVID-19 molecule 2-[(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluorophenyl)acetamide - dimer.

Spectrochim Acta A Mol Biomol Spectrosc 2021 Jan 12;244:118825. Epub 2020 Aug 12.

Register number 18113162132001, Department of Physics and Research Centre, Scott Christian College (Autonomous), Nagercoil- 629003, Tamil Nadu, Affiliated to Manonmaniam Sundarnar University, Abishekapatti, Tirunelveli 627012, India. Electronic address:

Novel antiviral active molecule 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl)acetamide has been synthesised and characterized by FT-IR and FT-Raman spectra. The equilibrium geometry, natural bond orbital calculations and vibrational assignments have been carried out using density functional B3LYP method with the 6-311G++(d,p) basis set. The complete vibrational assignments for all the vibrational modes have been supported by normal coordinate analysis, force constants and potential energy distributions. A detailed analysis of the intermolecular interactions has been performed based on the Hirshfeld surfaces. Drug likeness has been carried out based on Lipinski's rule and the absorption, distribution, metabolism, excretion and toxicity of the title molecule has been calculated. Antiviral potency of 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro-phenyl) acetamide has been investigated by docking against SARS-CoV-2 protein. The optimized geometry shows near-planarity between the phenyl ring and the pyrimidine ring. Differences in the geometries due to the substitution of the most electronegative fluorine atom and intermolecular contacts due to amino pyrimidine were analyzed. NBO analysis reveals the formation of two strong stable hydrogen bonded N-H···N intermolecular interactions and weak intramolecular interactions C-H···O and N-H···O. The Hirshfeld surfaces and consequently the 2D-fingerprint confirm the nature of intermolecular interactions and their quantitative contributions towards the crystal packing. The red shift in N-H stretching frequency exposed from IR substantiate the formation of N-H···N intermolecular hydrogen bond. Drug likeness and absorption, distribution, metabolism, excretion and toxicity properties analysis gives an idea about the pharmacokinetic properties of the title molecule. The binding energy -8.7 kcal/mol of the nonbonding interaction present a clear view that 2- [(4,6-diaminopyrimidin-2-yl)sulfanyl]-N-(4-fluoro- phenyl) acetamide can irreversibly interact with SARS-CoV-2 protease.
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http://dx.doi.org/10.1016/j.saa.2020.118825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7419267PMC
January 2021

Tissue sodium excess is not hypertonic and reflects extracellular volume expansion.

Nat Commun 2020 08 24;11(1):4222. Epub 2020 Aug 24.

Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK.

Our understanding of Na homeostasis has recently been reshaped by the notion of skin as a depot for Na accumulation in multiple cardiovascular diseases and risk factors. The proposed water-independent nature of tissue Na could induce local pathogenic changes, but lacks firm demonstration. Here, we show that tissue Na excess upon high Na intake is a systemic, rather than skin-specific, phenomenon reflecting architectural changes, i.e. a shift in the extracellular-to-intracellular compartments, due to a reduction of the intracellular or accumulation of water-paralleled Na in the extracellular space. We also demonstrate that this accumulation is unlikely to justify the observed development of experimental hypertension if it were water-independent. Finally, we show that this isotonic skin Na excess, reflecting subclinical oedema, occurs in hypertensive patients and in association with aging. The implications of our findings, questioning previous assumptions but also reinforcing the importance of tissue Na excess, are both mechanistic and clinical.
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http://dx.doi.org/10.1038/s41467-020-17820-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445299PMC
August 2020

Is there a role for proteomics in diabetic renal disease?

Nephrol Dial Transplant 2020 07;35(7):1133-1135

Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, UK.

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http://dx.doi.org/10.1093/ndt/gfz017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7417003PMC
July 2020

Temperature Variability at Local Scale in the Bordeaux Area. Relations With Environmental Factors and Impact on Vine Phenology.

Front Plant Sci 2020 20;11:515. Epub 2020 May 20.

EGFV, Bordeaux Sciences Agro, INRAE, Université de Bordeaux, ISVV, Bordeaux, France.

Climate is a major factor of the physical environment influencing terroir expression in viticulture. Thermal conditions strongly impact vine development and grape composition. Spatializing this parameter at local scale allows for more refined vineyard management. In this study, temperature variability was investigated over an area of 19,233 ha within the appellations of Saint-Émilion, Pomerol, and their satellites (Bordeaux, France). A network of 90 temperature sensors was deployed inside grapevine canopies of this area and temperatures were measured from 2012 through 2018. To determine the effect of temperature on vine development, the phenological stages (budbreak, flowering, and véraison) were recorded on 60 reference plots planted with L. cv. Merlot located near the temperature sensors. Results showed great spatial variability in temperature, especially minimum temperature, with an amplitude of up to 10°C on a given day. The spatial variability of the Winkler index measured in the canopy inside a given vintage was around 320 degree-days. This research explores the main factors affecting spatial variability in temperature, such as environmental factors and meteorological conditions. The impact of temperature on vine behavior was also analyzed. Observed phenological dates were compared to those estimated using the Grapevine Flowering Véraison model. Maps of temperatures and phenological observations were created over this area and provided a useful tool for improved adaptation of plant material and training systems to local temperature variability and change.
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http://dx.doi.org/10.3389/fpls.2020.00515DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7251079PMC
May 2020

Supported Housing as a recovery option for long-stay patients with severe mental illness in a psychiatric hospital in South India: Learning from an innovative de-hospitalization process.

PLoS One 2020 9;15(4):e0230074. Epub 2020 Apr 9.

Athena Institute, Faculty of Science, Vrije University, Amsterdam, Netherlands.

Individuals with severe mental illness have long been segregated from living in communities and participating in socio- cultural life. In recent years, owing to progressive legislations and declarations (in India and globally), there has been a growing movement towards promoting social inclusion and community participation, with emphasis on the need to develop alternative and inclusive care paradigms for persons with severe mental illness. However, transitions from inpatient care to community settings is a complex process involving implications at multiple levels involving diverse stakeholders such as mental health service users, care providers, local communities and policy makers. This article studies how the transition from a hospital setting to a community-based recovery model for personals with severe mental illness can be facilitated. It reflects on the innovative process of creating a Supported Housing model in South India, where 11 MH Service users transitioned from a psychiatric ECRC to independent living facilities. Experiences in various phases of the project development, including care provider- and community level responses and feedback were scrutinised to understand the strategies that were employed in enabling the transition. Qualitative methods (including in-depth interviews and naturalistic observations) were used with residents and staff members to explore the challenges they encountered in stabilizing the model, as well as the psychosocial benefits experienced by residents in the last phase. These were complemented with a Brief Psychiatric Rating Scale (BPRS) and WHO Quality of Life scale to compare baseline and post-assessment results and an increase of quality of life. Results display a significant reduction of psychiatric symptoms in patients (p< 0.5). It also describes the challenges encountered in the current context, and strategies that were used to respond and adapt the model to address these concerns effectively. Positive behavioural and psycho-emotional changes were observed amongst the residents, significant amongst those being enhanced in their mobility and participation. The article concludes by discussing the implications of this study for the development of innovative community-based models in wider contexts.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0230074PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7144972PMC
July 2020

Targeted delivery and apoptosis induction of trans-resveratrol-ferulic acid loaded chitosan coated folic acid conjugate solid lipid nanoparticles in colon cancer cells.

Carbohydr Polym 2020 Mar 26;231:115682. Epub 2019 Nov 26.

Centre for Academic and Research Excellence (CARE), CSIR-Central Leather Research Institute, Chennai, 600 020, Tamil Nadu, India. Electronic address:

The present study is aimed to study and to evaluate the colon cancer targeting efficacy of chitosan-coated-trans-resveratrol (RSV) and ferulic acid (FER) loaded SLNs (solid lipid nanoparticles) that conjugated with folic acid (FA) (C-RSV-FER-FA-SLNs) in suitable models (in vitro). The FA conjugation is performed using co-encapsulation method of stearic acid. Similarly, the prepared SLNs are exhibited better stability even under acidic conditions to exhibit their potentials to use as drug delivery system. Further, the optimized formulations (SLNs) are tested for physiochemical characterizations, which include FTIR, XRD, HNMR, particle size, zeta potential, and drug release. In vitro anti-cancer studies using HT-29 cells including, fluorescence staining, flow cytometry, and western blot analysis revealed that the C-RSV-FER-FA-SLNs effectively involved and increased cytotoxicity in cancer cells that leads to induction of apoptosis as compared to free RSV-FER. Thus, it is reported that, the good stability under acidic conditions of this C-RSV-FER-FA-SLNs may serve as a promising candidate for novel nanodrug formulations in cancer therapy.
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http://dx.doi.org/10.1016/j.carbpol.2019.115682DOI Listing
March 2020

Spectroscopic investigation of supramolecular organometallic compound L-threonine cadmium acetate monohydrate.

Spectrochim Acta A Mol Biomol Spectrosc 2020 Mar 15;228:117802. Epub 2019 Nov 15.

Research department of Physics, Holy Cross College (Autonomous), Nagercoil 629004, India.

Single crystal of L-Threonine cadmium acetate monohydrate, a supramolecular compounds crystallized by evaporation technique. The coordination network exhibit interesting structural property and have been investigated by Powder and single crystal X-ray diffraction. The functional group was identified using FTIR analysis. Dielectric property in a metal organic frame network plays an important role in the use of solid state as bioelectrical materials for various tissue engineering applications due to its relative permittivity. The UVVis studies suggested that UV absorption ability might be used as a corrosion resistant semiconductor material for optical device fabrication system. The non linear optical efficiency predicts that the material possesses good absorption coefficient.
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http://dx.doi.org/10.1016/j.saa.2019.117802DOI Listing
March 2020

Development of a novel fluorescent ligand of growth hormone secretagogue receptor based on the N-Terminal Leap2 region.

Mol Cell Endocrinol 2019 12 6;498:110573. Epub 2019 Sep 6.

Laboratory of Neurophysiology of the Multidisciplinary Institute of Cell Biology [IMBICE, Argentine Research Council (CONICET) and Scientific Research Commission, Province of Buenos Aires (CIC-PBA), National University of La Plata], 1900, La Plata, Buenos Aires, Argentina. Electronic address:

Liver-expressed antimicrobial peptide 2 (LEAP2) was recently recognized as an endogenous ligand for the growth hormone secretagogue receptor (GHSR), which also is a receptor for the hormone ghrelin. LEAP2 blocks ghrelin-induced activation of GHSR and inhibits GHSR constitutive activity. Since fluorescence-based imaging and pharmacological analyses to investigate the biology of GHSR require reliable probes, we developed a novel fluorescent GHSR ligand based on the N-terminal LEAP2 sequence, hereafter named F-LEAP2. In vitro, F-LEAP2 displayed binding affinity and inverse agonism to GHSR similar to LEAP2. In a heterologous expression system, F-LEAP2 labeling was specifically observed in the surface of GHSR-expressing cells, in contrast to fluorescent ghrelin labeling that was mainly observed inside the GHSR-expressing cells. In mice, centrally-injected F-LEAP2 reduced ghrelin-induced food intake, in a similar fashion to LEAP2, and specifically labeled cells in GHSR-expressing brain areas. Thus, F-LEAP2 represents a valuable tool to study the biology of GHSR in vitro and in vivo.
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http://dx.doi.org/10.1016/j.mce.2019.110573DOI Listing
December 2019

Resistin Mediates Sex-Dependent Effects of Perivascular Adipose Tissue on Vascular Function in the Shrsp.

Sci Rep 2019 05 3;9(1):6897. Epub 2019 May 3.

BHF Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, University of Glasgow, Glasgow, Scotland.

Premenopausal women are relatively protected from developing hypertension compared to men. Perivascular adipose tissue (PVAT) has been shown to mediate vasoactive effects; however, a sex-dependent difference in PVAT function in the setting of hypertension has not yet been explored. We investigated the effect of PVAT on resistance vessel biology in male and female 16 week old stroke prone spontaneously hypertensive rats (SHRSP). This preclinical model of hypertension exhibits a sex-dependent difference in the development of hypertension similar to humans. Wire myography was used to assess vascular function in third-order mesenteric arteries. K channel-mediated vasorelaxation by cromakalim was significantly impaired in vessels from SHRSP males + PVAT relative to females (maximum relaxation: male + PVAT 46.9 ± 3.9% vs. female + PVAT 97.3 ± 2.7%). A cross-over study assessing the function of male PVAT on female vessels confirmed the reduced vasorelaxation response to cromakalim associated with male PVAT (maximum relaxation: female + PVAT 90.6 ± 1.4% vs. female + PVAT 65.8 ± 3.5%). In order to explore the sex-dependent differences in PVAT at a molecular level, an adipokine array and subsequent western blot validation identified resistin expression to be increased approximately 2-fold in PVAT from male SHRSP vessels. Further wire myography experiments showed that pre-incubation with resistin (40 ng/ml) significantly impaired the ability of female + PVAT vessels to relax in response to cromakalim (maximum relaxation: female + PVAT 97.3 ± 0.9% vs. female + PVAT + resistin 36.8 ± 2.3%). These findings indicate a novel role for resistin in mediating sex-dependent vascular function in hypertension through a K channel-mediated mechanism.
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http://dx.doi.org/10.1038/s41598-019-43326-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6499830PMC
May 2019

N-Terminal Liver-Expressed Antimicrobial Peptide 2 (LEAP2) Region Exhibits Inverse Agonist Activity toward the Ghrelin Receptor.

J Med Chem 2019 01 24;62(2):965-973. Epub 2018 Dec 24.

Faculté de Pharmacie , Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, Université de Montpellier, Ecole Nationale Supérieure de Chimie de Montpellier , 15 avenue Charles Flahaut, BP 14491 , 34093 Montpellier cedex 5, France.

The ghrelin receptor or growth hormone secretagogue receptor (GHSR) is a G-protein-coupled receptor that controls growth hormone and insulin secretion, food intake, and reward-seeking behaviors. Liver-expressed antimicrobial peptide 2 (LEAP2) was recently described as an endogenous antagonist of GHSR. Here, we present a study aimed at delineating the structural determinants required for LEAP2 activity toward GHSR. We demonstrate that the entire sequence of LEAP2 is not necessary for its actions. Indeed, the N-terminal part alone confers receptor binding and activity to LEAP2. We found that both LEAP2 and its N-terminal part behave as inverse agonists of GHSR and as competitive antagonists of ghrelin-induced inositol phosphate production and calcium mobilization. Accordingly, the N-terminal region of LEAP2 is able to inhibit ghrelin-induced food intake in mice. These data demonstrate an unexpected pharmacological activity for LEAP2 that is likely to have an important role in the control of ghrelin response under normal and pathological conditions.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01644DOI Listing
January 2019

Genotoxic Effects of Silver Amalgam and Composite Restorations: Micronuclei-Based Cohort and Case-Control Study in Oral Exfoliated Cells.

Contemp Clin Dent 2018 Apr-Jun;9(2):249-254

Department of Oral Pathology, Sree Mookambika Institute of Dental Sciences, Kanyakumari, Tamil Nadu, India.

Context: A huge number of people carry dental fillings which contain either mercury-based amalgam and/or the recently introduced methacrylate-based resins. It has been shown that both these materials are known to be leached into the oral cavity and induce genotoxic alterations in the buccal mucosal cells. Because of its low cost and ease of manipulation, dental amalgam is still widely used as a restorative material in developing countries. The health risks associated with the components of this restorative material has always been a matter of concern. The present study was designed to assess the frequency of micronuclei (MN) in oral mucosal cells as it is a promising tool for studying the genotoxic effect of clastogenic agents on them.

Aims: The aim of this study is to evaluate the genotoxic effects of silver amalgam and composite restorations by measuring the mean number of MN in oral exfoliated cells.

Materials And Methods: The present study was a prospective cohort study which includes a study group consisting of 110 participants. The study sample was equally divided into 55 participants requiring only amalgam restoration and 55 participants requiring only composite restoration in any permanent molar teeth. The same participants before the restoration formed the control group. Smears were obtained from each patient before and 10 days after restoration and were stained with DNA-specific Feulgen stain. The number of cells containing MN out of 500 cells were counted and recorded. After the evaluation of the slides, the results were compiled and subjected to statistical analysis.

Results: There was a statistically significant ( < 0.01) variation in the mean number of MN after the restoration in both amalgam (5.41 ± 1.25) and composite (2.83 ± 0.85) restorations when compared to before the restoration. However, the mean number of MN in composite restoration was significantly less when compared to amalgam restoration. There was also a statistically significant difference in the mean number of MN in subjects with single restoration when compared with multiple restorations in both amalgam and composite restorations.

Conclusions: The observations from the present study showed the genotoxic effect of amalgam and composite restorations on the oral cavity. However, composite restorations were least cytotoxic when compared to amalgam restoration. Future research and technical advancements are needed for developing safer materials for use in humans.
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http://dx.doi.org/10.4103/ccd.ccd_849_17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5968691PMC
June 2018

GHSR-D2R heteromerization modulates dopamine signaling through an effect on G protein conformation.

Proc Natl Acad Sci U S A 2018 04 9;115(17):4501-4506. Epub 2018 Apr 9.

Institut des Biomolécules Max Mousseron, CNRS, Ecole Nationale Superieure de Chimie de Montepellier, Université Montpellier, 34093 Montpellier, France;

The growth hormone secretagogue receptor (GHSR) and dopamine receptor (D2R) have been shown to oligomerize in hypothalamic neurons with a significant effect on dopamine signaling, but the molecular processes underlying this effect are still obscure. We used here the purified GHSR and D2R to establish that these two receptors assemble in a lipid environment as a tetrameric complex composed of two each of the receptors. This complex further recruits G proteins to give rise to an assembly with only two G protein trimers bound to a receptor tetramer. We further demonstrate that receptor heteromerization directly impacts on dopamine-mediated Gi protein activation by modulating the conformation of its α-subunit. Indeed, association to the purified GHSR:D2R heteromer triggers a different active conformation of Gαi that is linked to a higher rate of GTP binding and a faster dissociation from the heteromeric receptor. This is an additional mechanism to expand the repertoire of GPCR signaling modulation that could have implications for the control of dopamine signaling in normal and physiopathological conditions.
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http://dx.doi.org/10.1073/pnas.1712725115DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5924877PMC
April 2018

Urinary proteomics for prediction of mortality in patients with type 2 diabetes and microalbuminuria.

Cardiovasc Diabetol 2018 04 6;17(1):50. Epub 2018 Apr 6.

Institute of Cardiovascular and Medical Sciences, University of Glasgow, 126 University Place, Glasgow, G12 8TA, UK.

Background: The urinary proteomic classifier CKD273 has shown promise for prediction of progressive diabetic nephropathy (DN). Whether it is also a determinant of mortality and cardiovascular disease in patients with microalbuminuria (MA) is unknown.

Methods: Urine samples were obtained from 155 patients with type 2 diabetes and confirmed microalbuminuria. Proteomic analysis was undertaken using capillary electrophoresis coupled to mass spectrometry to determine the CKD273 classifier score. A previously defined CKD273 threshold of 0.343 for identification of DN was used to categorise the cohort in Kaplan-Meier and Cox regression models with all-cause mortality as the primary endpoint. Outcomes were traced through national health registers after 6 years.

Results: CKD273 correlated with urine albumin excretion rate (UAER) (r = 0.481, p = <0.001), age (r = 0.238, p = 0.003), coronary artery calcium (CAC) score (r = 0.236, p = 0.003), N-terminal pro-brain natriuretic peptide (NT-proBNP) (r = 0.190, p = 0.018) and estimated glomerular filtration rate (eGFR) (r = 0.265, p = 0.001). On multivariate analysis only UAER (β = 0.402, p < 0.001) and eGFR (β = - 0.184, p = 0.039) were statistically significant determinants of CKD273. Twenty participants died during follow-up. CKD273 was a determinant of mortality (log rank [Mantel-Cox] p = 0.004), and retained significance (p = 0.048) after adjustment for age, sex, blood pressure, NT-proBNP and CAC score in a Cox regression model.

Conclusion: A multidimensional biomarker can provide information on outcomes associated with its primary diagnostic purpose. Here we demonstrate that the urinary proteomic classifier CKD273 is associated with mortality in individuals with type 2 diabetes and MA even when adjusted for other established cardiovascular and renal biomarkers.
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http://dx.doi.org/10.1186/s12933-018-0697-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5889591PMC
April 2018

Expanding the phenotypic spectrum associated with OPHN1 mutations: Report of 17 individuals with intellectual disability but no cerebellar hypoplasia.

Eur J Med Genet 2018 Aug 3;61(8):442-450. Epub 2018 Mar 3.

Centre de Génétique Humaine, Institut de Pathologie et de Génétique, Charleroi, Gosselies, Belgium.

Mutations in the oligophrenin 1 gene (OPHN1) have been identified in patients with X-linked intellectual disability (XLID) associated with cerebellar hypoplasia and ventriculomegaly, suggesting it could be a recognizable syndromic intellectual disability (ID). Affected individuals share additional clinical features including speech delay, seizures, strabismus, behavioral difficulties, and slight facial dysmorphism. OPHN1 is located in Xq12 and encodes a Rho-GTPase-activating protein involved in the regulation of the G-protein cycle. Rho protein members play an important role in dendritic growth and in plasticity of excitatory synapses. Here we report on 17 individuals from four unrelated families affected by mild to severe intellectual disability due to OPHN1 mutations without cerebellar anomaly on brain MRI. We describe clinical, genetic and neuroimaging data of affected patients. Among the identified OPHN1 mutations, we report for the first time a missense mutation occurring in a mosaic state. We discuss the intrafamilial clinical variability of the disease and compare our patients with those previously reported. We emphasize the power of next generation techniques (X-exome sequencing, whole-exome sequencing and targeted multi-gene panel) to expand the phenotypic and mutational spectrum of OPHN1-related ID.
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http://dx.doi.org/10.1016/j.ejmg.2018.03.002DOI Listing
August 2018

Acute oral toxicity evaluation of aqueous ethanolic extract of Roxb. roots in albino mice as per OECD 425 TG.

Toxicol Rep 2017 31;4:580-585. Epub 2017 Oct 31.

Riphah Institute of Pharmaceutical Sciences, Riphah International University, Lahore, Pakistan.

Background: roots have been used in ethno medicines for the treatment of different ailments. Despite its beneficial uses no studies on its toxicity potential have been reported.

Objective: The study was designed to evaluate acute toxic potential of aqueous ethanolic extract of roots according to OECD TG No. 425.

Material And Methods: Female mice were divided into two groups (n = 5). One group served as control while the other as treated group that received 2000 mg/kg b.w. of roots ethanolic extract orally. Then both groups were observed for 14 days. Then the blood samples were collected by cardiac puncture, under chloroform general anesthesia and were subjected to hematological and biochemical analyses. The vital organs of anesthetized animals were preserved for histopathological examination.

Results: The the data revealed that LD of the extract was greater than 2000 mg/kg b.w. There was no significant alteration found in body weight and organ to body mass index. In comparison with control group, there was significant increase in levels of ALT, AST, total proteins, globulin levels, serum urea, cholesterol, triglycerides, LDL, platelet count, MCV, MCH, WBC count and lymphocytes whereas ALP and MCHC levels were reduced significantly.

Conclusions: From the data obtained in this study, It can be concluded that though LD is greater than 2000 mg/kg b.w. but moderate toxicity signs appeared in liver, kidney, lipid profile and CBC also showed blood dyscresias at limit dose.
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http://dx.doi.org/10.1016/j.toxrep.2017.10.005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5671618PMC
October 2017

Differential accumulation of vimentin fragments in preeclamptic placenta.

Cytoskeleton (Hoboken) 2017 Nov 9;74(11):420-425. Epub 2017 Aug 9.

Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, Maharashtra, 411008, India.

Preeclampsia is a pregnancy complication that is the result of abnormal placentation because of inadequate trophoblast invasion into spiral arteries that prevent normal blood flow to the placenta. We report the alteration in vimentin protein proteolysis in placenta of normotensive and preeclamptic women, which is known to have a role in many physiological functions other than its major function in the structural integrity of the cell. Placental proteome from normotensive (n = 25) and preeclamptic pregnancies (n = 25) showed eight differentially accumulated protein spots of vimentin (proteolytic fragments) by two-dimensional electrophoresis. Immunoblots of normotensive and preeclamptic placenta revealed a difference in proteolytic processing of vimentin. In particular, lower molecular weight vimentin fragments of 32 and 20 kDa were 3.3 and 2.6-fold (p < 0.0001) higher, respectively, in preeclampsia compared with normotensive placenta.
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http://dx.doi.org/10.1002/cm.21390DOI Listing
November 2017

-related intellectual disability syndrome: a recognisable entity.

J Med Genet 2017 09 22;54(9):613-623. Epub 2017 Jul 22.

Center for Medical Genetics, Ghent University Hospital, Ghent, Belgium.

Background: Mutations in forkhead box protein P1 () cause intellectual disability (ID) and specific language impairment (SLI), with or without autistic features (MIM: 613670). Despite multiple case reports no specific phenotype emerged so far.

Methods: We correlate clinical and molecular data of 25 novel and 23 previously reported patients with defects. We evaluated FOXP1 activity by an in vitro luciferase model and assessed protein stability in vitro by western blotting.

Results: Patients show ID, SLI, neuromotor delay (NMD) and recurrent facial features including a high broad forehead, bent downslanting palpebral fissures, ptosis and/or blepharophimosis and a bulbous nasal tip. Behavioural problems and autistic features are common. Brain, cardiac and urogenital malformations can be associated. More severe ID and NMD, sensorineural hearing loss and feeding difficulties are more common in patients with interstitial 3p deletions (14 patients) versus patients with monogenic defects (34 patients). Mutations result in impaired transcriptional repression and/or reduced protein stability.

Conclusions: -related ID syndrome is a recognisable entity with a wide clinical spectrum and frequent systemic involvement. Our data will be helpful to evaluate genotype-phenotype correlations when interpreting next-generation sequencing data obtained in patients with ID and/or SLI and will guide clinical management.
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http://dx.doi.org/10.1136/jmedgenet-2017-104579DOI Listing
September 2017

Genetic and phenotypic heterogeneity suggest therapeutic implications in SCN2A-related disorders.

Brain 2017 May;140(5):1316-1336

The Danish Epilepsy Centre, Dianalund, Denmark.

Mutations in SCN2A, a gene encoding the voltage-gated sodium channel Nav1.2, have been associated with a spectrum of epilepsies and neurodevelopmental disorders. Here, we report the phenotypes of 71 patients and review 130 previously reported patients. We found that (i) encephalopathies with infantile/childhood onset epilepsies (≥3 months of age) occur almost as often as those with an early infantile onset (<3 months), and are thus more frequent than previously reported; (ii) distinct phenotypes can be seen within the late onset group, including myoclonic-atonic epilepsy (two patients), Lennox-Gastaut not emerging from West syndrome (two patients), and focal epilepsies with an electrical status epilepticus during slow sleep-like EEG pattern (six patients); and (iii) West syndrome constitutes a common phenotype with a major recurring mutation (p.Arg853Gln: two new and four previously reported children). Other known phenotypes include Ohtahara syndrome, epilepsy of infancy with migrating focal seizures, and intellectual disability or autism without epilepsy. To assess the response to antiepileptic therapy, we retrospectively reviewed the treatment regimen and the course of the epilepsy in 66 patients for which well-documented medical information was available. We find that the use of sodium channel blockers was often associated with clinically relevant seizure reduction or seizure freedom in children with early infantile epilepsies (<3 months), whereas other antiepileptic drugs were less effective. In contrast, sodium channel blockers were rarely effective in epilepsies with later onset (≥3 months) and sometimes induced seizure worsening. Regarding the genetic findings, truncating mutations were exclusively seen in patients with late onset epilepsies and lack of response to sodium channel blockers. Functional characterization of four selected missense mutations using whole cell patch-clamping in tsA201 cells-together with data from the literature-suggest that mutations associated with early infantile epilepsy result in increased sodium channel activity with gain-of-function, characterized by slowing of fast inactivation, acceleration of its recovery or increased persistent sodium current. Further, a good response to sodium channel blockers clinically was found to be associated with a relatively small gain-of-function. In contrast, mutations in patients with late-onset forms and an insufficient response to sodium channel blockers were associated with loss-of-function effects, including a depolarizing shift of voltage-dependent activation or a hyperpolarizing shift of channel availability (steady-state inactivation). Our clinical and experimental data suggest a correlation between age at disease onset, response to sodium channel blockers and the functional properties of mutations in children with SCN2A-related epilepsy.
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http://dx.doi.org/10.1093/brain/awx054DOI Listing
May 2017

Polymerization-Incompetent Uromodulin in the Pregnant Stroke-Prone Spontaneously Hypertensive Rat.

Hypertension 2017 05 27;69(5):910-918. Epub 2017 Mar 27.

From the BHF Glasgow Cardiovascular Research Centre, Institute of Cardiovascular and Medical Sciences, University of Glasgow, Scotland (S.M., H.Y.S., W.M., C.D.); Department of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, India (S.M., A.G.); and Mosaiques Diagnostics GmbH, Hannover, Germany (J.S.).

The kidney is centrally involved in blood pressure regulation and undergoes extensive changes during pregnancy. Hypertension during pregnancy may result in an altered urinary peptidome that could be used to indicate new targets of therapeutic or diagnostic interest. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of maternal chronic hypertension. Capillary electrophoresis-mass spectrometry was conducted to interrogate the urinary peptidome in SHRSP and the control Wistar-Kyoto strain at three time points: prepregnancy and gestational days 12 and 18. The comparison within and between the Wistar-Kyoto and SHRSP peptidome at all time points detected 123 differentially expressed peptides (fold change >1.5; <0.05). Sequencing of these peptides identified fragments of collagen α-chains, albumin, prothrombin, actin, serpin A3K, proepidermal growth factor, and uromodulin. Uromodulin peptides showed a pregnancy-specific alteration in SHRSP with a 7.8-fold (<0.01) and 8.8-fold (<0.05) increase at gestational days 12 and 18, respectively, relative to the Wistar-Kyoto. Further investigation revealed that these peptides belonged to the polymerization-inhibitory region of uromodulin. Two forms of uromodulin (polymerization competent and polymerization incompetent) were found in urine from both Wistar-Kyoto and SHRSP, where the polymerization-incompetent form was increased in a pregnancy-specific manner in SHRSP. Nifedipine-treated pregnant SHRSP showed only polymerization-competent uromodulin, indicating that calcium may be mechanistically involved in uromodulin polymerization. This study highlights, for the first time, a potential role of uromodulin and its polymerization in hypertensive pregnancy.
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http://dx.doi.org/10.1161/HYPERTENSIONAHA.116.08826DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5389592PMC
May 2017

Tubulointerstitial nephritis antigen-like 1 protein is downregulated in the placenta of pre-eclamptic women.

Clin Proteomics 2017 21;14. Epub 2017 Mar 21.

Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Pune, Maharashtra 411008 India.

Background: Tubulointerstitial nephritis antigen-like 1 protein (TINAGL1), is a matricellular protein, known to play role in cell adhesion and cell receptor interaction. Research related to TINAGL1 is limited to cell culture and animal models. Demonstration of TINAGL1 as a positive regulator of angiogenesis and its expression in the decidua of postimplantation mouse uterus, prompted us to validate its expression in human placenta during impaired angiogenesis in pre-eclamptic condition.

Methods: Placental tissue from normotensive (n = 25) and pre-eclamptic (n = 25) pregnancies were used to study the differentially expressed proteins by two-dimensional gel electrophoresis and TINAGL1 protein was validated with Western blotting.

Results: A total of 55 protein spots were differentially expressed (fold change >1.5, p < 0.05), of which 27 were upregulated and 28 were downregulated in the pre-eclamptic placenta. TINAGL1 was found to be downregulated in pre-eclamptic compared to normotensive pregnant women.

Conclusion: This is the first study reporting TINAGL1 to be present in human placenta and differentially expressed in pre-eclamptic condition. The functional role of TINAGL1 in association to human pregnancy needs to be explored further.
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http://dx.doi.org/10.1186/s12014-017-9144-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5361709PMC
March 2017

Placental Proteomics Provides Insights into Pathophysiology of Pre-Eclampsia and Predicts Possible Markers in Plasma.

J Proteome Res 2017 02 26;16(2):1050-1060. Epub 2017 Jan 26.

Division of Biochemical Sciences, CSIR-National Chemical Laboratory , Homi Bhabha Road, Pune, Maharashtra 411008, India.

Pre-eclampsia is a hypertensive disorder characterized by the new onset of hypertension >140/90 mmHg and proteinuria after the 20th week of gestation. The disorder is multifactorial and originates with abnormal placentation. Comparison of the placental proteome of normotensive (n = 25) and pre-eclamptic (n = 25) patients by gel-free proteomic techniques identified a total of 2145 proteins in the placenta of which 180 were differentially expressed (>1.3 fold, p < 0.05). Gene ontology enrichment analysis of biological process suggested that the differentially expressed proteins belonged to various physiological processes such as angiogenesis, apoptosis, oxidative stress, hypoxia, and placental development, which are implicated in the pathophysiology of pre-eclampsia. Some of the differentially expressed proteins were monitored in the plasma by multiple reaction monitoring analysis, which showed an increase in apolipoproteins A-I and A-II in gestational weeks 26-30 (2-fold, p < 0.01), while haptoglobin and hemopexin decreased in gestational weeks 26-30 and week 40/at delivery (1.8 fold, p < 0.01) in pre-eclamptic patients. This study provides a proteomic insight into the pathophysiology of pre-eclampsia. Identified candidate proteins can be evaluated further for the development of potential biomarkers associated with pre-eclampsia pathogenesis.
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http://dx.doi.org/10.1021/acs.jproteome.6b00955DOI Listing
February 2017

Undergraduate students' contributions to health service delivery through community-based education: A qualitative study by the MESAU Consortium in Uganda.

BMC Med Educ 2016 Apr 25;16:123. Epub 2016 Apr 25.

Office of the Principal, Makerere University College of Health Sciences, Kampala, Uganda.

Background: It has been realised that there is need to have medical training closer to communities where the majority of the population lives in order to orient the trainees' attitudes towards future practice in such communities. Although community based education (CBE) has increasingly been integrated into health professions curricula since the 1990s, the contribution students make to service delivery during CBE remains largely undocumented. In this study, we examined undergraduate health professions students' contribution to primary health care during their CBE placements.

Methods: This was a qualitative study involving the Medical Education for Equitable Services to All Ugandans consortium (MESAU). Overall, we conducted 36 Focus Group Discussions (FGDs): one each with youth, men and women at each of 12 CBE sites. Additionally, we interviewed 64 community key-informants. All data were audio-recorded, transcribed and analysed using qualitative data analysis software Atlas.ti Ver7.

Results: Two themes emerged: students' contribution at health facility level and students' contribution at community level. Under theme one, we established that students were not only learning; they also contributed to delivery of health services at the facilities. Their contribution was highly appreciated especially by community members. Students were described as caring and compassionate, available on time and anytime, and as participating in patient care. They were willing to share their knowledge and skills, and stimulated discussion on work ethics. Under the second theme, students were reported to have participated in water, sanitation, and hygiene education in the community. Students contributed to maintenance of safe water sources, educated communities on drinking safe water and on good sanitation practices (hand washing and proper waste disposal). Hygiene promotion was done at household level (food hygiene, hand washing, cleanliness) and to the public. Public health education was extended to institutions. School pupils were sensitised on various health-related issues including sexuality and sexual health.

Conclusion: Health professions students at the MESAU institutions contribute meaningfully to primary health care delivery. We recommend CBE to all health training programs in sub-Saharan Africa.
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http://dx.doi.org/10.1186/s12909-016-0626-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4843200PMC
April 2016

New ligands of the ghrelin receptor based on the 1,2,4-triazole scaffold by introduction of a second chiral center.

Bioorg Med Chem Lett 2016 05 4;26(10):2408-2412. Epub 2016 Apr 4.

Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, CNRS, ENSCM, Université de Montpellier, BP 14491, Faculté de Pharmacie, bât. E, 3(ème) étage, 15 avenue Charles Flahault, 34093 Montpellier Cedex 5, France. Electronic address:

Introducing a second chiral center on our previously described 1,2,4-triazole, allowed us to increase diversity and elongate the 'C-terminal part' of the molecule. Therefore, we were able to explore mimics of the substance P analogs described as inverse agonists. Some compounds presented affinities in the nanomolar range and potent biological activities, while one exhibited a partial inverse agonist behavior similar to a Substance P analog.
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http://dx.doi.org/10.1016/j.bmcl.2016.04.003DOI Listing
May 2016

Development of new quantitative mass spectrometry and semi-automatic isofocusing methods for the determination of Apolipoprotein E typing.

Clin Chim Acta 2016 Feb 19;454:33-8. Epub 2015 Dec 19.

CHRU de Montpellier, Hôpital St Eloi Université de Montpellier, INSERM U1183, IRMB, Laboratoire de Biochimie Protéomique Clinique, Montpellier, France. Electronic address:

Background: Apolipoprotein E (Apo E) is a 36 Kda glycoprotein involved in lipid transport. It exists in 3 major isoforms: E2, E3 and E4. ApoE status is known to be a major risk factor for late-onset Alzheimer's and cardiovascular diseases. Genotyping is commonly used to obtain ApoE status but can show technical issues with ambiguous determinations. Phenotyping can be an alternative, not requiring genetic material. We evaluated the ability to accurately type ApoE isoforms by 2 phenotyping tests in comparison with genotyping.

Methods: Two phenotyping techniques were used: (1) LC-MS/MS detection of 4 ApoE specific peptides (6490 Agilent triple quadripole): After its denaturation, serum was either reduced and alkylated, or only diluted, and then trypsin digested. Before analysis, desalting, evaporation and resuspension were performed. (2) Isoelectric focusing and immunoprecipitation: serum samples were neuraminidase digested, delipidated and electrophoresed on Hydragel ApoE (Sebia agarose gel) using Hydrasys 2 Scan instrument (Sebia, Lisses, France). ApoE isoforms bands were directly immunofixed in the gel using a polyclonal anti human ApoE antibody. Then, incubation of the gel with HRP secondary antibody followed by TTF1/TTF2 substrate allowed the visualization of ApoE bands. The results of the two techniques were compared to genotyping.

Results: Sera from 35 patients previously genotyped were analyzed with the 2 phenotyping techniques. 100% concordance between both phenotyping assays was obtained for the tested phenotypes (E2/E2, E2/E3, E2/E4, E3/E3, E3/E4, E4/E4). When compared to genotyping, 3 samples were discordant. After reanalyzing them by both phenotyping tests and DNA sequencing, 2/3 discrepancies were confirmed. Those can be explained by variants or rare ApoE alleles or by unidentified technical issues. 102 additional samples were then tested on LC-MS/MS only and compared to genotyping. The data showed 100% concordance.

Conclusion: Our 2 phenotyping methods represent a valuable alternative to genotyping. LC-MS/MS has the advantage of being fully specific, with identification of the different isoforms and can be considered as a reference method. Sebia isofocusing technique was concordant with LC-MS/MS. Plus, it is a rapid, semi-automated assay that can be easily implemented in clinical laboratories.
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http://dx.doi.org/10.1016/j.cca.2015.12.020DOI Listing
February 2016

Detergent-free Isolation of Functional G Protein-Coupled Receptors into Nanometric Lipid Particles.

Biochemistry 2016 Jan 24;55(1):38-48. Epub 2015 Dec 24.

Faculté de Pharmacie, Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Université Montpellier-ENSCM , 15 Avenue C. Flahault, F-34093 Montpellier, France.

G protein-coupled receptors (GPCRs) are integral membrane proteins that play a pivotal role in signal transduction. Understanding their dynamics is absolutely required to get a clear picture of how signaling proceeds. Molecular characterization of GPCRs isolated in detergents nevertheless stumbles over the deleterious effect of these compounds on receptor function and stability. We explored here the potential of a styrene-maleic acid polymer to solubilize receptors directly from their lipid environment. To this end, we used two GPCRs, the melatonin and ghrelin receptors, embedded in two membrane systems of increasing complexity, liposomes and membranes from Pichia pastoris. The styrene-maleic acid polymer was able, in both cases, to extract membrane patches of a well-defined size. GPCRs in SMA-stabilized lipid discs not only recognized their ligand but also transmitted a signal, as evidenced by their ability to activate their cognate G proteins and recruit arrestins in an agonist-dependent manner. Besides, the purified receptor in lipid discs undergoes all specific changes in conformation associated with ligand-mediated activation, as demonstrated in the case of the ghrelin receptor with fluorescent conformational reporters and compounds from distinct pharmacological classes. Altogether, these data highlight the potential of styrene-maleic stabilized lipid discs for analyzing the molecular bases of GPCR-mediated signaling in a well-controlled membrane-like environment.
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http://dx.doi.org/10.1021/acs.biochem.5b01040DOI Listing
January 2016