Chemical characterization (LC-MS-ESI), cytotoxic activity and intracellular localization of PAMAM G4 in leukemia cells.

Generation 4 of polyamidoamine dendrimer (G4-PAMAM) has several biological effects due to its tridimensional globular structure, repetitive branched amides, tertiary amines, and amino-terminal subunit groups liked to a common core. G4-PAMAM is cytotoxic due to its positive charges. However, its cytotoxicity could increase in cancer cells due to the excessive intracellular negative charges in these cells. Furthermore, this work reports G4-PAMAM chemical structural characterization using UHPLC-QTOF-MS/MS (LC-MS) by electrospray ionization to measure its population according to its positive charges. Additionally, the antiproliferative effects and intracellular localization were explored in the HMC-1 and K-562 cell lines by confocal microscopy. The LC-MS results show that G4-PAMAM generated multivalent mass spectrum values, and its protonated terminal amino groups produced numerous positive charges, which allowed us to determine its exact mass despite having a high molecular weight. Additionally, G4-PAMAM showed antiproliferative activity in the HMC-1 tumor cell line after 24 h (IC50 = 16.97 µM), 48 h (IC50 = 7.02 µM) and 72 h (IC50 = 5.98 µM) and in the K-562 cell line after 24 h (IC50 = 15.14 µM), 48 h (IC50 = 14.18 µM) and 72 h (IC50 = 9.91 µM). Finally, our results showed that the G4-PAMAM dendrimers were located in the cytoplasm and nucleus in both tumor cell lines studied.

Molecular modeling and LC-MS-based metabolomics of a glutamine-valproic acid (Gln-VPA) derivative on HeLa cells.

Glutaminase plays an important role in carcinogenesis and cancer cell growth. This biological target is interesting against cancer cells. Therefore, in this work, in silico [docking and molecular dynamics (MD) simulations] and in vitro methods (antiproliferative and LC-MS metabolomics) were employed to assay a hybrid compound derived from glutamine and valproic acid (Gln-VPA), which was compared with 6-diazo-5-oxo-L-norleucine (DON, a glutaminase inhibitor) and VPA (contained in Gln-VPA structure). Docking results from some snapshots retrieved from MD simulations show that glutaminase recognized Gln-VPA and DON. Additionally, Gln-VPA showed antiproliferative effects in HeLa cells and inhibited glutaminase activity. Finally, the LC-MS-based metabolomics studies on HeLa cells treated with either Gln-VPA (IC60 = 8 mM) or DON (IC50 = 3.5 mM) show different metabolomics behaviors, suggesting that they modulate different biological targets of the cell death mechanism. In conclusion, Gln-VPA is capable of interfering with more than one pharmacological target of cancer, making it an interesting drug that can be used to avoid multitherapy of classic anticancer drugs.

AMP-activated protein kinase (AMPK) signaling in GnRH neurons links energy status and reproduction.

BACKGROUND: Reproduction is tightly coupled to body energy and metabolic status. GnRH neurons, master elements and final output pathway for the brain control of reproduction, directly or indirectly receive and integrate multiple metabolic cues to regulate reproductive function. Yet, the molecular underpinnings of such phenomenon remain largely unfolded. AMP-activated protein kinase (AMPK), the fundamental cellular sensor that becomes activated in conditions of energy deficit, has been recently shown to participate in the control of Kiss1 neurons, essential gatekeepers of the reproductive axis, by driving an inhibitory valence in situations of energy scarcity at puberty. However, the contribution of AMPK signaling specifically in GnRH neurons to the metabolic control of reproduction remains unknown. METHODS: Double immunohistochemistry (IHC) was applied to evaluate expression of active (phosphorylated) AMPK in GnRH neurons and a novel mouse line, named GAMKO, with conditional ablation of the AMPK α1 subunit in GnRH neurons, was generated. GAMKO mice of both sexes were subjected to reproductive characterization, with attention to puberty and gonadotropic responses to kisspeptin and metabolic stress. RESULTS: A vast majority (>95%) of GnRH neurons co-expressed pAMPK. Female (but not male) GAMKO mice displayed earlier puberty onset and exaggerated LH (as surrogate marker of GnRH) responses to kisspeptin-10 at the prepubertal age. In adulthood, GAMKO females retained increased LH responsiveness to kisspeptin and showed partial resilience to the inhibitory effects of conditions of negative energy balance on the gonadotropic axis. The modulatory role of AMPK in GnRH neurons required preserved ovarian function, since the differences in LH pulsatility detected between GAMKO and control mice subjected to fasting were abolished in ovariectomized animals. CONCLUSIONS: Altogether, our data document a sex-biased, physiological role of AMPK signaling in GnRH neurons, as molecular conduit of the inhibitory actions of conditions of energy deficit on the female reproductive axis.

Early overnutrition sensitizes the growth hormone axis to the impact of diet-induced obesity via sex-divergent mechanisms.

In addition to its essential role in the physiological control of longitudinal growth, growth-hormone (GH) is endowed with relevant metabolic functions, including anabolic actions in muscle, lipolysis in adipose-tissue and glycemic modulation. Adult obesity is known to negatively impact GH-axis, thereby promoting a vicious circle that may contribute to the exacerbation of the metabolic complications of overweight. Yet, to what extent early-overnutrition sensitizes the somatotropic-axis to the deleterious effects of obesity remains largely unexplored. Using a rat-model of sequential exposure to obesogenic insults, namely postnatal-overfeeding during lactation and high-fat diet (HFD) after weaning, we evaluated in both sexes the individual and combined impact of these nutritional challenges upon key elements of the somatotropic-axis. While feeding HFD per se had a modest impact on the adult GH-axis, early overnutrition had durable effects on key elements of the somatotropic-system, which were sexually different, with a significant inhibition of pituitary gene expression of GH-releasing hormone-receptor (GHRH-R) and somatostatin receptor-5 (SST5) in males, but an increase in pituitary GHRH-R, SST2, SST5, GH secretagogue-receptor (GHS-R) and ghrelin expression in females. Notably, early-overnutrition sensitized the GH-axis to the deleterious impact of HFD, with a significant suppression of pituitary GH expression in both sexes and lowering of circulating GH levels in females. Yet, despite their similar metabolic perturbations, males and females displayed rather distinct alterations of key somatotropic-regulators/ mediators. Our data document a synergistic effect of postnatal-overnutrition on the detrimental impact of HFD-induced obesity on key elements of the adult GH-axis, which is conducted via mechanisms that are sexually-divergent.

Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome.

STUDY QUESTION: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. WHAT IS KNOWN ALREADY: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown. STUDY DESIGN, SIZE, DURATION: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS. PARTICIPANTS/MATERIALS, SETTING, METHODS: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 µg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria. MAIN RESULTS AND THE ROLE OF CHANCE: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women. LIMITATIONS, REASONS FOR CAUTION: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions. WIDER IMPLICATIONS OF THE FINDINGS: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

Novel mechanisms for the metabolic control of puberty: implications for pubertal alterations in early-onset obesity and malnutrition.

Puberty is driven by sophisticated neuroendocrine networks that timely activate the brain centers governing the reproductive axis. The timing of puberty is genetically determined; yet, puberty is also sensitive to numerous internal and external cues, among which metabolic/nutritional signals are especially prominent. Compelling epidemiological evidence suggests that alterations of the age of puberty are becoming more frequent; the underlying mechanisms remain largely unknown, but the escalating prevalence of obesity and other metabolic/feeding disorders is possibly a major contributing factor. This phenomenon may have clinical implications, since alterations in pubertal timing have been associated to adverse health outcomes, including higher risk of earlier all-cause mortality. This urges for a better understanding of the neurohormonal basis of normal puberty and its deviations. Compelling evidence has recently documented the master role of hypothalamic neurons producing kisspeptins, encoded by Kiss1, in the neuroendocrine pathways controlling puberty. Kiss1 neurons seemingly participate in transmitting the regulatory actions of metabolic cues on pubertal maturation. Key cellular metabolic sensors, as the mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK) and the fuel-sensing deacetylase, SIRT1, have been recently shown to participate also in the metabolic modulation of puberty. Recently, we have documented that AMPK and SIRT1 operate as major molecular effectors for the metabolic control of Kiss1 neurons and, thereby, puberty onset. Alterations of these molecular pathways may contribute to the perturbation of pubertal timing linked to conditions of metabolic stress in humans, such as subnutrition or obesity and might become druggable targets for better management of pubertal disorders.

In silico search, chemical characterization and immunogenic evaluation of amino-terminated G4-PAMAM-HIV peptide complexes using three-dimensional models of the HIV-1 gp120 protein.

Peptide epitopes have been widely used to develop synthetic vaccines and immunotherapies. However, peptide epitopes may exhibit poor absorption or immunogenicity due to their low molecular weights. Conversely, fourth-generation polyamidoamine (G4-PAMAM) dendrimers are nonimmunogenic and relatively nontoxic synthetic nanoparticles that have been used as adjuvants and nanocarriers of small peptides and to improve nasal absorption. Based on this information, we hypothesized that the combination of intranasal immunization and G4-PAMAM dendrimers would be useful for enhancing the antibody responses of HIV-1 gp120 peptide epitopes. Therefore, we first used structural data, peptide epitope predictors and docking and MD simulations on MHC-II to identify two peptide epitopes on the CD4 binding site of HIV-1 gp120. The formation of G4-PAMAM-peptide complexes was evaluated in silico (molecular docking studies using different G4-PAMAM conformations retrieved from MD simulations as well as the MMGBSA approach) and validated experimentally (electrophoresis, 1H NMR and cryo-TEM). Next, the G4-PAMAM dendrimer-peptide complexes were administered intranasally to groups of female BALB/cJ mice. The results showed that both peptides were immunogenic at the systemic and mucosal levels (nasal and vaginal), and G4-PAMAM dendrimer-peptide complexes improved IgG and IgA responses in serum and nasal washes. Thus, G4-PAMAM dendrimers have potential for use as adjuvants and nanocarriers of peptides.

SIRT1 mediates obesity- and nutrient-dependent perturbation of pubertal timing by epigenetically controlling Kiss1 expression.

Puberty is regulated by epigenetic mechanisms and is highly sensitive to metabolic and nutritional cues. However, the epigenetic pathways mediating the effects of nutrition and obesity on pubertal timing are unknown. Here, we identify Sirtuin 1 (SIRT1), a fuel-sensing deacetylase, as a molecule that restrains female puberty via epigenetic repression of the puberty-activating gene, Kiss1. SIRT1 is expressed in hypothalamic Kiss1 neurons and suppresses Kiss1 expression. SIRT1 interacts with the Polycomb silencing complex to decrease Kiss1 promoter activity. As puberty approaches, SIRT1 is evicted from the Kiss1 promoter facilitating a repressive-to-permissive switch in chromatin landscape. Early-onset overnutrition accelerates these changes, enhances Kiss1 expression and advances puberty. In contrast, undernutrition raises SIRT1 levels, protracts Kiss1 repression and delays puberty. This delay is mimicked by central pharmacological activation of SIRT1 or SIRT1 overexpression, achieved via transgenesis or virogenetic targeting to the ARC. Our results identify SIRT1-mediated inhibition of Kiss1 as key epigenetic mechanism by which nutritional cues and obesity influence mammalian puberty.

Computational Study of the Binding Modes of Diverse DPN Analogues on Estrogen Receptors (ER) and the Biological Evaluation of a New Potential Antiestrogenic Ligand.

Estrogen (17ß-estradiol) is essential for normal growth and differentiation in the mammary gland. In the last three decades, previous investigations have revealed that Estrogen Receptor Alpha (ERα) plays a critical role in breast cancer. More recently, observations regarding the widespread expression of ERß-like proteins in normal and neoplastic mammary tissues have suggested that ERß is also involved in the mentioned pathology. Design of new drugs both steroidal and nonsteroidal that target any of these receptors represents a promise to treat breast cancer although it remains a challenge due to the sequence similarity between their catalytic domains. In this work, we propose a new set of compounds that could effectively target the estrogen receptors ERα and ERß. These ligands were designed based on the chemical structure of the ERß-selective agonist Diarylpropionitrile (DPN). The designed ligands were submitted to in silico ADMET studies, yielding in a filtered list of ligands that showed better drug-like properties. Molecular dynamics simulations of both estrogen receptors and docking analysis were carried-out employing the designed compounds, from which two were chosen due to their promising characteristics retrieved from theoretical results (docking analysis or targeting receptor predictions). They were chemically synthetized and during the process, two precursor ligands were also obtained. These four ligands were subjected to biological studies from which it could be detected that compound mol60b dislplayed inhibitory activity and its ability to activate the transcription via an estrogenic mechanism of action was also determined. Interestinly, this observation can be related to theoretical binding free energy calculations, where the complex: ERß-mol60b showed the highest energy ΔGbind value in comparison to others.

Disentangling puberty: novel neuroendocrine pathways and mechanisms for the control of mammalian puberty.

BACKGROUND: Puberty is a complex developmental event, controlled by sophisticated regulatory networks that integrate peripheral and internal cues and impinge at the brain centers driving the reproductive axis. The tempo of puberty is genetically determined but is also sensitive to numerous modifiers, from metabolic and sex steroid signals to environmental factors. Recent epidemiological evidence suggests that the onset of puberty is advancing in humans, through as yet unknown mechanisms. In fact, while much knowledge has been gleaned recently on the mechanisms responsible for the control of mammalian puberty, fundamental questions regarding the intimate molecular and neuroendocrine pathways responsible for the precise timing of puberty and its deviations remain unsolved. OBJECTIVE AND RATIONALE: By combining data from suitable model species and humans, we aim to provide a comprehensive summary of our current understanding of the neuroendocrine mechanisms governing puberty, with particular focus on its central regulatory pathways, underlying molecular basis and mechanisms for metabolic control. SEARCH METHODS: A comprehensive MEDLINE search of articles published mostly from 2003 to 2017 has been carried out. Data from cellular and animal models (including our own results) as well as clinical studies focusing on the pathophysiology of puberty in mammals were considered and cross-referenced with terms related with central neuroendocrine mechanisms, metabolic control and epigenetic/miRNA regulation. OUTCOMES: Studies conducted during the last decade have revealed the essential role of novel central neuroendocrine pathways in the control of puberty, with a prominent role of kisspeptins in the precise regulation of the pubertal activation of GnRH neurosecretory activity. In addition, different transmitters, including neurokinin-B (NKB) and, possibly, melanocortins, have been shown to interplay with kisspeptins in tuning puberty onset. Alike, recent studies have documented the role of epigenetic mechanisms, involving mainly modulation of repressors that target kisspeptins and NKB pathways, as well as microRNAs and the related binding protein, Lin28B, in the central control of puberty. These novel pathways provide the molecular and neuroendocrine basis for the modulation of puberty by different endogenous and environmental cues, including nutritional and metabolic factors, such as leptin, ghrelin and insulin, which are known to play an important role in pubertal timing. WIDER IMPLICATIONS: Despite recent advancements, our understanding of the basis of mammalian puberty remains incomplete. Complete elucidation of the novel neuropeptidergic and molecular mechanisms summarized in this review will not only expand our knowledge of the intimate mechanisms responsible for puberty onset in humans, but might also provide new tools and targets for better prevention and management of pubertal deviations in the clinical setting.

Current Status and Future Prospects to Achieve Foot-and-Mouth Disease Eradication in South America.

South America has a favourable position with respect to foot-and-mouth disease (FMD) compared with other FMD-affected regions due to the elimination of endemic clinical presentation of the disease. South America has reached the final stage of control and aims to eradicate the disease in the region under the provisions of the Hemispheric Program for the Eradication of FMD 2011-2020 (PHEFA). This programme aims at bringing eradication to completion, thereby eliminating the pool of foot-and-mouth disease genotypes active in South America. This plan includes a regional political agreement that provides strategies and technical guidelines for the eradication of foot-and-mouth disease from South America. It incorporates knowledge and experience regarding the disease's history and its connection with the different production systems, animal movement and trade. The Pan American Foot and Mouth Disease Center has led the control and eradication programmes, providing the framework for designing national and subregional programmes that have led to significant progress in controlling the disease in South America. The current situation is the result of several factors, including the proper implementation of a national control programmes, good veterinary infrastructure in most countries and public-private participation in the process of eradicating the disease. Notwithstanding the favourable health status, there are significant challenges for the goal of eradication. At this stage, South American countries should enhance their surveillance strategies particularly through the use of target or risk-based surveys that contribute to increase the degree of sensitivity in the search for viral circulation in the context of absence of clinical occurrence of FMD.

Docking Studies of Glutamine Valproic Acid Derivative (S)-5- amino-2-(heptan-4-ylamino)-5-oxopentanoic Acid (Gln-VPA) on HDAC8 with Biological Evaluation in HeLa Cells.

In this contribution, we focused on evaluating a novel compound developed by our group. This molecule, derived from glutamine (Gln) and valproic acid (VPA), denominated (S)- 5-amino-2-(heptan-4-ylamino)-5-oxopentanoic acid (Gln-VPA), was submitted to docking studies on histone deacetylase 8 (HDAC8) to explore its non-bonded interactions. The theoretical results were validated in HeLa cells as a cancer cell model and in human dermal fibroblasts as a normal cell model. The effects of Gln-VPA on HeLa and normal fibroblasts in terms of cell survival and the ability to inhibit HDAC activity in nude nuclear proteins and in nuclear proteins of whole cells treated for 24 h were analyzed. The HeLa cell cycle was analyzed after 24 and 48 h of treatment with Gln-VPA. The docking studies show that Gln-VPA can reach the catalytic site of HDAC8. Gln-VPA was organically synthesized with a purity greater than 97%, and its structure was validated using mass spectrometry, nuclear magnetic resonance and infrared spectroscopy. Gln-VPA showed a similar effect to VPA as an HDAC inhibitor but with less toxicity to fibroblasts. Although Gln-VPA was less efficient than VPA in reducing the survival of HeLa cells, it could be studied for use as a cancer cell sensitizer.

Deciphering the GPER/GPR30-agonist and antagonists interactions using molecular modeling studies, molecular dynamics, and docking simulations.

The G-protein coupled estrogen receptor 1 GPER/GPR30 is a transmembrane seven-helix (7TM) receptor involved in the growth and proliferation of breast cancer. Due to the absence of a crystal structure of GPER/GPR30, in this work, molecular modeling studies have been carried out to build a three-dimensional structure, which was subsequently refined by molecular dynamics (MD) simulations (up to 120 ns). Furthermore, we explored GPER/GPR30's molecular recognition properties by using reported agonist ligands (G1, estradiol (E2), tamoxifen, and fulvestrant) and the antagonist ligands (G15 and G36) in subsequent docking studies. Our results identified the E2 binding site on GPER/GPR30, as well as other receptor cavities for accepting large volume ligands, through GPER/GPR30 π-π, hydrophobic, and hydrogen bond interactions. Snapshots of the MD trajectory at 14 and 70 ns showed almost identical binding motifs for G1 and G15. It was also observed that C107 interacts with the acetyl oxygen of G1 (at 14 ns) and that at 70 ns the residue E275 interacts with the acetyl group and with the oxygen from the other agonist whereas the isopropyl group of G36 is oriented toward Met141, suggesting that both C107 and E275 could be involved in the protein activation. This contribution suggest that GPER1 has great structural changes which explain its great capacity to accept diverse ligands, and also, the same ligand could be recognized in different binding pose according to GPER structural conformations.

Cystic adventitial disease of the popliteal artery: Two case reports and a review of the literature.

Two cases of cystic adventitial disease treated at our institution over the last year are presented. They were middle-aged and apparently healthy patients, and the symptoms begin with a sudden onset of unilateral claudication. After performing a magnetic resonance angiography, a cystic formation attached to the adventitia of the popliteal artery was identified. Both patients were treated in the same manner, with resection of the affected arterial segment and vein bypass interposition. Both remain asymptomatic after one year of follow-up in one case and six months in the other. Cystic adventitial disease is a rare entity, which presents in patients without cardiovascular risk factors, so sometimes it takes long to reach a definitive diagnosis. Concerning the different treatment options, cyst excision together with the affected arterial segment seems to offer better mid- and long-term results when compared with other treatment options such as cyst aspiration or endovascular techniques, although there are no multicenter trials evidencing the superiority of one against the others.

Resistin regulates pituitary lipid metabolism and inflammation in vivo and in vitro.

The adipokine resistin is an insulin-antagonizing factor that also plays a regulatory role in inflammation, immunity, food intake, and gonadal function and also regulates growth hormone (GH) secretion in rat adenopituitary cells cultures with the adipokine. Although adipose tissue is the primary source of resistin, it is also expressed in other tissues, including the pituitary. The aim of this study is to investigate the possible action of resistin on the lipid metabolism in the pituitary gland in vivo (rats in two different nutritional status, fed and fast, treated with resistin on acute and a chronic way) and in vitro (adenopituitary cell cultures treated with the adipokine). Here, by a combination of in vivo and in vitro experimental models, we demonstrated that central acute and chronic administration of resistin enhance mRNA levels of the lipid metabolic enzymes which participated on lipolysis and moreover inhibiting mRNA levels of the lipid metabolic enzymes involved in lipogenesis. Taken together, our results demonstrate for the first time that resistin has a regulatory role on lipid metabolism in the pituitary gland providing a novel insight in relation to the mechanism by which this adipokine can participate in the integrated control of lipid metabolism.

Using Bayesian networks to explore the role of weather as a potential determinant of disease in pigs.

Many pathogens are sensitive to climatic variables and this is reflected in their seasonality of occurrence and transmission. The identification of environmental conditions that influence disease occurrence can be subtle, particularly considering their complex interdependencies in addition to those relationships between climate and disease. Statistical treatment of environmental variables is often dependent on their correlations and thus descriptions of climate are often restricted to means rather than accounting for the more precise aspects (including mean, maximum, minimum, variability). Here we utilize a novel multivariate statistical modelling approach, additive Bayesian network (ABN) analyses, to identify the inter-linkages of different weather variables to better capture short-term environmental conditions that are important drivers of disease. We present a case study that explores weather as a driver of disease in livestock systems. We utilize quality assurance health scheme data on ten major diseases of pigs from 875 finishing pig herds distributed across the United Kingdom over 7 years (2005-2011). We examine the relationship between the occurrence of these pathologies and contemporary weather conditions measured by local meteorological stations. All ten pathologies were associated with at least 2 other pathologies (maximum 6). Three pathologies were associated directly with temperature variables: papular dermatitis, enzootic pneumonia and milk spots. Latitude was strongly associated with multiple pathologies, though associations with longitude were eliminated when clustering for repeated observations of farms was assessed. The identification of relationships between climatic factors and different (potentially related) diseases offers a more comprehensive insight into the complex role of seasonal drivers and herd health status than traditional analytical methods.

Cellular distribution, regulated expression, and functional role of the anorexigenic peptide, NUCB2/nesfatin-1, in the testis.

Nesfatin-1, product of the precursor NEFA/nucleobindin2 (NUCB2), was initially identified as anorectic hypothalamic neuropeptide, acting in a leptin-independent manner. In addition to its central role in the control of energy homeostasis, evidence has mounted recently that nesfatin-1 is also produced in peripheral metabolic tissues, such as pancreas, adipose, and gut. Moreover, nesfatin-1 has been shown to participate in the control of body functions gated by whole-body energy homeostasis, including puberty onset. Yet, whether, as is the case for other metabolic neuropeptides, NUCB2/nesfatin-1 participates in the direct control of gonadal function remains unexplored. We document here for the first time the expression of NUCB2 mRNA in rat, mouse, and human testes, where NUCB2/nesfatin-1 protein was identified in interstitial mature Leydig cells. Yet in rats, NUCB2/nesfatin-1 became expressed in Sertoli cells upon Leydig cell elimination and was also detected in Leydig cell progenitors. Although NUCB2 mRNA levels did not overtly change in rat testis during pubertal maturation and after short-term fasting, NUCB2/nesfatin-1 content significantly increased along the puberty-to-adult transition and was markedly suppressed after fasting. In addition, testicular NUCB2/nesfatin-1 expression was up-regulated by pituitary LH, because hypophysectomy decreased, whereas human choriogonadotropin (super-agonist of LH receptors) replacement enhanced, NUCB2/nesfatin-1 mRNA and peptide levels. Finally, nesfatin-1 increased human choriogonadotropin-stimulated testosterone secretion by rat testicular explants ex vivo. Our data are the first to disclose the presence and functional role of NUCB2/nesfatin-1 in the testis, where its expression is regulated by developmental, metabolic, and hormonal cues as well as by Leydig cell-derived factors. Our observations expand the reproductive dimension of nesfatin-1, which may operate directly at the testicular level to link energy homeostasis, puberty onset, and gonadal function.

The atypical cannabinoid O-1602 stimulates food intake and adiposity in rats.

AIMS: Cannabinoids are known to control energy homeostasis. Atypical cannabinoids produce pharmacological effects via unidentified targets. We sought to investigate whether the atypical cannabinoid O-1602 controls food intake and body weight. METHODS: The rats were injected acutely or subchronically with O-1602, and the expression of several factors involved in adipocyte metabolism was assessed by real-time polymerase chain reaction. In vivo findings were corroborated with in vitro studies incubating 3T3-L1 adipocytes with O-1602, and measuring intracellular calcium and lipid accumulation. Finally, as some reports suggest that O-1602 is an agonist of the putative cannabinoid receptor GPR55, we tested it in mice lacking GPR55. RESULTS: Central and peripheral administration of O-1602 acutely stimulates food intake, and chronically increases adiposity. The hyperphagic action of O-1602 is mediated by the downregulation of mRNA and protein levels of the anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript. The effects on fat mass are independent of food intake, and involve a decrease in the expression of lipolytic enzymes such as hormone sensitive lipase and adipose triglyceride lipase in white adipose tissue. Consistently, in vitro data showed that O-1602 increased the levels of intracellular calcium and lipid accumulation in adipocytes. Finally, we injected O-1602 in GPR55 -/- mice and found that O-1602 was able to induce feeding behaviour in GPR55-deficient mice. CONCLUSIONS: These findings show that O-1602 modulates food intake and adiposity independently of GPR55 receptor. Thus atypical cannabinoids may represent a novel class of molecules involved in energy balance.

Efficacy of chronic hepatitis C therapy with pegylated interferon and ribavirin in patients on methadone maintenance treatment.

A considerable number of patients undergoing methadone maintenance treatment (MMT) are not considered for treatment against hepatitis C virus (HCV) infection due to a possible lower adherence and efficacy in this population. We aimed to compare the response rates to HCV treatment in patients with or without MMT. HCV-infected patients who initiated pegylated interferon plus ribavirin were included in this prospective cohort study. The relation between sustained virologic response (SVR) and MMT was analyzed. A total of 214 patients were included in the study [81 (37.9%) with and 133 (62.1%) without MMT]. No differences in HCV and interleukin 28B (rs12979860) genotype distribution were observed between the two groups. Of these patients, 103 (48.1%) achieved SVR. Among the patients who received MMT, 39 (48.1%) reached SVR compared to 64 (48.1%) subjects without MMT (p = 0.99). The frequency of voluntary drop-out and treatment discontinuations due to adverse events was comparable between the patients with and without MMT [10 (12.3%) versus 14 (10.5%), p = 0.68, and 4 (4.9%) versus 9 (6.8%), p = 0.59, respectively]. The efficacy of HCV therapy in MMT patients is similar to that found in subjects not taking methadone. MMT patients should be equally considered for treatment with pegylated interferon plus ribavirin in HCV-infected patients.

Association between covariates and disease occurrence in the presence of diagnostic error.

Identification of covariates associated with disease is a key part of epidemiological research. Yet, while adjustment for imperfect diagnostic accuracy is well established when estimating disease prevalence, similar adjustment when estimating covariate effects is far less common, although of important practical relevance due to the sensitivity of such analyses to misclassification error. Case-study data exploring evidence for seasonal differences in Salmonella prevalence using serological testing is presented, in addition simulated data with known properties are analysed. It is demonstrated that: (i) adjusting for misclassification error in models comprising continuous covariates can have a very substantial impact on the resulting conclusions which can then be drawn from any analyses; and (ii) incorporating prior knowledge through Bayesian estimation can provide potentially more informative assessments of covariates while removing the assumption of perfect diagnostic accuracy. The method presented is widely applicable and easily generalized to many types of epidemiological studies.