Ecopharmacology: Deliberated or casual dispersion of pharmaceutical principles, phytosanitary, personal health care and veterinary products in environment needs a multivariate analysis or expert systems for the control, the measure and the remediation

.The increasing human and animal use and abuse of drugs as well as of personalhealthcare and gross domestic products, involve disposal and waste problems and, as a consequence, affect the environmental condition. Actually most of the active principles are complex synthesised organic molecules that react, inside human or animal body, by specific biochemical reactions that in no case can reach a 100% yield and produce residues that could be more noxious of the starting compounds. Just reading the indication sheet accompanying any drugs, it is easy to state that no drug can be considered healthy, so, their use constitutes a serious pollution source. The full awareness of this relatively new environmental problem let many researchers to face it from different point of view. Current studies are considering the sources of these substances in the environment, the effects on human health as well as on the flora and fauna species, the recalcitrance and possible degradation methods, analytical techniques able to determine them and their metabolites even at low concentrations and in complex matrices. Literature on the subject continuously increases and a comparison of all data became more and more difficult both for a single drug and for different ones based on the same or different active principles. This is a typical case in which chemometrics can extract a full information in the easier way, so the design of a European database coupled to suitable expertsystem software should be strongly suggested

First synthesis of a fully active spin-labeled peptide hormone

For the first time in the electron spin resonance (ESR) and peptide synthesis fields, a fully active spin-labeled peptide hormone was reported. the ESR spectra of this alpha-melanocyte stimulating hormone (alpha-MSH) analogue (acetyl-Toac(0)-alpha-MSH) where Toac is the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid, suggested a pH-independent conformation and a more restricted movement comparatively to the free Toac, Owing to its equivalent biological potency in a skin pigmentation assay as compared to the native alpha-MSH and its unique characteristic (paramagnetic, naturally fluorescent and fully active), this analogue is of great potential for investigation of relevant physiological roles reported for a-MSH, (C) 1999 Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-01498 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-01498 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

The expression of melanopsin and clock genes in Xenopus laevis melanophores and their modulation by melatonin

Vertebrates have a central clock and also several peripheral clocks. Light responses might result from the integration of light signals by these clocks. The dermal melanophores of Xenopus laevis have a photoreceptor molecule denominated melanopsin (OPN4x). The mechanisms of the circadian clock involve positive and negative feedback. We hypothesize that these dermal melanophores also present peripheral clock characteristics. Using quantitative PCR, we analyzed the pattern of temporal expression of Opn4x and the clock genes Per1, Per2, Bmal1, and Clock in these cells, subjected to a 14-h light:10-h dark (14L:10D) regime or constant darkness (DD). Also, in view of the physiological role of melatonin in the dermal melanophores of X. laevis, we determined whether melatonin modulates the expression of these clock genes. These genes show a time-dependent expression pattern when these cells are exposed to 14L:10D, which differs from the pattern observed under DD. Cells kept in DD for 5 days exhibited overall increased mRNA expression for Opn4x and Clock, and a lower expression for Per1, Per2, and Bmal1. When the cells were kept in DD for 5 days and treated with melatonin for 1 h, 24 h before extraction, the mRNA levels tended to decrease for Opn4x and Clock, did not change for Bmal1, and increased for Per1 and Per2 at different Zeitgeber times (ZT). Although these data are limited to one-day data collection, and therefore preliminary, we suggest that the dermal melanophores of X. laevis might have some characteristics of a peripheral clock, and that melatonin modulates, to a certain extent, melanopsin and clock gene expression

Comparative EPR and fluorescence conformational studies of fully active spin-labeled melanotropic peptides

Similar to melanocyte stimulating hormone (alpha -MSH), its potent and long-acting analogue, [Nle(4), D-Phe(7)]alpha -MSH, when labeled with the paramagnetic amino acid probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac), maintains its full biological potency, thus validating any comparative structural investigations between the two labeled peptides, Correlation times, calculated from tire electron paramagnetic resonance signal of Toac bound to the peptides, and Toac-Trp distances, estimated from the Toac fluorescence quenching of the Trp residue present in the peptides, indicate a more rigid and folded structure for the potent analogue as compared to the hormone, in aqueous medium. (C) 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilUniv São Paulo, Inst Fis, BR-66318 São Paulo, BrazilUniv São Paulo, Inst Biociencias, Dept Fisiol, BR-11176 São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Dissecting T cell lineage relationships by cellular barcoding

T cells, as well as other cell types, are composed of phenotypically and functionally distinct subsets. However, for many of these populations it is unclear whether they develop from common or separate progenitors. To address such issues, we developed a novel approach, termed cellular barcoding, that allows the dissection of lineage relationships. We demonstrate that the labeling of cells with unique identifiers coupled to a microarray-based detection system can be used to analyze family relationships between the progeny of such cells. To exemplify the potential of this technique, we studied migration patterns of families of antigen-specific CD8+ T cells in vivo. We demonstrate that progeny of individual T cells rapidly seed independent lymph nodes and that antigen-specific CD8+ T cells present at different effector sites are largely derived from a common pool of precursors. These data show how locally primed T cells disperse and provide a technology for kinship analysis with wider utility

Environmental control of biological rhythms: effects on development, fertility and metabolism

Internal temporal organisation properly synchronised to the environment is crucial for health maintenance. This organisation is provided at the cellular level by the molecular clock, a macromolecular transcription-based oscillator formed by the clock and the clock-controlled genes that is present in both central and peripheral tissues. In mammals, melanopsin in light-sensitive retinal ganglion cells plays a considerable role in the synchronisation of the circadian timing system to the daily light/dark cycle. Melatonin, a hormone synthesised in the pineal gland exclusively at night and an output of the central clock, has a fundamental role in regulating/timing several physiological functions, including glucose homeostasis, insulin secretion and energy metabolism. As such, metabolism is severely impaired after a reduction in melatonin production. Furthermore, light pollution during the night and shift work schedules can abrogate melatonin synthesis and impair homeostasis. Chronodisruption during pregnancy has deleterious effects on the health of progeny, including metabolic, cardiovascular and cognitive dysfunction. Developmental programming by steroids or steroid-mimetic compounds also produces internal circadian disorganisation that may be a significant factor in the aetiology of fertility disorders such as polycystic ovary syndrome. Thus, both early and late in life, pernicious alterations of the endogenous temporal order by environmental factors can disrupt the homeostatic function of the circadian timing system, leading to pathophysiology and/or disease.This work was supported by grants 1110220 from FONDECYT and ACT1116 from CONICYT, Chile (to HGR); Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP) and Conselho Nacional de Desenvolvimento Tecnológico e Científico (CNPq; Brazil, to AMC, FGA and JCN); Pro-Reitoria de Pesquisa Universidade Federal de Minas Gerais, Brazil (to MOP); fellowship 21120472 from CONICYT (Chile) to (NM) and an NIEHS sponsored Pilot Grant from the Department of Environmental Medicine at the University of Rochester School of Medicine to MTS

Haemagglutination inhibition and virus microneutralisation serology assays: use of harmonised protocols and biological standards in seasonal influenza serology testing and their impact on inter-laboratory variation and assay correlation: A FLUCOP collaborative study

Introduction: The haemagglutination inhibition assay (HAI) and the virus microneutralisation assay (MN) are long-established methods for quantifying antibodies against influenza viruses. Despite their widespread use, both assays require standardisation to improve inter-laboratory agreement in testing. The FLUCOP consortium aims to develop a toolbox of standardised serology assays for seasonal influenza. Building upon previous collaborative studies to harmonise the HAI, in this study the FLUCOP consortium carried out a head-to-head comparison of harmonised HAI and MN protocols to better understand the relationship between HAI and MN titres, and the impact of assay harmonisation and standardisation on inter-laboratory variability and agreement between these methods. Methods: In this paper, we present two large international collaborative studies testing harmonised HAI and MN protocols across 10 participating laboratories. In the first, we expanded on previously published work, carrying out HAI testing using egg and cell isolated and propagated wild-type (WT) viruses in addition to high-growth reassortants typically used influenza vaccines strains using HAI. In the second we tested two MN protocols: an overnight ELISA-based format and a 3-5 day format, using reassortant viruses and a WT H3N2 cell isolated virus. As serum panels tested in both studies included many overlapping samples, we were able to look at the correlation of HAI and MN titres across different methods and for different influenza subtypes. Results: We showed that the overnight ELISA and 3-5 day MN formats are not comparable, with titre ratios varying across the dynamic range of the assay. However, the ELISA MN and HAI are comparable, and a conversion factor could possibly be calculated. In both studies, the impact of normalising using a study standard was investigated, and we showed that for almost every strain and assay format tested, normalisation significantly reduced inter-laboratory variation, supporting the continued development of antibody standards for seasonal influenza viruses. Normalisation had no impact on the correlation between overnight ELISA and 3-5 day MN formats.publishedVersio

Integrase defective lentiviral vector as a vaccine platform for delivering Influenza antigens

Viral vectors represent an attractive technology for vaccine delivery. We exploited the integrase defective lentiviral vector (IDLV) as a platform for delivering relevant antigens within the context of the ADITEC collaborative research program. In particular, Influenza virus hemagglutinin (HA) and nucleoprotein (NP) were delivered by IDLVs while H1N1 A/California/7/2009 subunit vaccine (HAp) with or without adjuvant was used to compare the immune response in a murine model of immunization. In order to maximize the antibody response against HA, both IDLVs were also pseudotyped with HA (IDLV-HA/HA and IDLV-NP/HA, respectively). Groups of CB6F1 mice were immunized intramuscularly with a single dose of IDLV-NP/HA, IDLV-HA/HA, HAp alone or with HAp together with the systemic adjuvant MF59. Six months after the vaccine prime all groups were boosted with HAp alone. Cellular and antibody responses to influenza antigens were measured at different time points after the immunizations. Mice immunized with HA-pseudotyped IDLVs showed similar levels of anti-H1N1 IgG over time, evaluated by ELISA, which were comparable to those induced by HAp+MF59 vaccination, but significantly higher than those induced by HAp alone. The boost with HAp alone induced an increase of antibodies in all groups, and the responses were maintained at higher levels up to 18 weeks post-boost. The antibody response was functional and persistent overtime, capable of neutralizing virus infectivity, as evaluated by hemagglutination inhibition and microneutralization assays. Moreover, since neuraminidase (NA)-expressing plasmid was included during IDLV preparation, immunization with IDLV-NP/HA and IDLV-HA/HA also induced functional anti-NA antibodies, evaluated by Enzyme-Linked Lectine Assay (ELLA). IFN?-ELISPOT showed evidence of HA-specific response in IDLV-HA/HA immunized animals and persistent NP-specific CD8+ T cell response in IDLV-NP/HA immunized mice. Taken together our results indicate that IDLV can be harnessed for producing a vaccine able to induce a comprehensive immune response, including functional antibodies directed towards HA and NA proteins present on the vector particles in addition to a functional T cell response directed to the protein transcribed from the vector

Epidemiological characteristics of COVID-19 cases and estimates of the reproductive numbers 1 month into the epidemic, Italy, 28 January to 31 March 2020

BackgroundOn 20 February 2020, a locally acquired coronavirus disease (COVID-19) case was detected in Lombardy, Italy. This was the first signal of ongoing transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in the country. The number of cases in Italy increased rapidly and the country became the first in Europe to experience a SARS-CoV-2 outbreak.AimOur aim was to describe the epidemiology and transmission dynamics of the first COVID-19 cases in Italy amid ongoing control measures.MethodsWe analysed all RT-PCR-confirmed COVID-19 cases reported to the national integrated surveillance system until 31 March 2020. We provide a descriptive epidemiological summary and estimate the basic and net reproductive numbers by region.ResultsOf the 98,716 cases of COVID-19 analysed, 9,512 were healthcare workers. Of the 10,943 reported COVID-19-associated deaths (crude case fatality ratio: 11.1%) 49.5% occurred in cases older than 80 years. Male sex and age were independent risk factors for COVID-19 death. Estimates of R0 varied between 2.50 (95% confidence interval (CI): 2.18-2.83) in Tuscany and 3.00 (95% CI: 2.68-3.33) in Lazio. The net reproduction number Rt in northern regions started decreasing immediately after the first detection.ConclusionThe COVID-19 outbreak in Italy showed a clustering onset similar to the one in Wuhan, China. R0 at 2.96 in Lombardy combined with delayed detection explains the high case load and rapid geographical spread. Overall, Rt in Italian regions showed early signs of decrease, with large diversity in incidence, supporting the importance of combined non-pharmacological control measures