The role of Tyr(605) and Ala(607) of thimet oligopeptidase and Tyr(606) and Gly(608) of neurolysin in substrate hydrolysis and inhibitor binding

The physicochemical properties of TOP (thimet oligopeptidase) and NEL (neurolysin) and their hydrolytic activities towards the FRET (fluorescence resonance energy transfer) peptide series AbzGFSXFRQ-EDDnp [where Abz is o-aminobenzoyl; X = Ala, Ile, Leu, Phe, Tyr, Trp, Ser, Gln, Glu, His, Arg or Pro; and EDDnp is N-(2,4-dinitrophenyl)-ethylenediamine] were compared with those of site-mutated analogues. Mutations at Tyr(605) and Ala(607) in TOP and at Tyr(606) and Gly(608) in NEL did not affect the overall folding of the two peptidases, as indicated by their thermal stability, CD analysis and the pH-dependence of the intrinsic fluorescence of the protein. the kinetic parameters for the hydrolysis of substrates with systematic variations at position P-1 showed that Tyr(605) and Tyr(606) of TOP and NEL respectively, played a role in subsite S-1. Ala(607) of TOP and Gly(608) of NEL contributed to the flexibility of the loops formed by residues 600-612 (GHLAGGYDGQYYG; one-letter amino acid codes used) in NEL and 599-611 (GHLAGGYDAQYYG; one-letter amino acid codes used) in TOP contributing to the distinct substrate specificities, particularly with an isoleucine residue at P-1. TOP Y605A was inhibited less efficiently by JA-2 {N-[1-(R,S)-carboxy-3-phenylpropyl]Ala-Aib-Tyr-p-aminobenzoate}, which suggested that the aromatic ring of Ty,105 was an important anchor for its interaction with wild-type TOP. the hydroxy groups of Tyr 605 and Tyr.. did not contribute to the pH-activity profiles, since the pKs obtained in the assays of mutants TOP Y605F and NEL Y606F were similar to those of wild-type peptidases. However, the pH-k(cat)/K-m dependence curve of TOP Y605A differed from that of wild-type TOP and from TOP Y606F. These results provide insights into the residues involved in the substrate specificities of TOP and NEL and how they select cytosolic peptides for hydrolysis.Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilInst Butantan, Lab Especial Toxinol Aplicada, CAT, CEPID, BR-05467010 São Paulo, BrazilUniv São Paulo, Inst Ciencias Biomed, Dept Biol Celular & Desenvolvimento, Programa Biol Celular, BR-05508900 São Paulo, BrazilUniv São Paulo, Lab Neurociencias, BR-03071000 São Paulo, BrazilUniv Mogi das Cruzes, CIIB, BR-08780911 Mogi Das Cruzes, SP, BrazilUniversidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, BrazilWeb of Scienc

Revisión y análisis de técnicas y métodos computacionales para la evaluación de la condición corporal en vacas

BCS (del inglés "Body Condition Score") es un método que permite estimar la grasa corporal como indicador del status energético de las vacas. El monitoreo de esta variable es muy importante porque influye en la producción de leche, reproducción y salud de las vacas. El BCS se evalúa visualmente con la intervención de personal calificado, y puede estar sujeto a variaciones entre operadores. Para minimizar esta variación y disponer de más agilidad durante el registro de los valores, en la bibliografía se encuentran diferentes trabajos que la automatizan total o parcialmente aplicando técnicas de análisis de imágenes y aprendizaje máquina. En este documento se analizan dichos trabajos, señalando las principales ventajas y desventajas, que derivan en la identificación de oportunidades de investigación y desarrollo de nuevas alternativas que mejoren el tiempo de respuesta y precisión de las estimaciones.Sociedad Argentina de Informática e Investigación Operativa (SADIO

Revisión y análisis de técnicas y métodos computacionales para la evaluación de la condición corporal en vacas

BCS (del inglés "Body Condition Score") es un método que permite estimar la grasa corporal como indicador del status energético de las vacas. El monitoreo de esta variable es muy importante porque influye en la producción de leche, reproducción y salud de las vacas. El BCS se evalúa visualmente con la intervención de personal calificado, y puede estar sujeto a variaciones entre operadores. Para minimizar esta variación y disponer de más agilidad durante el registro de los valores, en la bibliografía se encuentran diferentes trabajos que la automatizan total o parcialmente aplicando técnicas de análisis de imágenes y aprendizaje máquina. En este documento se analizan dichos trabajos, señalando las principales ventajas y desventajas, que derivan en la identificación de oportunidades de investigación y desarrollo de nuevas alternativas que mejoren el tiempo de respuesta y precisión de las estimaciones.Sociedad Argentina de Informática e Investigación Operativa (SADIO

Grouping crossbred Holstein x Gyr heifers according to different feed efficiency indexes and its effects on energy and nitrogen partitioning, blood metabolic variables and gas exchanges.

The objectives of this study were: i) to classify animals into groups of high and low feed efficiency (FE) using three FE indexes (Residual feed intake (RFI), Residual weight gain (RG) and Feed conversion efficiency (FCE)), and ii) to evaluate whether crossbreed Holstein x Gyr heifers divergent for FE indexes exhibit differences in nutrient intake and digestibility, energy partitioning, heat production, methane emissions, nitrogen partitioning and blood parameters. Thirty-five heifers were housed in a tie-stall, received ad libitum TMR (75:25, corn silage: concentrate) and were ranked and classified into high (HE) or low efficiency (LE) for RFI, RG and FCE. The number of animals for each HE group were 13 ( 0.5 SD) and for the LE were 10 (> 0.5 SD) for RFI, 11 for RG and 12 for FCE (< 0.5 SD). Gas exchanges (O2 consumption, CO2 and CH4 production) in open-circuit respiratory chambers and whole tract digestibility trial was performed. A completely randomized experimental design was used and the data were analyzed by ANOVA and correlation study. High efficiency animals for RFI produced less CO2, consumed less O2 and had lower heat production (HP). Methane production was positively correlated with RFI. High efficiency RG had higher O2 consumption and CO2 production in relation to LE-RG. High efficiency FCE had greater NFC digestibility, higher positive energy balance (EB) and excreted (11.4 g/d) less nitrogen in urine. High efficiency RG and FCE groups emitted less CH4 per kg of weight gain than LE animals. Animals HE for RFI and FCE had lower &#946;-hydroxybutyrate and higher glucose concentrations, respectively. The differences in intake, digestibility, energy and nitrogen partition, CH4 emission, blood metabolic variables and heat production between the HE and LE groups varied according to the efficiency indexes adopted. The HP (kcal/d/BW0.75) was lower for HE animals for RFI and FCE indexes

Exogenous amylase increases gas production and improves in vitro ruminal digestion kinetics of sorghum and corn grains.

ABSTRACT - The aim of this study was to evaluate the effect of exogenous amylase on gas production, in vitro dry matter digestibility (IVDMD), and in vitro digestion kinetics of sorghum (Sorghum vulgaris) and two corn hybrids of different grain textures. Ruminal fluid was collected from two rumen-fistulated cows receiving or not exogenous amylase (0.7g kg-1 of dry matter (DM basis)), provided to achieve 396 kilo Novo units kg-1 for amylase activity (DM basis). Gas production was measured after 1, 3, 6, 9, 12, 15, 18, 21, 24, 30, 36, 42 e 48 hours of incubation. Amylase increased gas production (mL) by 5.4%. Corn hybrids have higher in vitro dry matter digestibility than sorghum. Exogenous amylase increased the potential of gas production (A) (P=0.01). There was an effect of hybrid for IVDMD (P<0.01). The addition of exogenous amylase increases the in vitro gas production, improves fermentation kinetics, and increases the production of the ammonia nitrogen of corn and sorghum grains, but does not affect in vitro and dry matter digestibility or the short-chain fatty acids production. RESUMO - O objetivo deste estudo foi avaliar o efeito da amilase exógena na produção de gases, a digestibilidade in vitro da matéria seca (DIVMS) e a cinética de digestão in vitro de sorgo (Sorghum vulgaris) e de dois híbridos de milho de diferentes texturas de grãos. O líquido ruminal foi coletado de duas vacas fistuladas no rúmen recebendo ou não amilase exógena (0,7g kg-1 de matéria seca (MS)), fornecida para atingir 396 kg Novo unidades kg-1 para atividade de amilase (base na MS). A produção de gás foi medida após uma, três, seis, nove, 12, 15, 18, 21, 24, 30, 36, 42 e 48 horas de incubação. A amilase aumentou a produção de gás (mL) em 5,4%. Híbridos de milho apresentam maior DIVMS que o sorgo. A amilase exógena aumentou o potencial de produção de gás (A) (P=0,01). Houve efeito de híbrido para DIVMS (P<0,01). Amilase exógena aumenta a produção de gás in vitro, melhora a cinética da fermentação e aumenta a produção de nitrogênio amoniacal de grãos de milho e sorgo, mas não afeta a digestibilidade in vitro da matéria seca ou a produção de ácidos graxos de cadeia curta

Co-Administration of a Plasmid DNA Encoding IL-15 Improves Long-Term Protection of a Genetic Vaccine against Trypanosoma cruzi

Background: Immunization of mice with the Trypanosoma cruzi trans-sialidase (TS) gene using plasmid DNA, adenoviral vector, and CpG-adjuvanted protein delivery has proven highly immunogenic and provides protection against acute lethal challenge. However, long-term protection induced by TS DNA vaccines has not been reported. the goal of the present work was to test whether the co-administration of a plasmid encoding IL-15 (pIL-15) could improve the duration of protection achieved through genetic vaccination with plasmid encoding TS (pTS) alone.Methodology: We immunized BALB/c mice with pTS in the presence or absence of pIL-15 and studied immune responses [with TS-specific IFN-gamma ELISPOT, serum IgG ELISAs, intracellular cytokine staining (IFN-gamma, TNF-alpha, and IL-2), tetramer staining, and CFSE dilution assays] and protection against lethal systemic challenge at 1 to 6 months post vaccination. Mice receiving pTS alone developed robust TS-specific IFN-gamma responses and survived a lethal challenge given within the first 3 months following immunization. the addition of pIL-15 to pTS vaccination did not significantly alter T cell responses or protection during this early post-vaccination period. However, mice vaccinated with both pTS and pIL-15 challenged 6 months post-vaccination were significantly more protected against lethal T. cruzi challenges than mice vaccinated with pTS alone (P6 months post immunization. Also, these TS-specific T cells were better able to expand after in vitro restimulation.Conclusion: Addition of pIL-15 during genetic vaccination greatly improved long-term T cell survival, memory T cell expansion, and long-term protection against the important human parasite, T. cruzi.National Institutes of HealthFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Millennium Institute for Gene TherapySt Louis Univ, Dept Internal Med, St Louis, MO 63103 USAUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol, Escola Paulista Med, São Paulo, BrazilSt Louis Univ, Dept Mol Microbiol, St Louis, MO 63103 USAUniv Fed Minas Gerais, Inst Ciencias Biol, Dept Microbiol, Belo Horizonte, MG, BrazilUniversidade Federal de São Paulo, Ctr Terapia Celular & Mol, Escola Paulista Med, São Paulo, BrazilNational Institutes of Health: RO1 AI040196CNPq: 420067/2005-1Web of Scienc

Mortality among over 6 million internal and international migrants in Brazil: a study using the 100 Million Brazilian Cohort

BACKGROUND: To understand if migrants living in poverty in low and middle-income countries (LMICs) have mortality advantages over the non-migrant population, we investigated mortality risk patterns among internal and international migrants in Brazil over their life course. METHODS: We linked socio-economic and mortality data from 1st January 2011 to 31st December 2018 in the 100 Million Brazilian Cohort and calculated all-cause and cause-specific age-standardised mortality rates according to individuals' migration status for men and women. Using Cox regression models, we estimated the age- and sex-adjusted mortality hazard ratios (HR) for internal migrants (i.e., Brazilian-born individuals living in a different Brazilian state than their birth) compared to Brazilian-born non-migrants; and for international migrants (i.e., people born in another country) compared to Brazilian-born individuals. FINDINGS: The study followed up 45,051,476 individuals, of whom 6,057,814 were internal migrants, and 277,230 were international migrants. Internal migrants had similar all-cause mortality compared to Brazilian non-migrants (aHR = 0.99, 95% CI = 0.98-0.99), marginally higher mortality for ischaemic heart diseases (aHR = 1.04, 95% CI = 1.03-1.05) and higher for stroke (aHR = 1.11, 95% CI = 1.09-1.13). Compared to Brazilian-born individuals, international migrants had 18% lower all-cause mortality (aHR = 0.82, 95% CI = 0.80-0.84), with up to 50% lower mortality from interpersonal violence among men (aHR = 0.50, 95% CI = 0.40-0.64), but higher mortality from avoidable causes related to maternal health (aHR = 2.17, 95% CI = 1.17-4.05). INTERPRETATION: Although internal migrants had similar all-cause mortality, international migrants had lower all-cause mortality compared to non-migrants. Further investigations using intersectional approaches are warranted to understand the marked variations by migration status, age, and sex for specific causes of death, such as elevated maternal mortality and male lower interpersonal violence-related mortality among international migrants. FUNDING: The Wellcome Trust

Subdominant/Cryptic CD8 T Cell Epitopes Contribute to Resistance against Experimental Infection with a Human Protozoan Parasite

During adaptive immune response, pathogen-specific CD8+ T cells recognize preferentially a small number of epitopes, a phenomenon known as immunodominance. Its biological implications during natural or vaccine-induced immune responses are still unclear. Earlier, we have shown that during experimental infection, the human intracellular pathogen Trypanosoma cruzi restricts the repertoire of CD8+ T cells generating strong immunodominance. We hypothesized that this phenomenon could be a mechanism used by the parasite to reduce the breath and magnitude of the immune response, favoring parasitism, and thus that artificially broadening the T cell repertoire could favor the host. Here, we confirmed our previous observation by showing that CD8+ T cells of H-2a infected mice recognized a single epitope of an immunodominant antigen of the trans-sialidase super-family. In sharp contrast, CD8+ T cells from mice immunized with recombinant genetic vaccines (plasmid DNA and adenovirus) expressing this same T. cruzi antigen recognized, in addition to the immunodominant epitope, two other subdominant epitopes. This unexpected observation allowed us to test the protective role of the immune response to subdominant epitopes. This was accomplished by genetic vaccination of mice with mutated genes that did not express a functional immunodominant epitope. We found that these mice developed immune responses directed solely to the subdominant/cryptic CD8 T cell epitopes and a significant degree of protective immunity against infection mediated by CD8+ T cells. We concluded that artificially broadening the T cell repertoire contributes to host resistance against infection, a finding that has implications for the host-parasite relationship and vaccine development