DM intake by feedlot beef cattle: factors that impact intake patterns.

DM intake (DMI) for individual pens of cattle is recorded daily or averaged across each week by most commercial feedlots as an index of performance. Numerous factors impact DMI by feedlot cattle. Some are available at the start of the feedlot period (initial BW, sex), and others become available early in the feeding period (daily DMI during adaptation) or more continuously (daily DMI from the previous week). To evaluate the relative impact of these factors on daily DMI during individual weeks within the feedlot period, we employed a dataset compiled from 2009 to 2014 from one commercial feedlot, including 4 132 pens (485 458 cattle), which were split into two fractions: 80% were used to calculate DMI regressions on these factors to develop a prediction equation for mean DMI for each week of the feeding period, and 20% were reserved to test the adequacy of these prediction equations. Correlations were used to determine the relationship between all available variables with observed DMI. These variables were then included in the generalized least squares regression models. A veracity test of the model was performed against the reserved data. Daily DMI from previous week was the factor most highly correlated with daily DMI (P < 0.10) during from week 6 to week 31, accounting for approximately 70% of the variation, followed by mean daily DMI during adaptation period (weeks 1-4), including in the prediction model from weeks 5 to 12. Initial shrunk BW (ISBW) was the third most correlated factor, which was included in prediction equations from week 5 to week 20. Sex entered the prediction model only after week 8. Daily DMI for each test week within the feeding period was predicted closely (r2 = 0.98) by these four factors (RMSE = 0.155 kg). In conclusion, the mean daily DMI during each week of the finishing period for a pen of cattle could be predicted closely based on mean daily DMI intake during the previous week plus other variables available early in a feedlot period (daily DMI during adaptation period, ISBW and sex).

Feedlot performance and rumen morphometrics of Nellore cattle adapted to high-concentrate diets over periods of 6, 9, 14 and 21 days.

The energy content of finishing diets offered to feedlot cattle may vary across countries. We assumed that the lower is the energy content of the finishing diet, the shorter can be the adaptation period to high-concentrate diets without negatively impacting rumen health while still improving feedlot performance. This study was designed to determine the effects of adaptation periods of 6, 9, 14 and 21 days on feedlot performance, feeding behaviour, blood gas profile, carcass characteristics and rumen morphometrics of Nellore cattle. The experiment was designed as a completely randomised block, replicated 6 times, in which 96 20-month-old yearling Nellore bulls (391.1 ± 30.9 kg) were fed in 24 pens (4 animals/pen) according to the adaptation period adopted: 6, 9, 14 or 21 days. The adaptation diets contained 70%, 75% and 80.5% concentrate, and the finishing diet contained 86% concentrate. After adaptation, one animal per pen was slaughtered (n = 24) for rumen morphometric evaluations and the remaining 72 animals were harvested after 88 days on feed. Orthogonal contrasts were used to assess linear, quadratic and cubic relationships between days of adaptation and the dependent variable. Overall, as days of adaptation increased, final BW (P = 0.06), average daily gain (ADG) (P = 0.07), hot carcass weight (P = 0.04) and gain to feed ratio (G : F) (P = 0.07) were affected quadratically, in which yearling bulls adapted by 14 days presented greater final BW, ADG, hot carcass weight and improved G : F. No significant (P > 0.10) days of adaptation effect was observed for any of feeding behaviour variables. As days of adaptation increased, the absorptive surface area of the rumen was affected cubically, where yearling bulls adapted by 14 days presented greater absorptive surface area (P = 0.03). Thus, Nellore yearling bulls should be adapted by 14 days because it led to improved feedlot performance and greater development of rumen epithelium without increasing rumenitis scores.

Synthesis, structure-activity relationship and trypanocidal activity of pyrazole-imidazoline and new pyrazole-tetrahydropyrimidine hybrids as promising chemotherapeutic agents for Chagas disease.

Drug therapy for Chagas disease remains a major challenge as potential candidate drugs have failed clinical trials. Currently available drugs have limited efficacy and induce serious side effects. Thus, the discovery of new drugs is urgently needed in the fight against Chagas' disease. Here, we synthesized and evaluated the biological effect of pyrazole-imidazoline (1a-i) and pyrazole-tetrahydropyrimidine (2a-i) derivatives against relevant clinical forms of Trypanosoma cruzi. The structure-activity relationship (SAR), drug-target search, physicochemical and ADMET properties of the major active compounds in vitro were also assessed in silico. Pyrazole derivatives showed no toxicity in Vero cells and also no cardiotoxicity. Phenotypic screening revealed two dichlorinated pyrazole-imidazoline derivatives (1c and 1d) with trypanocidal activity higher than that of benznidazole (Bz) against trypomastigotes; these were also the most potent compounds against intracellular amastigotes. Replacement of imidazoline with tetrahydropyrimidine in the pyrazole compounds completely abolished the trypanocidal activity of series 2(a-i) derivatives. The physicochemical and ADMET properties of the compounds predicted good permeability, good oral bioavailability, no toxicity and mutagenicity of 1c and 1d. Pyrazole nucleus had high frequency hits for cruzipain in drug-target search and structure activity relationship (SAR) analysis of pyrazole-imidazoline derivatives revealed enhanced activity when chlorine atom was inserted in meta-positions of the benzene ring. Additionally, we found evidence that both compounds (1c and 1d) have the potential to interact non-covalently with the active site of cruzipain and also inhibit the cysteine proteinase activity of T. cruzi. Collectively, the data presented here reveal pyrazole derivatives with promise for further optimization in the therapy of Chagas disease.

Efficacy of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinone derivatives against different Trypanosoma cruzi discrete type units: Identification of a promising hit compound.

The limited efficacy of benznidazole (Bz) indicated by failures of current Phase II clinical trials emphasizes the urgent need to identify new drugs with improved safety and efficacy for treatment of Chagas disease (CD). Herein, we analyzed the efficacy of a series of 2-hydroxy-3-phenylsulfanylmethyl-[1,4]-naphthoquinones against different Trypanosoma cruzi discrete type units (DTUs) of relevant clinical forms of CD. Cytotoxic and trypanocidal effect of naphthoquinone derivatives were assessed in mammalian cells, trypomastigotes and intracellular amastigotes using, luminescent assays (CellTiter-Glo and T. cruzi Dm28c-luciferase) and/or counting with a light microscope. Reactive oxygen species (ROS) production and intracellular targets of promising compounds were assessed with 2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) probe and ultrastructural analysis, respectively. ADMET properties were analyzed by in silico modeling. Most of the compounds showed low cytotoxic effect. Only two compounds (Compounds 2 and 11) had IC50 values lower than Bz, showing higher susceptibility of bloodstream trypomastigotes. Compound 2 exhibited greater efficacy against trypomastigotes from different T. cruzi DTUs, even better than Bz against Brazil and CL strains. Ultrastructural analysis revealed changes in intracellular compartments, suggesting autophagy as one possible mechanism of action. Oxidative stress, induced by Compound 2, resulted in elevated level of ROS, leading to parasite death. Compound 2 was also effective against intracellular amastigotes, showing high selectivity index. ADMET analysis predicted good oral bioavailability, reduced drug metabolism and no carcinogenic potential for Compound 2. The data highlight Compound 2 as a hit compound and stimulate further structural and pharmacological optimization to potentiate its trypanocidal activity and selectivity.

Relationships of feedlot performance, feeding behavior, rumen morphometrics, and carcass characteristics of Nellore cattle differing in phenotypic residual feed intake.

The objective of this study was to examine the relationship of DMI fluctuation, feedlot performance, feeding behavior, rumen morphometrics, and carcass characteristics in Nellore cattle classified by residual feed intake (RFI). One experiment was conducted in 2 consecutive years using individual pens (1.0 × 7.0 m) at the São Paulo State University feedlot, Dracena campus, Brazil. The experiment in year 1 started in June of 2012 with forty-eight 20-mo-old Nellore yearling bulls with an initial BW of 358.2 ± 19.4 kg. The experiment in year 2 started in January of 2013 with sixty 20-mo-old Nellore yearling bulls with an initial BW of 402.5 ± 33.0 kg. Experiments in years 1 and 2 lasted 94 and 84 d, respectively. All yearling bulls were categorized as high RFI (>0.5 SD above the mean, = 25), medium RFI (±0.5 SD from the mean, = 56), and low RFI (<0.5 SD below the mean, = 27). Visual appraisal to collect behavior data was made on d 40 (finishing period) of both years. Yearling bulls were harvested when average across treatment groups achieved a fat thickness of 4 mm at the 12th rib. Low-RFI yearling bulls had lower daily DMI, expressed either in kilograms ( < 0.01) or as percentage of BW ( < 0.01), and improved G:F ( < 0.01) when compared to high-RFI animals. No differences were observed ( > 0.10) for ADG, final BW, or HCW among RFI groups. Also, low-RFI yearling bulls had thinner final 12th rib ( < 0.01) and biceps femoris (P8) fat thickness ( < 0.01). Low-RFI yearling bulls were slower to consume ( = 0.03) and ruminate ( < 0.01) 1 kg of either DM or NDF. No significant ( > 0.10) RFI effect was observed for any ruminal morphometrics variables evaluated, with the exception of papillae area, in which low-RFI Nellore yearling bulls tended to have smaller ( = 0.07) papillae area than medium-RFI animals. In general, low-RFI Nellore yearling bulls consumed more particles larger than 19 and 8 mm and had a similar performance when compared to both medium- and high-RFI bulls; however, carcass fat composition was negatively impacted.

Trypanosoma cruzi heparin-binding proteins present a flagellar membrane localization and serine proteinase activity.

Heparin-binding proteins (HBPs) play a key role in Trypanosoma cruzi-host cell interactions. HBPs recognize heparan sulfate (HS) at the host cell surface and are able to induce the cytoadherence and invasion of this parasite. Herein, we analysed the biochemical properties of the HBPs and also evaluated the expression and subcellular localization of HBPs in T. cruzi trypomastigotes. A flow cytometry analysis revealed that HBPs are highly expressed at the surface of trypomastigotes, and their peculiar localization mainly at the flagellar membrane, which is known as an important signalling domain, may enhance their binding to HS and elicit the parasite invasion. The plasmon surface resonance results demonstrated the stability of HBPs and their affinity to HS and heparin. Additionally, gelatinolytic activities of 70 kDa, 65·8 kDa and 59 kDa HBPs over a broad pH range (5·5-8·0) were revealed using a zymography assay. These proteolytic activities were sensitive to serine proteinase inhibitors, such as aprotinin and phenylmethylsulfonyl fluoride, suggesting that HBPs have the properties of trypsin-like proteinases.

Integrated solutions for urban runoff pollution control in Brazilian metropolitan regions.

One of the most important causes for poor water quality in urban rivers in Brazil is the low collection efficiency of the sewer system due to unforeseen interconnections with the stormwater drainage system. Since the beginning of the 20th century, Brazilian cities have adopted separate systems for sanitary sewers and stormwater runoff. Gradually these two systems became interconnected. A major challenge faced today by water managers in Brazil is to find efficient and low cost solutions to deal with this mixed system. The current situation poses an important threat to the improvement of the water quality in urban rivers and lakes. This article presents an evaluation of the water quality parameters and the diffuse pollution loads during rain events in the Pinheiros River, a tributary of the Tietê River in São Paulo. It also presents different types of integrated solutions for reducing the pollution impact of combined systems, based on the European experience in urban water management. An evaluation of their performance and a comparison with the separate system used in most Brazilian cities is also presented. The study is based on an extensive water quality monitoring program that was developed for a special investigation in the Pinheiros River and lasted 2.5 years. Samples were collected on a daily basis and water quality variables were analyzed on a daily, weekly or monthly basis. Two hundred water quality variables were monitored at 53 sampling points. During rain events, additional monitoring was carried out using an automated sampler. Pinheiros River is one of the most important rivers in the São Paulo Metropolitan Region and it is also a heavily polluted one.

Trypanosoma cruzi heparin-binding proteins mediate the adherence of epimastigotes to the midgut epithelial cells of Rhodnius prolixus.

Heparin-binding proteins (HBPs) have been demonstrated in both infective forms of Trypanosoma cruzi and are involved in the recognition and invasion of mammalian cells. In this study, we evaluated the potential biological function of these proteins during the parasite-vector interaction. HBPs, with molecular masses of 65·8 kDa and 59 kDa, were isolated from epimastigotes by heparin affinity chromatography and identified by biotin-conjugated sulfated glycosaminoglycans (GAGs). Surface plasmon resonance biosensor analysis demonstrated stable receptor-ligand binding based on the association and dissociation values. Pre-incubation of epimastigotes with GAGs led to an inhibition of parasite binding to immobilized heparin. Competition assays were performed to evaluate the role of the HBP-GAG interaction in the recognition and adhesion of epimastigotes to midgut epithelial cells of Rhodnius prolixus. Epithelial cells pre-incubated with HBPs yielded a 3·8-fold inhibition in the adhesion of epimastigotes. The pre-treatment of epimastigotes with heparin, heparan sulfate and chondroitin sulfate significantly inhibited parasite adhesion to midgut epithelial cells, which was confirmed by scanning electron microscopy. We provide evidence that heparin-binding proteins are found on the surface of T. cruzi epimastigotes and demonstrate their key role in the recognition of sulfated GAGs on the surface of midgut epithelial cells of the insect vector.

Involvement of host cell heparan sulfate proteoglycan in Trypanosoma cruzi amastigote attachment and invasion.

Cell surface glycosaminoglycans (GAGs) play an important role in the attachment and invasion process of a variety of intracellular pathogens. We have previously demonstrated that heparan sulfate proteoglycans (HSPG) mediate the invasion of trypomastigote forms of Trypanosoma cruzi in cardiomyocytes. Herein, we analysed whether GAGs are also implicated in amastigote invasion. Competition assays with soluble GAGs revealed that treatment of T. cruzi amastigotes with heparin and heparan sulfate leads to a reduction in the infection ratio, achieving 82% and 65% inhibition of invasion, respectively. Other sulfated GAGs, such as chondroitin sulfate, dermatan sulfate and keratan sulfate, had no effect on the invasion process. In addition, a significant decrease in infection occurred after interaction of amastigotes with GAG-deficient Chinese Hamster Ovary (CHO) cells, decreasing from 20% and 28% in wild-type CHO cells to 5% and 9% in the mutant cells after 2 h and 4 h of infection, respectively. These findings suggest that amastigote invasion also involves host cell surface heparan sulfate proteoglycans. The knowledge of the mechanism triggered by heparan sulfate-binding T. cruzi proteins may provide new potential candidates for Chagas disease therapy.

Heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes.

We have examined the heparin binding proteins from Leishmania (Viannia) braziliensis promastigotes (HBP-Lb) by chromatography assays. The proposed strategy to isolate an enriched fraction of the HBP-Lb consisted of an association of the Triton X-114 method with affinity chromatography in heparin-Sepharose 4B column. SDS-PAGE analysis of the eluted proteins showed two main protein bands (65.0 and 54.5 kDa), while a single protein band was observed in native electrophoresis gel. The hemagglutination property of HBP-Lb over rabbit erythrocytes was confirmed up to 6.3+/-0.5 microg of protein mL(-1). Additionally, we have assayed the potential of HBP-Lb labeled with sulfo-NHS-LC-biotin in binding to nitrocellulose-immobilized gut proteins extracted of Lutzomyia intermedia and Lutzomyia whitmani. The results indicated a similar profile of five ligands (67.0, 62.1, 59.5, 56.0 and 47.5 kDa) in both studied Lutzomyia species. This is the first direct description of this class of protein in L. (V.) braziliensis with a suggestion of its biological activity in the interaction of Leishmania with Lutzomyia gut cells, which maybe a crucial step during this parasite's life cycle.

Disarray of sarcomeric alpha-actinin in cardiomyocytes infected by Trypanosoma cruzi.

Infection with Trypanosoma cruzi causes acute myocarditis and chronic cardiomyopathy. Remarkable changes have been demonstrated in the structure and physiology of cardiomyocytes during infection by this parasite that may contribute to the cardiac dysfunction observed in Chagas' disease. We have investigated the expression of alpha-actinin, an actin-binding protein that plays a key role in the formation and maintenance of Z-lines, during the T. cruzi-cardiomyocyte interaction in vitro. Immunolocalization of alpha-actinin in control cardiomyocytes demonstrated a typical periodicity in the Z line of cardiac myofibrils, as well as its distribution at focal adhesion sites and along the cell-cell junctions. No significant changes were observed in the localization of alpha-actinin after 24 h of infection. In contrast, depletion of sarcomeric distribution of alpha-actinin occurred after 72 h in T. cruzi-infected cardiomyocytes, while no change occurred at focal adhesion contacts. Biochemical assays demonstrated a reduction of 46% and 32% in the expression of alpha-actinin after 24 h and 72 h of infection, respectively. Intracellular parasites were also stained with an anti-alpha-actinin antibody that recognized a protein of 78 kDa by Western blot. Taken together, our data demonstrate a degeneration of the myofibrils in cardiomyocytes induced by T. cruzi infection, rather than a disassembly of the I bands within sarcomeres.

Trypanosoma cruzi-cardiomyocyte interaction: role of fibronectin in the recognition process and extracellular matrix expression in vitro and in vivo.

We investigated the involvement of fibronectin (FN) in Trypanosoma cruzi-cardiomyocyte invasion and the extracellular matrix (ECM) components expression during T. cruzi infection in vivo and in vitro. Treatment of trypomastigotes with FN or a synthetic peptide (MRGDS) prior to cardiomyocyte interaction reduced T. cruzi infection, indicating that FN mediates the parasite invasion through its RGD sequence. In murine experimental Chagas' disease, an enhancement of the ECM components was detected in the myocardium during the late acute infection, coinciding with inflammatory infiltrates accumulation. In contrast, highly infected cardiomyocytes displayed a reduction in FN expression in vitro, while laminin spatial distribution was altered. Although it has been demonstrated that cardiomyocytes are able to synthesize cytokines upon T. cruzi infection, our data suggest that matrix remodeling is dependent on cytokines secreted by inflammatory cells recruited in immune response.

Trypanosoma cruzi Infection Impairs the Endocytosis of Zymosan A by Cardiomyocytes.

OBJECTIVE: We have previously reported that mannose receptors participate and are regulated during Trypanosoma cruzi cardiomyocyte (CM) infection. Our present aim is to characterize the endocytosis of mannosylated ligands like zymosan A (Zy) in uninfected and T. cruzi-infected CM. METHODS: CM infected or not by T. cruzi were incubated with Zy for different periods of time and their internalization was analyzed at light microscopy level. Fluorescent approaches were performed by treating Zy with concanavalin-A-TRITC and washing it exhaustively prior to incubation with CM. The cultures were further stained with phalloidin-FITC and DAPI for actin and DNA visualization, respectively. RESULTS: CM internalized Zy particles in a time-dependent fashion. The ligand specificity was confirmed by the addition of mannan, which efficiently blocked the Zy endocytosis. Designed fluorescent approaches extended and confirmed the Zy internalization by striated cells. Infected cultures displayed impairment in Zy endocytosis, which seems to be directly related to host infection rates. CONCLUSIONS: Altogether, our results show the ability of CM to ingest large particles such as the mannosylated ligand Zy. During their infection with T. cruzi, there is a loss in Zy internalization possibly due to the negative modulation of mannose receptors.

Increased Trypanosoma cruzi invasion and heart fibrosis associated with high transforming growth factor beta levels in mice deficient in alpha(2)-macroglobulin.

Trypanosoma cruzi proteinases are involved in host cell invasion in human patients and in mouse models. In mice, murine alpha(2)-macroglobulin (MAM) and murinoglobulin are circulating plasma proteinase inhibitors that also have important roles in inflammation and immune modulation. To define their role in experimental Chagas disease, we investigated the susceptibility to T. cruzi infection of mice that are deficient only in alpha2-macroglobulins (AM-KO) or in both MAM and monomeric murinoglobulin-1 (MM-KO), relative to the wild type (WT). Despite the high parasite load, parasitemia was lower in AM-KO and MM-KO mice than in WT mice. Nevertheless, we observed a significantly higher parasite load in the hearts of AM-KO and MM-KO mice, i.e., more amastigote nests and inflammatory infiltrates than in WT mice. This result demonstrates a protective role for MAM in the acute phase of murine T. cruzi infection. We further demonstrated in vitro that human alpha2-macroglobulins altered the trypomastigote morphology and motility in a dose-dependent way, and that also impaired T. cruzi invasion in cardiomyocytes. Finally, we demonstrated that the levels of transforming growth factor beta in AM-KO mice increased significantly in the third week postinfection, concomitant with high amastigote burden and important fibrosis. Combined, these in vivo and in vitro findings demonstrate that the MAM contribute to the resistance of mice to acute myocarditis induced by experimental T. cruzi infection.