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In Brazil, around 80% of snakebites are caused by snakes of the genus Bothrops. A three-dimensional culture model was standardized and used to perform treatments with Bothrops erythromelas venom (BeV) and its antivenom (AV). The MRC-5 and L929 cell lines were cultured at increasing cell densities. Morphometric parameters were evaluated through images obtained from an inverted microscope: solidity, circularity, and Feret diameter. L929 microtissues (MT) showed better morphometric data, and thus they were used for further analysis. MT viability was assessed using the acridine orange and ethidium bromide staining method, which showed viable cells in the MT on days 5, 7, and 10 of cultivation. Histochemical and histological analyses were performed, including hematoxylin/eosin staining, which showed a good structure of the spheroids. Alcian blue staining revealed the presence of acid proteoglycans. Immunohistochemical analysis with ki-67 showed different patterns of cell proliferation. The MT were also subjected to pharmacological tests using the BeV, in the presence or absence of its AV. The results showed that the venom was not cytotoxic, but it caused morphological changes. The MT showed cell detachment, losing their structure. The antivenom was able to partially prevent the venom activities.

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The objective of this study was to evaluate the effects of amino resin-treated soybean meal (SBM) on ruminal fermentation, nutrient digestion, and N partitioning. Treatments were: (1) untreated solvent-extracted SBM, (2) amino resin-treated SBM (AR-SBM), and (3) heat-treated SBM (HT-SBM). The experimental design was arranged as a replicated 3 × 3 Latin square with 6 fermenters in a dual-flow continuous culture system. Treatments were randomly assigned to fermenters within a Latin square for each period. Each fermenter was fed 106 g/d of diet DM equally distributed in 2 feeding times daily at 0800 and 1800. Diets were formulated to contain 16% CP, 30% NDF, and 30% starch across treatments. The experiment consisted of 3 experimental periods, each lasting for 10 d. The first 7 d of each period were considered adaptation, and the last 3 d were used for sampling and data collection. On d 8 and 9, samples were collected for analysis of diurnal variation in concentrations of NH3-N, pH, and VFA during the first 8 h after feeding. On d 8, 9, and 10, samples were collected from the liquid and solid effluents accumulated over 24 h for analysis of daily averages of NH3-N and VFA pools, and true ruminal digestibility estimates. Data were analyzed using the MIXED procedure of SAS and significance was declared when P ≤ 0.05. The model included the fixed effect of treatment and random effects of square, period, and fermenter within square, while time and interaction treatment × time were included for analyses of diurnal variation, with time as repeated measures. Compared with SBM, the cultured ruminal contents of AR-SBM and HT-SBM had lower NH3-N concentrations, indicating lower microbial fermentation of protein. Molar proportions of isovalerate and isobutyrate were greater in SBM than AR-SBM and HT-SBM, with greater molar proportion of isobutyrate for SBM particularly during the first 2 h after feeding. Flow of NH3-N was greater for SBM compared with AR-SBM and HT-SBM, whereas NAN flow, bacterial N flow, and N efficiency were greater for AR-SBM and HT-SBM compared with SBM. Our results indicate that both the amino resin and heat treatments of SBM allow for similar decrease in microbial degradation of CP without limiting microbial protein synthesis in diets with 16% CP. Amino resin treatment may be effective in reducing microbial fermentation of protein in the rumen without adverse effects on digestibility or fermentation parameters as compared with SBM.

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Rumen-protected choline (RPC) promotes benefits in milk production, immunity, and health in dairy cows by optimizing lipid metabolism during transition period management and early lactation. However, the RPC success in dairy cows depends on choline bioavailability, which is affected by the type of protection used in rumen-protected choline. Therefore, our objectives were to determine the effects of a novel RPC on dry matter intake (DMI), identify markers of metabolism and immunity, and evaluate lactation performance. Dry Holstein (n = 48) cows at 245 ± 3 d of gestation were blocked by parity and assigned to control or RPC treatment within each block. Cows enrolled in the RPC treatment received 15 g/d of CholiGEM (Kemin Industries, Cavriago RE, Italy) from 21 d prepartum and 30 g/d of CholiGEM from calving to 21 d postpartum. During the transition period, DMI was measured daily, and blood was sampled weekly for energy-related metabolites such as ß-hydroxybutyrate (BHB), glucose, and nonesterified fatty acids (NEFA), as well as immune function markers such as haptoglobin (Hp) and lipopolysaccharide-binding protein (LPB). Vaginal discharge samples were collected at the calving and 7 d postpartum and stored in microcentrifuge tubes at -80°C until 16S rRNA sequencing. The main responses of body condition score, body weight, DMI, milk yield, milk components, and immune function markers were analyzed using the GLIMMIX procedure of SAS with the effects of treatment, time, parity, and relevant covariates added to the models. The relative abundance of microbiome α-diversity was evaluated by 3 indexes (Chao1, Shannon, and Simpson) and ß-diversity by principal coordinate analysis and permutational multivariate ANOVA. We found no differences in DMI in the pre- and postpartum periods. Cows fed RPC increased the yields of energy- and 3.5% fat-corrected milk and fat yield in primiparous and multiparous cows, with an interaction between treatment and parity for these lactation variables. However, we found no differences in milk protein and lactose up to 150 DIM between treatments. Glucose, NEFA, and BHB had no differences between the treatments. However, RPC decreased BHB numerically (control = 1.07 ± 0.13 vs. RPC = 0.63 ± 0.13) in multiparous on the third week postpartum and tended to reduce the incidence of subclinical ketosis (12.7% vs. 4.2%). No effects for Hp and LPB were found in cows fed RPC. Chao1, Shannon, and Simpson indexes were lower at calving in the RPC treatment than in the Control. However, no differences were found 7 d later for Chao1, Shannon, and Simpson indexes. The vaginal discharge microbiome was altered in cows fed RPC at 7 d postpartum. Fusobacterium, a common pathogen associated with metritis, was reduced in cows fed RPC. Rumen-protected choline enhanced lactation performance and health and altered the vaginal discharge microbiome which is a potential proxy for uterine healthy in dairy cows. The current study's findings corroborate that RPC is a tool to support adaptation to lactation and shed light on opportunities for further research in reproductive health.

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Our objective was to evaluate the effects of unprotected choline chloride (Cho) on the ruminal microbiome at 2 dietary neutral detergent fiber (NDF) concentrations. We hypothesized that the effects of Cho on ruminal bacterial populations would depend on NDF. Eight dual-flow continuous-culture fermentors were arranged in a duplicated 4 × 4 Latin square as a 2 × 2 factorial with the following treatments: (1) 30% NDF-control (30% NDF diet, no supplemental choline); (2) 30% NDF-Cho (30% NDF diet plus 1.9 g of choline ion per kg of dry matter); (3) 40% NDF-control (40% NDF diet, no supplemental choline); and (4) 40% NDF-Cho (40% NDF diet plus 1.9 g of choline ion per kg of dry matter). We did 4 fermentation periods of 10 d each and used the last 3 d for collection of samples of solid and liquid digesta effluents for DNA extraction. Overall, 32 solid and 32 liquid samples were analyzed by amplification of the V4 variable region of bacterial 16S rRNA. Data were analyzed with R (R Project for Statistical Computing) and SAS (SAS Institute Inc.) to determine effects of Cho, NDF, and NDF × Cho on taxa relative abundance. The correlation of propionate molar proportion with taxa relative abundance was also analyzed. At the phylum level, relative abundance of Firmicutes in the liquid fraction tended to be greater when Cho was supplemented with a 30% NDF diet. At the order level, Cho increased Coriobacteriales in solid fraction and decreased Fibrobacterales in liquid fraction. Moreover, Cho decreased abundance of Clostridiales and increased Selenomonadales in the solid fraction, only with the 30% NDF diet. For genera, lower abundance of Pseudobutyrivibrio resulted from Cho in solid and liquid fractions. Greater abundance of Succinivibrio in solid and Selenomonas and Selenomonas 1 in liquid resulted from Cho with the 30% NDF diet. Propionate molar proportion was positively correlated with relative abundance of order Selenomonadales in solid and liquid fractions, and with genus Succinivibrio in solid and genera Selenomonas and Selenomonas 1 in liquid. Our results indicate that Cho primarily decreases abundance of bacteria involved in fiber degradation and increases abundance of bacteria mainly involved in nonstructural carbohydrate degradation and synthesis of propionate, particularly when a diet with 30% NDF is provided.

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BACKGROUND: Scorpion venom causes renal injury and affects vascular ion-channels function. Centruroides margaritatus scorpion is found in Colombia and is frequently the cause of envenomation accidents; however, its renal impact has never been investigated. OBJECTIVE: To evaluate the effects of C. margaritatus venom (CmV) on renal parameters using isolated rat kidney and renal cell culture models. METHODS: Wistar rats (n = 5, weighing 240-300 g) were first perfused with Krebs-Henseleit solution containing 6 g 100 mL-1 bovine serum albumin. After 30 minutes, the kidneys were perfused with CmV to a final concentration of 10 µgmL-1; evaluation was performed by measuring Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Moreover, kidney histological analyses and cell cytotoxicity in renal tubule epithelial cells (MDCK) and proximal tubular cells (LLC-MK2) were assessed. RESULTS: CmV increased PP and RVR 60 min after perfusion. On the other hand, UF, GFR, and the percentages of sodium, potassium and chloride tubular transport decreased after experimental envenomation. UF dropped after 120 min, while GFR and percentage of electrolyte tubular transport diminished after 60, 90 and 120 min. CmV was not toxic to MDCK cell line but reduced the viability of LLC-MK2 cells at concentrations ranging from 6.25 to 200 µgmL-1. Histological analyses disclosed hydropic degeneration, edema, and protein deposits. Flow cytometry disclosed that cell death occurred predominantly by necrosis. CONCLUSION: Our results suggest that C. margaritatus venom can trigger renal impairment, mainly in the proximal kidney tubule.

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BACKGROUND: Envenomation caused by Bothrops alternatus is common in Southern Brazil. Acute Kidney Injury occurs after Bothrops snakebite and more information is necessaryrequired to understand its mechanism. OBJECTIVE: The objective was to evaluate the effect of Bothrops alternatus venom (BaV) on renal cells and rat isolated kidney function. METHODS: Wistar rats (n = 6, weighing 260-320 g) were perfused with a Krebs-Henseleit solution containing 6 g 100 mL-1 of bovine serum albumin. After 30 minutes, the kidneys were perfused with BaV to a final concentration of 1 and 3 µgmL-1; and subsequently were evaluated for Perfusion Pressure (PP), Renal Vascular Resistance (RVR), Urinary Flow (UF), Glomerular Filtration Rate (GFR), and percentage of electrolyte tubular transport. Renal histological analysis, cytokine release, oxidative stress and cytotoxicity in renal proximal tubular cells were assessed. RESULTS: BaV reduced PP, RVR, GFR, UF, total and proximal sodium transport (%TNa+), and chloride (%TCl-) in the isolated kidney perfusion model. Histological analysis of perfused kidneys disclosed the presence of proteinaceous material in the glomeruli and renal tubules, vacuolar tubular epithelial cell degeneration, Bowman's capsule degeneration, swelling of glomerular epithelial cells, glomerular atrophy and degeneration, and the presence of intratubular protein. Cytokine release (TNF-α, IL-1ß, IL-10) and oxidative stress were increased in the kidneys. The viability of LLC-MK2 cells (IC50: 221.3 µg/mL) was decreased by BaV and necrosis was involved in cell death. CONCLUSION: These findings indicate that BaV modifies functional parameters in an isolated perfused kidney model and has cytotoxic effects on renal lineage cells.

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OBJECTIVES: To examine the association of sedentary behavior (SB) with metabolic syndrome (MetS) in adults, as well as to identify the impact of physical inactivity and economic condition on such association. STUDY DESIGN: This is a cross-sectional study. METHODS: This study was conducted in the city of Bauru, São Paulo State, Brazil (n = 970). Television (TV) viewing and physical activity (PA) were assessed by questionnaire. MetS was assessed via medical records. Descriptive statistics and binary logistic regression were used in data analyses. RESULTS: Participants with moderate TV viewing were associated with MetS when compared with low TV viewing group, even after adjustments for age, sex, economic status, smoking, and PA (odds ratio [OR] = 1.49 [95% confidence interval {CI}: 1.01-2.20]). The fully adjusted model showed an association between high exposure to TV viewing with MetS (OR = 1.77 [95%CI: 1.11-2.82]). The combination of physical inactivity and high TV viewing boosted the likelihood of having MetS (OR = 1.89 [95%CI: 1.08-3.29]). CONCLUSION: The results of the present study suggest that moderate and high TV viewing time is associated with the presence of MetS, mainly in insufficiently active adults and those living in lower economic condition.

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Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3′,4′-OH flavonoids are known inhibitors of phospholipases A2.

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Bredemeyera floribunda roots are popularly used to treat snakebites in the semiarid region of Northeast Brazil, and previous studies indicate the anti-ophidian actions of triterpenoid saponins found in its roots. To assess B. floribunda root extract (BFRE) activity against the effects of Bothrops jararacussu venom (BjuV), antiphospholipasic, antiproteolytic, antihemorrhagic, antinecrotic, and anti-edematogenic activities were investigated in mice. Phytochemical analysis revealed the presence of saponins, flavonoids, and sugars, with rutin and saccharose being the major constituents of BFRE. Acute toxicity was determined and BFRE was nontoxic to mice. Phospholipase A2 and proteolytic activities induced by BjuV were inhibited in vitro by BFRE at all concentrations tested herein. BFRE (150 mg/kg) inhibited paw edema induced by BjuV (50 µg/animal), reducing total edema calculated by area under the curve, but carrageenan-induced paw edema was unchanged. Hemorrhagic and necrotizing actions of BjuV (50 µg/animal) were considerably decreased by BFRE treatment. Thus, BFRE blocked the toxic actions of B. jararacussu venom despite having no anti-inflammatory activity, which points to a direct inhibition of venom's toxins, as demonstrated in the in vitro assays. The larger amounts of rutin found in BFRE may play a role in this inhibition, since 3',4'-OH flavonoids are known inhibitors of phospholipases A2.

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The purpose of this study was to investigate the association between television (TV) viewing and all-cause mortality among Brazilian adults after 6 years of follow-up. This longitudinal study started in 2010 in the city of Bauru, SP, Brazil, and involved 970 adults aged ≥50 years. Mortality was reported by relatives and confirmed in medical records of the Brazilian National Health System. Physical activity (PA) and TV viewing were assessed by the Baecke questionnaire. Health status, sociodemographic and behavioral covariates were considered as potential confounders. After 6 years of follow-up, 89 deaths were registered (9.2% [95% CI=7.4%-11%]). Type 2 diabetes mellitus was associated with higher risk of mortality (P-value=.012). Deaths correlated significantly with age (ρ=.188; P-value=.001), overall PA score (ρ=-.128; P-value=.001) and TV viewing (ρ=.086; P-value=.007). Lower percentage of participants reported TV viewing time as often (16%) and very often (5.7%), but there was an association between higher TV viewing time ("often" and "very often" grouped together) and increased mortality after 6 years of follow-up (P-value=.006). The higher TV viewing time was associated with a 44.7% increase in all-cause mortality (HR=1.447 [1.019-2.055]), independently of other potential confounders. In conclusion, the findings from this cohort study identified increased risk of mortality among adults with higher TV viewing time, independently of PA and other variables.

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