A new 3D model of L929 fibroblasts microtissues uncovers the effects of Bothrops erythromelas venom and its antivenom.

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.

ACE gene polymorphisms (rs4340) II and DI are more responsive to the ergogenic effect of caffeine than DD on aerobic power, heart rate, and perceived exertion in a homogeneous Brazilian group of adolescent athletes.

The purpose of this study was to verify the association between angiotensin-converting enzyme (ACE) genotypes DD, DI, and II and caffeine (CAF) ingestion on endurance performance, heart rate, ratio of perceived exertion (RPE), and habitual caffeine intake (HCI) of adolescent athletes. Seventy-four male adolescent athletes (age: DD=16±1.7; DI=16±2.0; II=15±1.7 years) ingested CAF (6 mg/kg) or placebo (PLA) one hour before performing the Yo-Yo Intermittent Recovery level 1 (Yo-Yo IR1) test. No difference was found among groups for HCI. However, CAF increased the maximal distance covered and VO2max in DI and II genotype carriers compared to PLA (DD: Δ=31 m and 0.3 mL·kg-1·min-1; DI: Δ=286 m and 1.1 mL·kg-1·min-1; II: Δ=160 m and 1.4 mL·kg-1·min-1). Heart rate of DI and II genotype carriers increased with CAF compared to PLA, while RPE was higher in the II and lower in the DD genotypes. The correlations between HCI and maximal distance covered or VO2max were significant in the II genotype carriers with CAF. CAF increased endurance capacity, heart rate, and RPE in adolescent athletes with allele I, while endurance performance and aerobic power had a positive correlation to HCI in the II genotype group. These findings suggested that DD genotype were less responsive to CAF and that genetic variations should be taken into account when using CAF supplementation to enhance exercise performance.

Trastuzumab deruxtecan in human epidermal growth factor receptor 2-positive breast cancer brain metastases: a systematic review and meta-analysis.

BACKGROUND: Trastuzumab deruxtecan (T-DXd) has shown promising results in patients with breast cancer brain metastases (BCBMs). We conducted a systematic review and meta-analysis to evaluate the effectiveness and safety of T-DXd in the human epidermal growth factor receptor 2 (HER2)-positive BCBM population. PATIENTS AND METHODS: We searched PubMed, Embase, and Cochrane Library databases as well as American Society of Clinical Oncology (ASCO), European Society for Medical Oncology (ESMO), and San Antonio Breast Cancer Symposium (SABCS) websites for clinical trials (CTs) and observational studies evaluating T-DXd in patients with HER2-positive BCBM. Heterogeneity was assessed with I2 statistics. Random effects models were used for all statistical analyses, which were carried out using R software (version 4.2.2). RESULTS: Ten studies were included, six CTs (n = 189) and four observational studies (n = 130), with a total of 319 patients. The median progression-free survival was 15 months [95% confidence interval (CI) 13.9-16.1 months]. The objective response rate (ORR) was 61% (95% CI 52% to 70%), and the intracranial (IC)-ORR was 61% (95% CI 54% to 69%). No significant differences in ORR and IC-ORR were observed between CTs and observational studies (P = 0.31 and 0.58, respectively). The clinical benefit rate (CBR) was 80% (95% CI 52% to 94%), and the IC-CBR was 70% (95% CI 54% to 82%). The ORR was 68% (95% CI 57% to 77%) in the subgroup of patients with stable BMs and 60% (95% CI 48%-72%) in patients with active BM, with no significant difference between groups (P = 0.35). CONCLUSIONS: Our systematic review and meta-analysis supports the IC activity of T-DXd in patients with stable BM and active BM. TRIAL REGISTRATION: International Prospective Register of Systematic Reviews (PROSPERO) under the protocol number CRD42023422589.

ACE gene polymorphisms (rs4340) II and DI are more responsive to the ergogenic effect of caffeine than DD on aerobic power, heart rate, and perceived exertion in a homogeneous Brazilian group of adolescent athletes

The purpose of this study was to verify the association between angiotensin-converting enzyme (ACE) genotypes DD, DI, and II and caffeine (CAF) ingestion on endurance performance, heart rate, ratio of perceived exertion (RPE), and habitual caffeine intake (HCI) of adolescent athletes. Seventy-four male adolescent athletes (age: DD=16±1.7; DI=16±2.0; II=15±1.7 years) ingested CAF (6 mg/kg) or placebo (PLA) one hour before performing the Yo-Yo Intermittent Recovery level 1 (Yo-Yo IR1) test. No difference was found among groups for HCI. However, CAF increased the maximal distance covered and VO2max in DI and II genotype carriers compared to PLA (DD: Δ=31 m and 0.3 mL·kg-1·min-1; DI: Δ=286 m and 1.1 mL·kg-1·min-1; II: Δ=160 m and 1.4 mL·kg-1·min-1). Heart rate of DI and II genotype carriers increased with CAF compared to PLA, while RPE was higher in the II and lower in the DD genotypes. The correlations between HCI and maximal distance covered or VO2max were significant in the II genotype carriers with CAF. CAF increased endurance capacity, heart rate, and RPE in adolescent athletes with allele I, while endurance performance and aerobic power had a positive correlation to HCI in the II genotype group. These findings suggested that DD genotype were less responsive to CAF and that genetic variations should be taken into account when using CAF supplementation to enhance exercise performance.

Enrichment diets of pigs with oil blends and its effects on performance, carcass characteristics and fatty acid profile / Dietas enriquecidas com blends de óleos para suínos em terminação e seus efeitos sobre o desempenho, as características de carcaça e o perfil de ácidos graxos

The addition of different oil blends in the feed of finishing pigs was evaluated. Twenty-four castrated male finishing pigs were used in a randomized block design containing four treatments and six replicates. The treatments consisted of: Reference ration (RR) - 100% soybean oil feed; and the combination of the different oils: Blend1 - 50.0% soybean oil (SO), 25.0% flaxseed oil (FO), 12.5% olive oil (OO) and 12.5% canola oil (CO); Blend2 - 25.0% SO, 50.0% FO, 12.5% OO and 12.5% CO; and Blend3 - 25.0% SO, 12.5% FO, 12.5% OO and 50.0% CO. The performance, quantitative and qualitative carcass parameters, fatty acids profile and economic feasibility of the diets were evaluated. The use of blends in the diets did not influence the performance or carcass quality, but increased marbling and carcass yield. The fatty acid profile of the loin presented greater amounts of stearic acid in Blend3 and higher percentage of unsaturated fatty acids in animals fed with Blend1. The fatty tissue presented greater amounts of myristic acid in Blend1 and oleic acid in Blend3. The reference ration was the most economic. The Blends did not affect performance or carcass characteristics and improved the fatty acid profile.(AU)

Foi avaliada a utilização de diferentes blends de óleo em dietas de suínos em terminação. Foram utilizados 24 suínos, machos, castrados, distribuídos em delineamento de blocos ao acaso, com quatro tratamentos e seis repetições. Os tratamentos consistiram de: ração referência (RR) - 100% de ração com utilização de óleo de soja; e a combinação de diferentes óleos: Blend1 - 50,0% de óleo de soja (OS), 25,0% de óleo de linhaça (OL), 12,5% de óleo de oliva (OO) e 12,5% de óleo de canola (OC); Blend2 - 25,0% OS; 50,0% OL; 12,5% OO e 12,5% OC; e Blend3 - 25,0% OS; 12,5% OL; 12,5% OO e 50,0% OC. Foram avaliados os parâmetros de desempenho, a qualidade de carcaça, o perfil de ácidos graxos e a viabilidade econômica. O uso de blends nas dietas não influenciou o desempenho ou a qualidade da carcaça, mas aumentou o marmoreio e o rendimento de carcaça. O perfil de ácidos graxos do lombo apresentou maiores quantidades de ácido esteárico com a utilização do Blend3 e maior porcentagem de ácidos graxos insaturados nos animais alimentados com o Blend1. O tecido adiposo apresentou maiores quantidades de ácido mirístico quando se forneceu o Blend1 e de ácido oleico com o Blend3. A ração testemunha foi a mais econômica. As misturas não afetaram o desempenho e as características de carcaça e melhoraram o perfil de ácidos graxos da carne.(AU)

Enrichment diets of pigs with oil blends and its effects on performance, carcass characteristics and fatty acid profile / Dietas enriquecidas com blends de óleos para suínos em terminação e seus efeitos sobre o desempenho, as características de carcaça e o perfil de ácidos graxos

The addition of different oil blends in the feed of finishing pigs was evaluated. Twenty-four castrated male finishing pigs were used in a randomized block design containing four treatments and six replicates. The treatments consisted of: Reference ration (RR) - 100% soybean oil feed; and the combination of the different oils: Blend1 - 50.0% soybean oil (SO), 25.0% flaxseed oil (FO), 12.5% olive oil (OO) and 12.5% canola oil (CO); Blend2 - 25.0% SO, 50.0% FO, 12.5% OO and 12.5% CO; and Blend3 - 25.0% SO, 12.5% FO, 12.5% OO and 50.0% CO. The performance, quantitative and qualitative carcass parameters, fatty acids profile and economic feasibility of the diets were evaluated. The use of blends in the diets did not influence the performance or carcass quality, but increased marbling and carcass yield. The fatty acid profile of the loin presented greater amounts of stearic acid in Blend3 and higher percentage of unsaturated fatty acids in animals fed with Blend1. The fatty tissue presented greater amounts of myristic acid in Blend1 and oleic acid in Blend3. The reference ration was the most economic. The Blends did not affect performance or carcass characteristics and improved the fatty acid profile.(AU)

Foi avaliada a utilização de diferentes blends de óleo em dietas de suínos em terminação. Foram utilizados 24 suínos, machos, castrados, distribuídos em delineamento de blocos ao acaso, com quatro tratamentos e seis repetições. Os tratamentos consistiram de: ração referência (RR) - 100% de ração com utilização de óleo de soja; e a combinação de diferentes óleos: Blend1 - 50,0% de óleo de soja (OS), 25,0% de óleo de linhaça (OL), 12,5% de óleo de oliva (OO) e 12,5% de óleo de canola (OC); Blend2 - 25,0% OS; 50,0% OL; 12,5% OO e 12,5% OC; e Blend3 - 25,0% OS; 12,5% OL; 12,5% OO e 50,0% OC. Foram avaliados os parâmetros de desempenho, a qualidade de carcaça, o perfil de ácidos graxos e a viabilidade econômica. O uso de blends nas dietas não influenciou o desempenho ou a qualidade da carcaça, mas aumentou o marmoreio e o rendimento de carcaça. O perfil de ácidos graxos do lombo apresentou maiores quantidades de ácido esteárico com a utilização do Blend3 e maior porcentagem de ácidos graxos insaturados nos animais alimentados com o Blend1. O tecido adiposo apresentou maiores quantidades de ácido mirístico quando se forneceu o Blend1 e de ácido oleico com o Blend3. A ração testemunha foi a mais econômica. As misturas não afetaram o desempenho e as características de carcaça e melhoraram o perfil de ácidos graxos da carne.(AU)

Bothrops alternatus Snake Venom Induces Cytokine Expression and Oxidative Stress on Renal Function.

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.

Rotavirus analyses by SYBR Green real-time PCR and microbiological contamination in bivalves cultivated in coastal water of Amazonian Brazil.

The contamination of mussels and oysters by viruses and bacteria is often associated with water contamination and gastroenteritis in humans. The present study evaluated viral and bacterial contamination in 380 samples, from nine mollusk-producing regions in coastal water north of the Brazilian Amazon. Rotavirus contamination was studied for groups A to H, using a two-step SYBR Green RT-qPCR (quantitative reverse transcription polymerase chain reaction), and bacterial families Enterobacteriaceae, Vibrionaceae, and Aeromonadaceae by classical and molecular methods. From the 19 pools analyzed, 26.3% (5/19) were positive for group A Rotavirus, I2 genotype for VP6 region, without amplifications for groups B-H. Bacteriological analysis identified Escherichia coli isolates in 89.5% (17/19) with identification of atypical enteropathogenic E. coli aEPEC in 10.5% (2/19), Salmonella (Groups C1 and G) (10.5%, 2/19), Vibrio alginolyticus (57.9%, 11/19) V. parahaemolyticus (63.2%, 12/19), V. fluvialis (42.1%, 8/19), V. vulnificus (10.5%, 2/19), V. cholerae non-O1, non O139(10.5%, 2/19) and Aeromonas salmonicida (52.6%, 10/19). All the samples investigated presented some level of contamination by enterobacteria, rotavirus, or both, and these results may reflect the level of contamination in the Northern Amazon Region, due to the natural maintenance of some of these agents or by the proximity with human populations and their sewer.

Detection and function of lipopolysaccharide and its purified lipid A after treatment with auxiliary chemical substances and calcium hydroxide dressings used in root canal treatment.

AIM: To investigate the influence of auxiliary chemical substances (ACSs) and calcium hydroxide [Ca(OH)2 ] dressings on lipopolysaccharides (LPS)/lipid A detection and its functional ability in activating Toll-like receptor 4 (TLR4). METHODOLOGY: Fusobacterium nucleatum pellets were exposed to antimicrobial agents as following: (i) ACS: 5.25%, 2.5% and 1% sodium hypochlorite solutions (NaOCl), 2% chlorhexidine (CHX) (gel and solution) and 17% ethylenediaminetetraacetic acid (EDTA); (ii) intracanal medicament: Ca(OH)2 paste for various periods (1 h, 24 h, 7 days, 14 days and 30 days); (iii) combination of substances: (a) 2.5% NaOCl (1 h), followed by 17% EDTA (3 min) and Ca(OH)2 (7 days); (b) 2% CHX (1 h), afterwards, 17% EDTA (3 min) followed by Ca(OH)2 (7 days). Saline solution was the control. Samples were submitted to LPS isolation and lipid A purification. Lipid A peaks were assessed by matrix-assisted laser desorption ionization time-of-flight mass spectrom (MALDI-TOF MS) whilst LPS bands by SDS-PAGE separation and silver staining. TLR4 activation determined LPS function activities. Statistical comparisons were carried out using one-way anova with Tukey-Kramer post-hoc tests at the 5% significance level. RESULTS: Matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of control lipid A demonstrated the ion cluster at mass/charge (m/z) 1882 and an intense band in SDS-PAGE followed by silver staining of control LPS. In parallel, LPS control induced a robust TLR4 activation when compared to ACS (P ≤ .001). 5.25% NaOCl treatment led to the absence of lipid A peaks and LPS bands, whilst no changes occurred to lipid A/LPS after treatment with others ACS. Concomitantly, 5.25% NaOCl-treated LPS did not activate TLR4 (P < .0001). As for Ca(OH)2 , lipid A was not detected by MALDI-TOF nor by gel electrophoresis within 24 h. LPS treated with Ca(OH)2 was a weak TLR4 activator (P < .0001). From 24 h onwards, no significant differences were found amongst the time periods tested (P > 0.05). The addition of Ca(OH)2 for 7 days to cells treated either with 2.5% NaOCl or 2% CHX led to the absence of lipid A peaks and LPS bands, leading to a lower activation of TLR4. CONCLUSION: 5.25% NaOCl and Ca(OH)2 dressings from 24 h onwards were able to induce both, loss of lipid A peaks and no detection of LPS bands, rendering a diminished immunostimulatory activity through TLR4.

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.(AU)

Investigation of enteric viruses in the feces of neotropical migratory birds captured on the coast of the State of Pará, Brazil

Migratory birds can become long-distance vectors for a wide range of microorganisms and can cause human disease, being the Brazilian coast a gateway for northern migratory birds. These animals are considered natural reservoirs of different viruses that cause important diseases, being relevant research of viral pathogens in migratory birds to epidemiology surveillance. The objective of the study was to investigate the presence of avian rotavirus (AvRV), avian reovirus (ARV) and picobirnavirus (PBV) in Neotropical migratory birds captured on the coast of Brazil. A total of 23 individual fecal samples of the migratory birds species Calidris pusilla (20 birds), Numenius phaeopus (1 bird) and Charadrius semipalmatus (2 birds) were collected. Fecal suspensions were prepared from the collected samples for subsequent extraction of double-stranded RNA (dsRNA), which was subjected to polyacrylamide gel electrophoresis (PAGE) and reverse transcription polymerase chain reaction (RT-PCR). The electrophoretic profiles were not detected by PAGE, and the amplification for the studied viruses PBV, ARV and AvRV (specie D, gene VP6 and NSP4) were negative. Positivity for AvRVD, VP7 gene was of 4.35% (1/23) for the migratory bird Calidris pusilla. After sequencing and building the tree of phylogenetic relationships avian Rotavirus Group D identified in this study was phylogenetically related and grouped into one branch, together to previously reported AvRVD from Brazil in chicken flocks with 99.8% nucleotide and 100% amino acid similarities.

Cryopreservation and characterization of canine preantral follicles.

The aim of this study was to define the population, morphological and ultrastructural characteristics of bitch preantral follicles (PAFs) and to compare the effects on the morphology of PAF of two cryopreservation techniques - slow freezing (SF) and vitrification (V) - of bitches' ovarian tissue. The average population (number per ovary) of PAFs was 48,541 ±â€¯18,366, where 94.25% were primordial (45,145 ±â€¯16,076). The average diameter of the primordial follicles was 27.5 ±â€¯4.2 µm. The overall percentage of morphologically normal PAFs was 93.66 ±â€¯6.81% for the control group, 86.16 ±â€¯11.05% after SF and 68.14 ±â€¯12.75% after V. The percentage of normal primordial follicles was 96.69 ±â€¯4.72% in control, 89.51 ±â€¯10.39% in SF and 75.32 ±â€¯9.23% in V. There was no significant difference in the overall percentage of normal PAFs among SF and the control. However, slow frozen follicles presented ultrastructural damage, while vitrified primordial and primary follicles were well preserved. In conclusion, although slow freezing seemed to be a good preserving method, vitrification was more effective than slow freezing in preserving the ultrastructure of primordial and primary follicles of bitches.

Does the Reciproc file remove root canal bacteria and endotoxins as effectively as multifile rotary systems?

AIM: To evaluate the effectiveness of Reciproc for the removal of cultivable bacteria and endotoxins from root canals in comparison with multifile rotary systems. METHODOLOGY: The root canals of forty human single-rooted mandibular pre-molars were contaminated with an Escherichia coli suspension for 21 days and randomly assigned to four groups according to the instrumentation system: GI - Reciproc (VDW); GII - Mtwo (VDW); GIII - ProTaper Universal (Dentsply Maillefer); and GIV -FKG Race(™) (FKG Dentaire) (n = 10 per group). Bacterial and endotoxin samples were taken with a sterile/apyrogenic paper point before (s1) and after instrumentation (s2). Culture techniques determined the colony-forming units (CFU) and the Limulus Amebocyte Lysate assay was used for endotoxin quantification. Results were submitted to paired t-test and anova. RESULTS: At s1, bacteria and endotoxins were recovered in 100% of the root canals investigated (40/40). After instrumentation, all systems were associated with a highly significant reduction of the bacterial load and endotoxin levels, respectively: GI - Reciproc (99.34% and 91.69%); GII - Mtwo (99.86% and 83.11%); GIII - ProTaper (99.93% and 78.56%) and GIV - FKG Race(™) (99.99% and 82.52%) (P < 0.001). No statistical difference were found amongst the instrumentation systems regarding bacteria and endotoxin removal (P > 0.01). CONCLUSION: The reciprocating single file, Reciproc, was as effective as the multifile rotary systems for the removal of bacteria and endotoxins from root canals.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food.

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.(AU)

Biofilm formation on polystyrene under different temperatures by antibiotic resistant Enterococcus faecalis and Enterococcus faecium isolated from food

The ability of antibiotic resistant E. faecalis and E. faecium isolated from food to form biofilm at different temperatures in the absence or presence of 0.75% glucose was evaluated. A synergistic effect on biofilm at 10 °C, 28 °C, 37 °C and 45 °C and glucose was observed for E. faecalis and E. faecium.

A validated method for the simultaneous quantitation of bioactive alkaloid markers in the leaf ethanolic extract of Cissampelos sympodialis Eichl.: a phenological variation study.

INTRODUCTION: The leaf hydroalcoholic extract of Cissampelos sympodialis Eichl. (Menispermaceae) has shown promising activity in different animal models of asthma. Several alkaloids have been identified in the extract, including warifteine and methylwarifteine (bisbenzylisoquinoline), as well as milonine (morphinandienone). OBJECTIVE: To develop and validate an analytical method for the simultaneous quantitation of the bioactive markers of C. sympodialis hydroalcoholic leaf extract and to apply the method to a seasonal (phenological) study of the concentration of the alkaloid markers. METHODOLOGY: The method used reversed phase high performance liquid chromatography with UV detection and calibration by standard addition. Separation was achieved using a C18-column (250 × 4.6 mm, 5 µm) and a mobile phase consisting of a mixture of 0.05% aqueous (Et)3NH2 (A):MeOH(B) in gradient mode at a flow rate of 1.0 mL/min. RESULTS: The method proved to be linear in the concentration range tested (2-100 µg/mL, r² > 0,99), precise (RSD ≤ 15%), accurate (85-115%), selective and robust. Detection limits for warifteine, methyl-warifteine and milonine were 0.39, 1.10 and 1.77 µg/mL respectively. The highest concentration of total alkaloids (determined as the sum of the three alkaloids) in the hydroalcoholic extract of the leaves was 2.9 ± 0.2 mg/g extract (n = 3), prior to fruit development. Both warifteine and methylwarifteine were detected in the total alkaloid fraction of the ripened fruits. CONCLUSION: The results demonstrated that significant variations in the concentration of the biomarkers occurred throughout the vegetative cycle. The lowest concentration of the alkaloids in the leaves coincided with their appearance in the ripened fruits.

DNA sequencing of 13 cytokine gene fragments of Aotus infulatus and Saimiri sciureus, two non-human primate models for malaria.

Aotus and Saimiri are non-human primate models recommended by the World Health Organization for experimental studies in malaria, especially for vaccine pre-clinical trials. However, research using these primates is hindered by the lack of specific reagents to evaluate immune responses to infection or vaccination. As a step toward developing molecular tools for cytokine expression studies in these species, primer pairs for 18 cytokine gene fragments were designed based on human DNA sequences and used to amplify the corresponding genes in Aotus infulatus and Saimiri sciureus genomic DNA samples. IFNγ, TNFα, LTA, IL2, IL3, IL4, IL5, IL6, IL10, IL12, IL13, CSF2 and TGFß2 gene fragments were amplified and sequenced. Primer pairs for IL8, IL17, IL18, IL27 and MIF failed to generate amplification products. When compared to the available corresponding human and non-human primate sequences, most--except IL3 and IL4--showed identity degrees above 90%. Small variations in sequence can help to explain the failure to amplify certain genes or the amplification only at lower annealing temperatures as compared to human DNA samples for several primer pairs. The sequences made available provide the basis for designing molecular tools such as primers for real time PCR specific for A. infulatus and/or S. sciureus. The nucleotide sequences reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned accession numbers DQ985386 to DQ985389, DQ989356 to DQ989369, FJ89020 to FJ89024, and FJ89029.

Morphometry, estimation and ultrastructure of ovarian preantral follicle population in queens.

The aim of the present study was to estimate the population and morphometrically and ultrastructurally characterize preantral follicles from queen ovaries. Ovaries from 5 queens were collected and processed for light and electron microscopy. A total of 190 preantral follicles (100 primordial, 60 primary and 30 secondary) were analyzed by light microscopy. The diameters of the follicle, oocyte and oocyte nucleus were taken and the number of granulosa cells was counted using a computer program. Queen ovaries presented 37,853 +/- 6,118 preantral follicles on average, with 87% primordial, 10.4% primary and 2.3% secondary follicles. Significant differences were observed in the 3 follicular classes in regard to follicular, oocyte and oocyte nucleus diameters and granulosa cell number (p < 0.05). In regard to ultrastructure, queen preantral follicles presented many unique characteristics, such as early zona pellucida formation in primary follicles and the organization of mitochondria and other organelles in conglomerates and cortical granules aligned at the peripheral zone in secondary follicles. In conclusion, this study described the morphometry and ultrastructure of queen preantral follicles and the preantral follicle population in the ovaries, establishing a pattern for the species and consequently allowing comparisons with other species.

Sexual dimorphism in the squirrel monkey, Saimiri sciureus (Linnaeus, 1758) and Saimiri ustus (I. Geoffroy, 1844) (Primates, Cebidae).

Causes and implications of sexual dimorphism have been studied in several different primates using a variety of morphological characters such as body weight, canine length, coat color and ornamentation. Here we describe a peculiar coat color characteristic in the squirrel monkey that is present only in adult females over five years old and which increases with age. Neither males nor young animals manifest this phenomenon, which is characterized by a spot of black hairs located anteriorly to the external ear (pinna). This characteristic could be used to discriminate adult females of Saimiri sciureus in the wild without the need of capture techniques.