Regional distribution and integrity of equine ovarian pre-antral follicles.

The goal of this study was to determine the distribution of pre-antral follicles in the ovarian parenchyma of mares. For Experiment 1, each ovary was cut longitudinally at the greater curvature, performing two hemiovaries. After that, six fragments from each hemiovary were obtained, resulting in 12 fragments, which were divided into the innermost region of the parenchyma, the middle region and the outermost region. All the three obtained sections were cut transversally to obtain two fragments from each one. For Experiment 2, each ovary also submitted to a longitudinal cut on the greater curvature, forming two hemiovaries. Each hemiovary was sectioned into four symmetrical fragments, resulting in eight fragments per ovary. The fragments were related as being near to or far from the ovulatory fossa. The fragments of both experiments were immediately fixed in Carnoy for 12 hr and kept in 70% ethanol for 24 hr. Follicles were classified according to the stages of development and for morphological integrity according to oocyte morphology and granulosa cells. After the histological assessment, a total of 1,130 follicles were visualized from Experiment 1, being 1,054 (93.3%) primordial follicles and 76 (4.7%) follicles in development. The innermost region had the highest percentage of pre-antral follicles compared to the other regions (p < .05). The middle and outermost regions showed higher percentages of intact primordial and developing follicles than the innermost region (p < .05). Considering Experiment 2, 938 follicles were found, being 894 (95.3%) primordial and 44 (4.7%) follicles in development. The region near the ovulatory fossa presented higher (58.7%; 551 of 938) follicular concentration compared to the region far from the ovulatory fossa (41.3%; 387 of 938; p < .05). As a conclusion, distribution of pre-antral follicles in the equine ovary has a specific pattern through the parenchyma. Also, the follicular integrity differed in the studied ovarian areas.

The development and integrity of equine pre-antral follicles cultured in vitro with follicle-stimulating hormone (FSH) supplementation.

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre-antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre-antral follicles, the groups with 100 ng/ml FSH of 2-days culture as well as 50, 100 and 200 ng/ml FSH of 6-days culture provided the best results. In conclusion, the in vitro culture of abattoir-derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.

Hypercholesterolemia and Ecto-enzymes of Purinergic System: Effects of Paullinia cupana.

Hypercholesterolemia is a metabolic disorder characterized by high levels of low-density lipoprotein and blood cholesterol, causing inflammatory lesion. Purinergic signaling modulates the inflammatory and immune responses through adenine nucleotides and nucleoside. Guaraná has hypocholesterolemic and antiinflammatory properties. Considering that there are few studies demonstrating the effects of guaraná powder on the metabolism of adenine nucleotides, we investigated its effects on the activity of ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) and ecto-adenosine deaminase activity in lymphocytes of rats with diet-induced hypercholesterolemia. The rats were divided into hypercholesterolemic and normal diet groups. Each group was subdivided by treatment: saline, guaraná powder 12.5, 25, or 50 mg/kg/day and caffeine concentration equivalent to highest dose of guaraná, fed orally for 30 days. An increase in adenosine triphosphate hydrolysis was observed in the lymphocytes of rats with hypercholesterolemia and treated with 25 or 50 mg/kg/day when compared with the other groups. The hypercholesterolemic group treated with the highest concentration of guaraná powder showed decreased ecto-adenosine deaminase activity compared with the normal diet groups. Guaraná was able to reduce the total cholesterol and low-density lipoprotein cholesterol to basal levels in hypercholesterolemic rats. High concentrations of guaraná associated with a hypercholesterolemic diet are likely to have contributed to the reduction of the inflammatory process.

Trypanocidal activity of free and nanoencapsulated curcumin on Trypanosoma evansi.

This study aimed to evaluate in vitro and in vivo trypanocidal activity of free and nanoencapsulated curcumin against Trypanosoma evansi. In vitro efficacy of free curcumin (CURC) and curcumin-loaded in lipid-core nanocapsules (C-LNCs) was evaluated to verify their lethal effect on T. evansi. To perform the in vivo tests, T. evansi-infected animals were treated with CURC (10 and 100 mg kg(-1), intraperitoneally [i.p.]) and C-LNCs (10 mg kg(-1), i.p.) during 6 days, with the results showing that these treatments significantly attenuated the parasitaemia. Infected untreated rats showed protein peroxidation and an increase of nitrites/nitrates, whereas animals treated with curcumin showed a reduction on these variables. As a result, the activity of antioxidant enzymes (superoxide dismutase and catalase) differs between groups (P<0.05). Infected animals and treated with CURC exhibited a reduction in the levels of alanine aminotransferase and creatinine, when compared with the positive control group. The use of curcumin in vitro resulted in a better parasitaemia control, an antioxidant activity and a protective effect on liver and kidney functions of T. evansi-infected adult male Wistar rats.

Comparison of methods for estimates of molecular genetic diversity in genus Croton: influence of coefficients, clustering strategies and data projection.

We investigated 10 similarity (and disimilarity) coefficients in a set of 40 wild genotypes of Croton linearifolius subjected to analyses using hierarchical grouping methods, grouping methods by optimization and data projection in two-dimensional space. Genotypes were characterized by analyzing DNA polymorphism with the use of 15 ISSR and 12 RAPD markers. The distance measurements were compared by the Spearman correlation test, projection in two-dimensional space and grouping efficiency evaluation. The Spearman correlation coefficients between the 10 coefficients evaluated were significant (P < 0.001) and indicated significant changes in genotype ranking due to type of coefficient used (0.76 ≤ rs ≤ 1). Wide variation was also observed in the efficiency of clustering methods, where the unweighted pair group method with arithmetic mean was the most suitable (0.3 ≤ D ≤ 1.5 ; 0.41 ≤ rc ≤ 0.77; 5.99 ≤ S ≤ 12.61). Projection efficiencies in two-dimensional space showed high-stress values (65 < S < 89%). Similar to the results observed for hierarchical clustering methods and for projection in two-dimensional space, the formation of groups with grouping methods by optimization showed variations when using different coefficients. We believe that the results confirm the influence of coefficients in studies of genetic diversity, showing the need to use criteria and standards for selecting appropriate methods for genetic studies of the genus Croton.

Improved tretinoin photostability in a topical nanomedicine replacing original liquid suspension with spray-dried powder with no loss of effectiveness.

OBJECTIVE: The use of spray-dried powders containing tretinoin-loaded nanocapsules instead of the original liquid suspension, aimed at the preparation of dermatological nanomedicines with improved photostability, was investigated. METHODS: Powders were prepared using lactose as a drying adjuvant. Hydrogels were prepared using two approaches: dispersing Carbopol Ultrez 10 in an aqueous redispersion of the powder or incorporating the powder in previously formed hydrogels. RESULTS AND DISCUSSION: The photodegradation of tretinoin in hydrogels prepared with the powders showed similar half-life times (around 19.5 h) compared to preparations with the original liquid nanocapsules (20.7 ± 1.4 h), regardless of the preparation approach. In addition, the topical nanomedicines prepared with the spray-dried powders presented a significant improvement in tretinoin photostability compared to the formulation containing the non-encapsulated drug. CONCLUSION: This study verified that the addition of the spray-dried powders containing tretinoin-loaded lipid-core nanocapsules to hydrogels did not influence the photoprotection of the drug compared with the preparation procedure using the original liquid suspension.

Antibiofilm effect of cleaning agents for ocular prostheses.

Introduction: This study aimed to evaluate the antibiofilm effect of different agents (neutral soap, 4% chlorhexidine, Efferdent effervescent tablets, 1% triclosan, and citronella essential oil) used for ocular prosthesis cleaning. Material and Methods: Biofilms of S. aureus and S. epidermidis were formed on 60 ocular prosthesis acrylic resin specimens. The specimens were cleaned with the studied agents with different techniques. Microorganism counting was performed. Data were submitted to ANOVA and HSD Tukey-Kramer (p<.01). Results: When compared to the control group, all cleaning protocols promoted a reduction in growth of microorganisms. The 4% chlorhexidine, effervescent tablets, and 1% triclosan cleaning agents eliminated biofilm in all groups. Conclusion: Therefore, immersion in 4% chlorhexidine, effervescent tablets, and 1% triclosan could be the best protocols indicated for ocular prosthesis cleaning due to their ability to eliminate biofilm.

Genetic variation in a wild population of the 'sleep' passion fruit (Passiflora setacea) based on molecular markers.

Little is known about the molecular genetic diversity of most Passiflora species. We used RAPD markers to evaluate the genetic diversity of 24 genotypes of the 'sleep' passion fruit (Passiflora setacea). Twelve primers generated 95 markers, 88% of which were polymorphic. The genetic distance estimated by the complement of the Dice index ranged from 0.29 (among accessions Ps-G1 and Ps-G13) to 0.69 (among accessions Ps-G21 and Ps-G23). Genotype grouping based on the UPGMA algorithm showed considerable variability among genotypes. We conclude that P. setacea has a broad genetic base that could be exploited in breeding programs.

Comparison of efficiency of distance measurement methodologies in mango (Mangifera indica) progenies based on physicochemical descriptors.

We investigated seven distance measures in a set of observations of physicochemical variables of mango (Mangifera indica) submitted to multivariate analyses (distance, projection and grouping). To estimate the distance measurements, five mango progeny (total of 25 genotypes) were analyzed, using six fruit physicochemical descriptors (fruit weight, equatorial diameter, longitudinal diameter, total soluble solids in °Brix, total titratable acidity, and pH). The distance measurements were compared by the Spearman correlation test, projection in two-dimensional space and grouping efficiency. The Spearman correlation coefficients between the seven distance measurements were, except for the Mahalanobis' generalized distance (0.41 ≤ rs ≤ 0.63), high and significant (rs ≥ 0.91; P < 0.001). Regardless of the origin of the distance matrix, the unweighted pair group method with arithmetic mean grouping method proved to be the most adequate. The various distance measurements and grouping methods gave different values for distortion (-116.5 ≤ D ≤ 74.5), cophenetic correlation (0.26 ≤ rc ≤ 0.76) and stress (-1.9 ≤ S ≤ 58.9). Choice of distance measurement and analysis methods influence the.

Milk Contamination by Mycobacterium tuberculosis Complex, Implications for Public Health in Amazonas, Brazil.

ABSTRACT: In Brazil, contamination of raw milk with Mycobacterium tuberculosis complex (MTC) has been reported in several states. The highest rate of consumption of raw milk and its derivatives in Brazil occurs in Amazonas. This state also has the highest prevalence of tuberculosis in both humans and livestock. We assessed the contamination of cow's milk and buffalo's milk with MTC in Amazonas, focusing on Mycobacterium bovis, the species most commonly found in cattle and buffalo. In 2019, 250 samples of raw milk (91 from cattle, 159 from buffalo) were collected before processing from three milk plants in the state of Amazonas. The samples were placed into 21 pools and analyzed using shotgun metagenomic sequencing and taxonomic classification with Kraken 2 and MegaBLAST. To confirm the identity of mycobacterial species found, BLASTN was used to identify specific genomic positions in the TbD1 and RD1 regions and flanking RD4 region. MTC genetic material was identified in all pools of raw milk. Genetic material consistent with M. bovis was identified in seven pools of raw milk (1 from cattle, 6 from buffalo). Buffalo's milk had significantly higher MTC reads than did cow's milk. The common practice of consumption of raw milk and its derivatives in Amazonas presents a risk to public health. Urgent measures to prevent transmission of foodborne tuberculosis are needed in the Amazon region. Greater efforts and resources also should be directed toward elimination of bovine tuberculosis in cattle and buffalo herds in Amazonas and the rest of Brazil.

Activity of cholinesterases and adenosine deaminase in blood and serum of rats experimentally infected with Trypanosoma cruzi.

This study aimed to evaluate the activity of cholinesterases and adenosine deaminase (ADA) in blood and serum of rats infected with Trypanosoma cruzi. Twelve adult rats were used in the experiment divided into two uniform groups. Rodents from group A (control group) were non-infected and animals from group B served as infected, receiving intraperitoneally 3·3×10(7) trypomastigotes/each. Blood collection was performed at days 60 and 120 post-infection (PI) in order to evaluate the hemogram, blood activity of acetylcholinesterase, and serum butyrylcholinesterase and ADA activities. Hematological parameters did not differ between groups. A significant increase (P<0·05) of acetylcholinesterase activity was observed in blood while butyrylcholinesterase had a significant reduction (P<0·01) in serum of infected rats at days 60 and 120 PI. ADA activity in serum showed an inhibition in infected animals when compared to non-infected at day 120 PI. Based on these results, it is possible to conclude that the activity of cholinesterases and ADA were changed in animals infected with T. cruzi. The possible causes of these alterations will be discussed in this paper.

Endophytic and pathogenic isolates of the cacao fungal pathogen Moniliophthora perniciosa (Tricholomataceae) are indistinguishable based on genetic and physiological analysis.

We evaluated the genetic and physiological variability of Moniliophthora perniciosa obtained from healthy and diseased branches of cacao (Theobroma cacao) plants. The diversity of the isolates was evaluated by RAPD technique and by studies of virulence and exoenzyme production. The genetic variability of endophytic and pathogenic M. perniciosa was evaluated in association with pathogenicity assays. RAPD analysis showed eight genetic groups, which were not related to plant disease status (healthy versus diseased branches). Isolates from cacao were included in three groups, excluding isolates from other host plants. Pathogenicity and enzyme analysis showed that the virulence of the isolates is not related to exoenzyme production. This is the first evidence that M. perniciosa colonizes healthy parenchymatic tissues, showing that endophytic behavior may occur in this species.

Confirmation of cross-fertilization using molecular markers in ornamental passion flower hybrids.

Several interspecific Passiflora hybrids are produced in the northern hemisphere for the ornamental plant market. In Brazil, production of passion flower hybrids is limited to the introgression of genes into the main cultivated species, yellow passion fruit, to be used as rootstocks. Confirmation of hybridization in the initial developmental stage is important for breeding perennial and sub-perennial plants, such as passion flowers, reducing time and costs in plant stock maintenance. In order to obtain F1 hybrids with ornamental potential, four species of Passiflora (P. alata, P. gardneri, P. gibertii, and P. watsoniana) from the Active Germplasm Bank at UESC were hybridized. Flower buds, in pre-anthesis, of the genitors were previously protected, and the female buds were emasculated. To confirm hybridization, the genomic DNA of the genitor species and the supposed hybrids was extracted and RAPD primers were used to obtain molecular markers and select passion flower interspecific hybrids. Eight primers were used to confirm hybrids derived from P. gardneri with P. alata, P. watsoniana with P. alata, P. watsoniana with P. gardneri, and P. gardneri with P. gibertii; 75, 50, 45, and 46% of the informative bands, respectively, confirmed the hybrid nature of these plants. The RAPD technique was effective in the early identification of hybrids; this will be useful for development of hybrid Passiflora progeny.

Chemical composition and phytotoxicity of essential oils of Croton doctoris S. Moore (Euphorbiaceae).

Essential oils from the stems and leaves of Croton doctoris were analyzed by gas chromatography and mass spectrometry, resulting in 22 identified compounds. The effects of these essential oils on the germination, root and shoot growth, total chlorophyll content, potential root respiration, peroxidase activity, catalase, superoxide dismutase, and mitotic index in lettuce and onion were determined. Antioxidant, antimicrobial, and cytotoxic activity were also investigated. The results revealed that the stem oil consisted of 15 compounds, of which caryophyllene oxide (24.5%) and E-caryophyllene (13.3%) were the major constituents. The leaf oil contained E-caryophyllene (39.6%) and α-humulene (13.2%) as major compounds. The oils inhibited the germination and growth of lettuce and onion seedlings and reduced chlorophyll content, root respiration, and cell division. They also caused oxidative stress, indicated by the increased activity of the evaluated antioxidant enzymes. These abnormal physiological processes contributed to the inhibition of plant growth. The most pronounced phytotoxic effects were observed in the stem oil. The cytotoxicity tests indicated that leaf oil was more active than stem oil, resulting from the presence of biologically active sesquiterpenes that inhibit the growth of cancer cells.

Genetic variability in wild genotypes of Passiflora cincinnata based on RAPD markers.

The genetic diversity and characteristics of commercial interest of Passiflora species make it useful to characterize wild germplasm, because of their potential use for fruit, ornamental and medicinal purposes. We evaluated genetic diversity, using RAPD markers, of 32 genotypes of Passiflora cincinnata collected from the wild in the region of Vitória da Conquista, Bahia, Brazil. Thirteen primers generated 95 polymorphic markers and only one monomorphic marker. The mean genetic distance between the genotypes estimated by the complement of the Dice index was 0.51 (ranging from 0.20-0.85), and genotype grouping based on the UPGMA algorithm showed wide variability among the genotypes. This type of information contributes to identification and conservation of the biodiversity of this species and for the identification of pairs of divergent individuals for maximum exploitation of existing variability.

Genetic diversity in wild species of passion fruit (Passiflora trintae) based on molecular markers.

In spite of the importance of and the considerable variability observed in Passiflora (Passifloraceae), little is known about the genetic diversity of most of the species of this genus. We evaluated the genetic diversity by RAPD markers in 18 genotypes of Passiflora trintae. The 15 primers generated 112 markers, 84% of which were polymorphic. The genetic distance estimated by the complement of the Dice index (average dissimilarity = 0.30) and genotype grouping based on the UPGMA algorithm showed low variability among genotypes. More attention should be given to the study and conservation of the biodiversity of this economically important genus.

Hydrogel containing dexamethasone-loaded nanocapsules for cutaneous administration: preparation, characterization, and in vitro drug release study.

CONTEXT: Our group previously reported the development of dexamethasone-loaded polymeric nanocapsules as an alternative for topical dermatological treatments. OBJECTIVE: Our study aimed to prepare and characterize a hydrogel containing this system to improve the effectiveness of the glucocorticoid for cutaneous disorders. METHODS: For the antiproliferative activity assay, a dexamethasone solution and D-NC were tested on Allium cepa root meristem model. D-NC were prepared by the interfacial deposition of preformed polymer. Hydrogels were prepared using Carbopol Ultrez 10 NF, as polymer, and characterized according to the following characteristics: pH, drug content, spreadability, viscosity, and in vitro drug release. RESULTS AND DISCUSSION: Nanocapsules showed mean particle size and zeta potential of 201 +/- 6 and -5.73 +/- 0.42 nm, respectively. They demonstrated a lower mitotic index (4.62%) compared to free dexamethasone (8.60%). Semisolid formulations presented acidic pH values and adequate drug content (between 5.4% and 6.1% and 100% and 105%, respectively). The presence of nanocapsules in hydrogels led to a decrease in their spreadability factor. Intact nanoparticles were demonstrated by TEM as well as by dynamic light scattering (mean particle size < 300 nm). In vitro studies showed a controlled dexamethasone release from hydrogels containing the drug associated to the nanocapsules following the Higuchi's squared root model (k = 20.21 +/- 2.96 mg/cm(2)/h(1/2)) compared to the hydrogels containing the free drug (k = 26.65 +/- 2.09 mg/cm(2)/h(1/2)). CONCLUSION: Taking all these results together, the hydrogel containing D-NC represent a promising approach to treat antiproliferative-related dermatological disorders.

Microparticles containing lemongrass volatile oil: preparation, characterization and thermal stability.

Lemongrass volatile oil (LVO) is an important ingredient in cosmetics, presenting antimicrobial properties, in particular antifungal activity, and it is a promising raw material for the development of pharmaceutical products. However, its volatility and susceptibility to degradation are the major drawbacks for the use of Cymbopogon citratus oil in pharmaceutical compounding. Thus, the aim of this work was to develop and to characterize microparticles containing this oil viewing the stabilization of LVO. Two techniques of preparation were evaluated; spray drying and precipitation, and two encapsulation materials, beta-cyclodextrin (beta-CD) and hydroxypropyl-beta-cyclodextrin (HP-beta-CD) were tested. The microparticles were characterized in terms of content of water, yield, percentage of inclusion, infrared spectroscopy. Morphology was evaluated by scanning electronic microscopy. Studies of stability were also conducted. The content of citral (neral and geranial), major component of the oil, present in microparticles was assayed by a validated HPLC method. The percentage of inclusion of LVO into the microparticles was 56-60% and 26-29% using beta-CD and HP-beta-CD, respectively. The results showed that the use of the beta-CD as encapsulant material was more efficient. Additionally, an increased inclusion of lemongrass oil was observed with the precipitation technique.

High-pressure studies on l,l-dileucine crystals by Raman spectroscopy and synchrotron X-ray diffraction combined with DFT calculations.

The vibrational properties of the dipeptide l-leucyl-l-leucine hydrate were investigated through Raman and infrared spectroscopy. With the aid of first principle calculations using the density functional theory, the assignment of the vibrational modes from the material was furnished. In addition, the behavior of the crystal under high pressure was investigated using Raman spectroscopy (~8 GPa) and synchrotron X-ray diffraction (~26 GPa). The results show significant changes in both the X-ray diffractogram and the Raman spectra, suggesting that l-leucyl-l-leucine hydrate undergoes a phase transition between 2.3 and 2.9 GPa. Finally, for pressures above 16 GPa the broadening of X-ray peaks suggests a disorder in the crystal lattice induced by high-pressure effects.

Comparison of coefficients and distance measurements in passion fruit plants based on molecular markers and physicochemical descriptors.

We investigated seven distance measures and 14 similarity coefficients in a set of observations of variables of the 'yellow' passion fruit plant (Passiflora edulis Sims), submitted to multivariate analyses (distance, projection and grouping). Fourteen genotypes were characterized, based on DNA amplification with 16 random amplified polymorphic DNA primers and the assessment of nine fruit physical-chemical descriptors. The distance measurements and the similarity coefficients were compared by the Spearman correlation test, projection in two-dimensional space and grouping efficiency, using five grouping methods; the genotype ranking varied with the different techniques. Coler-Rodger distance measures, Euclidean distance square measures and Yule similarity coefficients proved to be inadequate for projection in two-dimensional space or for grouping matrices. Regardless of the origin of the distance matrix, the unweighted pair group method with arithmetic mean grouping method proved to be the most adequate. The various distance measurements, similarity coefficients and grouping methods gave different values of distortion, cophenetic correlation and stress; they influence the characterization of genetic variability and this should be taken into account for studies of yellow passion fruit plants.