Why Is It So Difficult To Have Competent Oocytes from In vitro Cultured Preantral Follicles?

The developmental competence of oocytes is acquired gradually during follicular development, mainly through oocyte accumulation of RNA molecules and proteins that will be used during fertilization and early embryonic development. Several attempts to develop in vitro culture systems to support preantral follicle development up to maturation are reported in the literature, but oocyte competence has not yet been achieved in human and domestic animals. The difficulties to have fertilizable oocytes are related to thousands of mRNAs and proteins that need to be synthesized, long-term duration of follicular development, size of preovulatory follicles, composition of in vitro culture medium, and the need of multi-step culture systems. The development of a culture system that maintains bidirectional communication between the oocyte and granulosa cells and that meets the metabolic demands of each stage of follicle growth is the key to sustain an extended culture period. This review discusses the physiological and molecular mechanisms that determine acquisition of oocyte competence in vitro, like oocyte transcriptional activity, follicle and oocyte sizes, and length and regulation of follicular development in murine, human, and domestic animal species. The state of art of in vitro follicular development and the challenges to have complete follicular development in vitro are also highlighted.

Platforms for Clinical-Grade CAR-T Cell Expansion.

Chimeric antigen receptor (CAR)-T cell therapy has revolutionized the immunotherapy field with high rate complete responses especially for hematological diseases. Despite the diversity of tumor specific-antigens, the manufacturing process is consistent and involves multiple steps, including selection of T cells, activation, genetic modification, and in vitro expansion. Among these complex manufacturing phases, the choice of culture system to generate a high number of functional cells needs to be evaluated and optimized. Flasks, bags, and rocking motion bioreactor are the most used platforms for CAR-T cell expansion in the current clinical trials but are far from being standardized. New processing options are available and a systematic effort seeking automation, standardization and the increase of production scale, would certainly help to bring the costs down and ultimately democratize this personalized therapy. In this review, we describe different cell expansion platforms available as well as the quality control requirements for clinical-grade production.

Explant Type, Culture System, 6-Benzyladenine, Meta-Topolin and Encapsulation Affect Indirect Somatic Embryogenesis and Regeneration in Carica papaya L.

A protocol to propagate papaya hybrid plants through indirect somatic embryogenesis was developed considering the effect of explant type, culture system, particular cytokinins and encapsulation, in different stages of the process. Optimal 2,4-dichlorophenoxyacetic acid (2,4-D) concentrations for non-embryogenic callus formation ranged between 9.0 and 27.1 µM in half-cut seeds, while higher concentrations were harmful. Non-embryogenic callus was also obtained with 22-158 µM 2,4-D from hypocotyl segments. Callus with embryogenic structures was only obtained in half-cut seeds cultured in the darkness on half-strength Murashige and Skoog culture medium supplemented with 2,4-D, while hypocotyl segments and isolated zygotic embryos failed to produce this type of callus regardless of the 2,4-D and sucrose (30 and 70 g l-1) concentrations tested in this study. Both, embryogenic callus development and quantity of somatic embryos formed per embryogenic callus, which ranged between 11 and 31 units after 14 months, required 2,4-D, but without any effect of the concentration. Histological studies confirmed the multicellular origin of the somatic embryos. In further steps, liquid medium induced over four times more somatic embryos than agar-gelled medium and showed significantly higher production of globular somatic embryos (85 vs. 57%). Both, 6-benzyladenine (BA) and meta-topolin (Mtop) stimulated sprouting (40-45%) of the somatic embryos (development of shoots only) in concentrations of up to 2.7 and 10 µM, respectively. Sprouting probability showed a 2nd order polynomial trend despite the range of concentration used for each cytokinin. This is the first report about the positive effect of Mtop on the apical shoot development of Carica papaya somatic embryos known to the authors. Radicle growth was observed in 5% or less of the cultivated embryos, regardless of the BA concentration. Finally, all encapsulation conditions tested (2.5, 3.5, and 4.5% sodium alginate, combined with 50 and 100 mM CaCl2) reduced sprouting of somatic embryos when compared to the non-encapsulated ones, whereas capsule hardness showed low correlation with embryo sprouting. Embryos were further cultivated until they became plantlets approximately 5 cm long. They were acclimatized and afterward planted in the field, where they flowered and produced fruit.

In vitro development of bovine secondary follicles in two- and three-dimensional culture systems using vascular endothelial growth factor, insulin-like growth factor-1, and growth hormone.

The aim of this study was to evaluate the development and estradiol production of isolated bovine secondary follicles in two-dimensional (2D, experiment 1) and three-dimensional (3D using alginate, experiment 2) long-term culture systems in the absence (control group; only α-MEM(+)) or presence of vascular endothelial growth factor (VEGF), insulin-like growth factor-1, or GH alone, or a combination of all. A total of 363 isolated secondary follicles were cultured individually for 32 days at 38.5 °C in 5% CO2 in a humidified incubator with addition of medium (5 µL) every other day. In 2D culture system, follicular growth and antrum formation rates were higher (P < 0.05) in VEGF treatment compared with the other treatments. In 3D culture system, only estradiol concentration was greater (P < 0.05) in the GH than in the control group, whereas the other end points were similar (P > 0.05). In summary, this study demonstrated that the benefits of using a certain type of medium supplement depended on the culture system (2D vs. 3D). Vascular endothelial growth factor was an effective supplement for the in vitro culture of bovine secondary follicles when the 2D culture system was used, whereas GH only affected estradiol production using the 3D culture system. This study sheds light on advancements in methodology to facilitate subsequent studies on bovine preantral follicle development.

Virulence potential and antibiotic susceptibility pattern of motile aeromonads associated with freshwater ornamental fish culture systems: a possible threat to public health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF-ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health.

Virulence potential and antibiotic susceptibility pattern of motile aeromonads associated with freshwater ornamental fish culture systems: a possible threat to public health.

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF-ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health.

Virulence potential and antibiotic susceptibility pattern of motile aeromonads associated with freshwater ornamental fish culture systems: a possible threat to public health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF-ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health.

Herdabilidades e correlações entre caracteres de linhagens de feijão obtidas em monocultivo e em consórcio com o milho

Hybrids from 'Ouro 1919' x 'Ouro Negro 1992', 'Ouro 1919' x Raça D and 'Ouro 1919' x Phaseolus aborigineus 283, beans were obtained to study heritability and genetic correlations between some yield responses, in two crop systems namely monocropping and intercropping with maize during both dry and rainy seasons. All estimates were obtained through field data of F1, F2 and F3 populations as well as those of their original parents. Main populations were defined by the Single Seed Descent (SSD) method. Heritabilities estimated under intercropping conditions were often higher than those under monocropping system. Among all studied responses the highest heritabilities corresponded to the number of legumes per plant. Genetic correlation coefficients within systems suggest that the selection procedure should mainly consider the number of legumes per plant when selecting under monocropping system aiming further intercropping use.

Foram realizados os cruzamentos entre 'Ouro 1919' x 'Ouro Negro 1992', 'Ouro 1919' x Raça D e 'Ouro 1919' x Phaseolus aborigineus 283, com o objetivo de estimar as herdabilidades, no sentido amplo e as correlações fenotípica e genotípica da produtividade de grãos e seus componentes de rendimento, nos sistemas de monocultivo e de consórcio com a cultura do milho nas épocas de semeadura das "águas" e da "seca'. Para obtenção dessas estimativas, utilizaram-se, no ensaio de campo, as populações, F1, F2 e F3 e seus genitores. As populações segregantes foram conduzidas pelo método de melhoramento "Single Seed Descent" (SSD). De modo geral, os coeficientes de herdabilidade foram maiores no sistema de consórcio do que no monocultivo. Entre os caracteres estudados, as maiores herdabilidades foram, de modo geral, obtidas para o número de vagens por planta. Os coeficientes de correlação entre os caracteres avaliados, em cada sistema de cultivo, mostraram que, no caso de seleção no monocultivo visando semeadura no consórcio, o número de vagens por planta é o caráter com maior potencial a ser utilizado no processo seletivo.

Herdabilidades e correlações entre caracteres de linhagens de feijão obtidas em monocultivo e em consórcio com o milho

Hybrids from 'Ouro 1919' x 'Ouro Negro 1992', 'Ouro 1919' x Raça D and 'Ouro 1919' x Phaseolus aborigineus 283, beans were obtained to study heritability and genetic correlations between some yield responses, in two crop systems namely monocropping and intercropping with maize during both dry and rainy seasons. All estimates were obtained through field data of F1, F2 and F3 populations as well as those of their original parents. Main populations were defined by the Single Seed Descent (SSD) method. Heritabilities estimated under intercropping conditions were often higher than those under monocropping system. Among all studied responses the highest heritabilities corresponded to the number of legumes per plant. Genetic correlation coefficients within systems suggest that the selection procedure should mainly consider the number of legumes per plant when selecting under monocropping system aiming further intercropping use.

Foram realizados os cruzamentos entre 'Ouro 1919' x 'Ouro Negro 1992', 'Ouro 1919' x Raça D e 'Ouro 1919' x Phaseolus aborigineus 283, com o objetivo de estimar as herdabilidades, no sentido amplo e as correlações fenotípica e genotípica da produtividade de grãos e seus componentes de rendimento, nos sistemas de monocultivo e de consórcio com a cultura do milho nas épocas de semeadura das "águas" e da "seca'. Para obtenção dessas estimativas, utilizaram-se, no ensaio de campo, as populações, F1, F2 e F3 e seus genitores. As populações segregantes foram conduzidas pelo método de melhoramento "Single Seed Descent" (SSD). De modo geral, os coeficientes de herdabilidade foram maiores no sistema de consórcio do que no monocultivo. Entre os caracteres estudados, as maiores herdabilidades foram, de modo geral, obtidas para o número de vagens por planta. Os coeficientes de correlação entre os caracteres avaliados, em cada sistema de cultivo, mostraram que, no caso de seleção no monocultivo visando semeadura no consórcio, o número de vagens por planta é o caráter com maior potencial a ser utilizado no processo seletivo.