Can Culture Media Impact Preimplantation Embryo Aneuploidy?

With continued improvements in blastocyst culture, cell sampling approaches, and genetic analysis platforms, the resulting improvements in embryo development and the resolution and accuracy of chromosome analysis have provided valuable insights into the preimplantation embryo. This includes the impact of in vitro culture conditions on chromosomal dynamics. Specifically, through analysis of embryo aneuploidy and mosaicism, a growing number of reports indicate that rates of chromosomal abnormalities can vary between IVF centers. Because differences in mosaicism reflect mitotic errors, this endpoint analysis suggests that IVF laboratory-controlled variables during embryo development may be influencing chromosome separation and segregation. A growing body of literature suggests that culture media may be one variable influencing preimplantation embryo aneuploidy and mosaicism. However, these data are far from definitive in demonstrating cause-and-effect. Whether reported differences may be due to media formulation, use of sequential media or single-step media, or uninterrupted culture approaches is unknown. Importantly, variables directly impacting media performance and embryo development, including pH, temperature, osmolality, and oxygen concentration, must also be considered and make it difficult to isolate the impact of culture media as the sole factor responsible. These IVF laboratory variables will be reviewed and literature suggesting a possible link to mitotic aneuploidy/mosaicism will be discussed.

Virological and molecular investigation on specimens from buffaloes suspected to be infected by buffalopox virus in Egypt 2019

In this study, skin lesions from buffaloes showing clinical signs of buffalopox infection were tested to isolate and identify the buffalopox virus (BPXV). Clinical examination of infected buffaloes was performed and visible clinical signs recorded. Skin scabs from infected buffaloes were collected and used for virus isolation on embryonated chicken egg (ECE) and tissue culture cell lines. The isolated BPXV was identified and characterized using polymerase chain reaction (PCR). The infected buffaloes displayed fever, skin eruptions, enlargement of superficial lymph nodes, emaciation and drop in milk yield. The ECE inoculated with the prepared skin scab samples showed clear raised white pock lesions on the chorioallantoic membrane (CAM). The inoculated tissue cultures (VERO and BHK cell lines) revealed a cytopathic effect (CPE) including rounding, clumping with cytoplasmic granulation and cluster formation. PCR for the C18L specific BPXV gene was carried out on the virus infected tissue culture produced 368 bp bands. Human infection with BPXV was also recorded. It was concluded that BPXV is circulating in Egyptian buffaloes, causing economical losses and infection in contact humans.

Paracrine activity of adipose derived stem cells on limbal epithelial stem cells

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of patients for the cornea transplantation all over the world and the list is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and has a potential role in mitigation of the adverse impact of inflammation induced by lipopolysaccharide.

Mature rat hepatocyte dedifferentiation into long lived proliferating hepatic progenitor cells

Objective: to obtain long-lived proliferating cells with progenitor features by dedifferentiation of mature rat hepatocytes using combinations of small molecules.Materials and Methods. Hepatocytes isolated from rat liver by perfusion were cultured in the presence of a cocktail of three small molecules – Wnt signaling pathway activator (CHIR99021), TGF-β inhibitors (A83-01) and ROCK kinase (Y27632). The morphological characteristics and growth features of the culture were assessed using fluorescence and phase-contrast microscopy during cell culture. Cell proliferative activity was analyzed using real-time time-lapse imaging. The expression of surface and intracellular markers was analyzed using flow cytometry and high-resolution fluorescence microscopy.Results. Using a cocktail of small molecules, Y-27632, A-83-01, and CHIR99021, long-lived proliferating cells that express progenitor cell markers, such as α-fetoprotein and HNF4α, were obtained from mature rat hepatocytes. The cells had hepatocyte-like morphology and formed discrete clusters of proliferating cells, forming a single cell layer during culturing. Removal of the small molecules from the medium led to expansion of fibroblast-like cells and elimination of potentially progenitor hepatocyte-like cells.Conclusion. Proliferating progenitor cells can be obtained by dedifferentiation of mature hepatocytes.

Paracrine activity of adipose derived stem cells on limbal epithelial stem cells

Limbal stem cells deficiency (LSCD) is an eye disease caused by the loss of stem cells in the corneal limbus as a succession of an injury due physical, biological, or chemical agents. Current therapies of LSCD are focused on the transplantation of donor corneas or tissue equivalents produced from autologous limbal stem cells. Every year there are waiting millions of people for the cornea transplantation all over the world and the list of waiting patients is growing due to the relatively low number of cornea donors. On the other hand, the transplantation of tissue or cells into the recipient’s body is associated with the higher risk of possible side effects. The possibility of the application of an indirect treatment using the properties of the paracrine activity of stem cells, would be beneficial for the patients with transplant failures. This study was to evaluate the paracrine effect of mesenchymal stem cells derived from adipose tissue (ADSC) on the viability of limbal epithelial stem cells (LESC). The paracrine effect was assessed by treating LESC with conditioned medium collected from ADSC culture. Cell viability, cytotoxicity, apoptosis and proliferation were evaluated using in vitro assays in standard conditions and induced inflammation. After the exposure to the examined conditions, the expression of genes related to pro- and anti- inflammatory factors was evaluated and compared to the secretion of selected cytokines by ELISA test. Moreover, the changes in LESC phenotype were assessed using of phenotype microarrays. Our findings suggest that paracrine activity of ADSC on LESC promotes its proliferation and mitigates the adverse impact of induced inflammation.

Biophysical and Biochemical Comparison of Extracellular Vesicles Produced by Infective and Non-Infective Stages of Trypanosoma cruzi

Extracellular vesicles (EVs) are small lipid vesicles released by either any prokaryotic or eukaryotic cell, or both, with a biological role in cell-to-cell communication. In this work, we characterize the proteomes and nanomechanical properties of EVs released by tissue-culture cell-derived trypomastigotes (mammalian infective stage; (TCT)) and epimastigotes (insect stage; (E)) of Trypanosoma cruzi, the etiologic agent of Chagas disease. EVs of each stage were isolated by differential centrifugation and analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS), dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), electron microscopy and atomic force microscopy (AFM). Measurements of zeta-potential were also included. Results show marked differences in the surface molecular cargos of EVs between both stages, with a noteworthy expansion of all groups of trans-sialidase proteins in trypomastigote’s EVs. In contrast, chromosomal locations of trans-sialidases of EVs of epimastigotes were dramatically reduced and restricted to subtelomeric regions, indicating a possible regulatable expression of these proteins between both stages of the parasite. Regarding mechanical properties, EVs of trypomastigotes showed higher adhesion compared to the EVs of epimastigotes. These findings demonstrate the remarkable surface remodeling throughout the life cycle of T. cruzi, which shapes the physicochemical composition of the extracellular vesicles and could have an impact in the ability of these vesicles to participate in cell communication in completely different niches of infection.

Probiotic Properties and Potentiality of Lactiplantibacillus plantarum Strains for the Biological Control of Chalkbrood Disease

Ascosphaera apis is an entomopathogenic fungus that affects honeybees. In stressful conditions, this fungus (due not only to its presence, but also to the combination of other biotic and abiotic stressors) can cause chalkbrood disease. In recent years, there has been increasing attention paid towards the use of lactic acid bacteria (LAB) in the honeybees’ diets to improve their health, productivity and ability to resist infections by pathogenic microorganisms. The screening of 22 strains of Lactiplantibacillus plantarum, isolated from the gastrointestinal tracts of honeybees and beebread, led to the selection of five strains possessing high antagonistic activity against A. apis. This study focused on the antifungal activity of these five strains against A. apis DSM 3116 and DSM 3117 using different matrices: cell lysate, broth culture, cell-free supernatant and cell pellet. In addition, some functional properties and the antioxidant activity of the five L. plantarum strains were evaluated. All five strains exhibited high antagonistic activity against A. apis, good surface cellular properties (extracellular polysaccharide (EPS) production and biofilm formation) and antioxidant activity. Although preliminary, these results are encouraging, and in future investigations, the effectiveness of these bacteria as probiotics in honeybee nutrition will be tested in vivo in the context of an eco-friendly strategy for the biological control of chalkbrood disease.

Interactivity of biochemical and physical stimuli during epigenetic conditioning and cardiomyocytic differentiation of stem and progenitor cells derived from adult hearts

Mixed populations of cardiosphere-derived stem and progenitor cells containing proliferative and cardiomyogenically committed cells were obtained from adult rat hearts. The cells were cultured in either static 2D monolayers or dynamic 3D scaffold systems with fluid flow. Cardiomyocyte lineage commitment in terms of GATA4 and Nkx2.5 expression was significantly enhanced in the dynamic 3D cultures compared with static 2D conditions. Treatment of the cells with 5-azacytidine (5-aza) produced different responses in the two culture systems, as activity of this chemical epigenetic conditioning agent depended on the cell attachment and hydrodynamic conditions provided during culture. Cell growth was unaffected by 5-aza in the static 2D cultures but was significantly reduced under dynamic 3D conditions relative to untreated controls. Myogenic differentiation measured as Mef2c expression was markedly upregulated by 5-aza in the dynamic 3D cultures but downregulated in the static 2D cultures. The ability of the physical environment to modulate the cellular cardiomyogenic response to 5-aza underscores the interactivity of biochemical and physical stimuli applied for cell differentiation. Accordingly, observations about the efficacy of 5-aza as a cardiomyocyte induction agent may not be applicable across different culture systems. Overall, use of dynamic 3D rather than static 2D culture was more beneficial for cardio-specific myogenesis than 5-aza treatment, which generated a more ambiguous differentiation response.

Antimicrobial activity of bacteria isolated from tissue of the coral Palythoa caribaeorum (Zoantharia: Sphenopidae) from Paraíba, Brazil coastal reefs

Introduction: The coral-associated bacteria with antimicrobial activity may be important to promote the health of their host through various interactions, and may be explored as a source of new bioactive compounds. Objective: To analyze the antimicrobial activity of bacteria associated with the zoanthid Palythoa caribaeorum from the coral reefs of Carapibus, Paraiba state, Brazil. Methods: The phylogenetic analysis of the bacteria was conducted based on partial sequences of the 16S rRNA gene using molecular and bioinformatics tools. The antimicrobial activity of the 49 isolates was tested against four bacterial strains and one yeast strain: Bacillus cereus (CCT0198), Escherichia coli (ATCC 25922), Staphylococcus aureus (ATCC 25923), Pseudomonas aeruginosa and Candida albicans (ATCC 10231). The antibiosis and antibiogram assays were conducted and the Minimal Inhibitory Concentration (MIC) was determined by the microdilution method. Results: The bacterial isolates belonged to Firmicutes phylum (84 % of the isolates) and the Proteobacteria phylum (16 % of the isolates). Among the 49 isolates five genera were found, with the Bacillus genus being the most abundant (82 % of the isolates), followed by Vibrio (10 %), Pseudomonas (4 %), Staphylococcus (2 %) and Alteromonas (2 %). Antibiosis test revealed that 16 isolates (33 %) showed antimicrobial activity against one or more of five tested reference strains. The highest number of antagonistic bacteria were found in the Bacillus genus (12 isolates), followed by Vibrio (three isolates) and Pseudomonas (one isolate) genera. The B. subtilis NC8 was the only isolate that inhibited all tested strains in the antibiosis assay. However, antibiogram test with post-culture cell-free supernatant of NC8 isolate showed the inhibition of only B. cereus, S. aureus and C. albicans, and the lyophilized and dialyzed material of this isolate inhibited only B. cereus. The lyophilized material showed bacteriostatic activity against B. cereus, with a MIC value of 125 μg/μl, and in the cytotoxicity assay, the hemolysis value was of 4.8 %, indicating its low cytotoxicity. Conclusions: The results show the antimicrobial potential of some bacterial isolates associated with the P. caribaeourum tissue, especially those belonged to Bacillus genus.