RNA sequencing and gene co-expression network of in vitro matured oocytes and blastocysts of buffalo.

In reproductive technologies, uncovering the molecular aspects of oocyte and embryo competence under different conditions is crucial for refining protocols and enhancing efficiency. RNA-seq generates high-throughput data and provides transcriptomes that can undergo additional computational analyses. This study presented the transcriptomic profiles of in vitro matured oocytes and blastocysts produced in vitro from buffalo crossbred (Bubalus bubalis), coupled with gene co-expression and module preservation analysis. Cumulus Oophorus Complexes, obtained from slaughterhouse-derived ovaries, were subjected to in vitro maturation to yield metaphase II oocytes (616) or followed in vitro fertilization and culture to yield blastocysts for sequencing (526). Oocyte maturation (72%, ±3.34 sd) and embryo development (21.3%, ±4.18 sd) rates were obtained from three in vitro embryo production routines following standard protocols. Sequencing of 410 metaphase II oocytes and 70 hatched blastocysts (grade 1 and 2) identified a total of 13,976 genes, with 62% being ubiquitously expressed (8,649). Among them, the differentially expressed genes (4,153) and the strongly variable genes with the higher expression (fold-change above 11) were highlighted in oocytes (BMP15, UCHL1, WEE1, NLRPs, KPNA7, ZP2, and ZP4) and blastocysts (APOA1, KRT18, ANXA2, S100A14, SLC34A2, PRSS8 and ANXA2) as representative indicators of molecular quality. Additionally, genes exclusively found in oocytes (224) and blastocysts (2,200) with specific biological functions were identified. Gene co-expression network and module preservation analysis revealed strong preservation of functional modules related to exosome components, steroid metabolism, cell proliferation, and morphogenesis. However, cell cycle and amino acid transport modules exhibited weak preservation, which may reflect differences in embryo development kinetics and the activation of cell signaling pathways between buffalo and bovine. This comprehensive transcriptomic profile serves as a valuable resource for assessing the molecular quality of buffalo oocytes and embryos in future in vitro embryo production assays.

Use of platelet-rich plasma on in vitro maturation during bovine embryo production.

One of the crucial aspects to be considered for successful in vitro production (IVP) of embryos is the composition of the various media used throughout the stages of this reproductive biotechnology. The cell culture media employed should fulfill the metabolic requirements of both gametes during oocyte maturation and sperm development, as well as the embryo during its initial cell divisions. Most IVP protocols incorporate blood serum into the media composition as a source of hormones, proteins, growth factors, and nutrients. Numerous studies have suggested Platelet-Rich Plasma (PRP) as a substitute for fetal sera in cell culture, particularly for stem cells. Therefore, the objective of this study is to assess the potential use of PRP as a replacement for fetal bovine serum (FBS) during oocyte maturation for in vitro production of bovine embryos. During in vitro maturation (IVM), cumulus-oocyte complexes (COCs) were allocated into the following experimental groups: Group G1 (IVM medium with 5% PRP); Group G2 (MIV medium with 5% PRP and 5% SFB); Group G3 (MIV medium with 5% SFB); and Group G4 (MIV medium without either PRP or SFB). Subsequently, the cumulus-oocyte complexes were fertilized with semen from a single bull, and the resulting zygotes were cultured for seven days. Cleavage and blastocyst formation rates were assessed on days 2 and 7 of embryonic development, respectively. The quality of matured COCs was also evaluated by analyzing the gene expression of HSP70, an important protein associated with cellular stress. The results demonstrated that there were no significant differences among the experimental groups in terms of embryo production rates, both in the initial cleavage stages and blastocyst formation (except for the G4 group, which exhibited a lower blastocyst formation rate on D7, as expected). This indicates that PRP could be a cost-effective alternative to SFB in the IVP of embryos.

Development of a mobile app for the evaluation of patients with chronic rhinosinusitis.

OBJECTIVE: To develop a mobile application with a standardized routine, to be used by general otolaryngologists for evaluating patients with chronic rhinosinusitis. METHODS: A systematic review was made to identify outcomes, recommendations and what tests that would be used in the routine evaluation of patients with chronic rhinosinusitis; establish an expert consensus on items to be included in this routine evaluation of patients with chronic rhinosinusitis using the Delphi method; development of an application for use on a mobile device, with the routine evaluation of patients with chronic rhinosinusitis. RESULTS: Based on the systematic review, the outcomes used in studies about chronic rhinosinusitis were identified, as well as guidelines recommendations, which showed discrepancies between them. These recommendations and outcomes were presented to specialists in chronic rhinosinusitis, until a consensus was reached. As a result of the Delphi method, the flowchart of the routine evaluation of patients with chronic rhinosinusitis was defined, and then was used for the development of the mobile application. CONCLUSION: The creation of the mobile application for evaluating patients with chronic rhinosinusitis followed an adequate methodology of elaboration made by specialists in the field of chronic rhinosinusitis, standardizing the investigation of these patients. LEVEL OF EVIDENCE: Level 5.

Development of a mobile app for the evaluation of patients with chronic rhinosinusitis

Abstract Objective To develop a mobile application with a standardized routine, to be used by general otolaryngologists for evaluating patients with chronic rhinosinusitis. Methods A systematic review was made to identify outcomes, recommendations and what tests that would be used in the routine evaluation of patients with chronic rhinosinusitis; establish an expert consensus on items to be included in this routine evaluation of patients with chronic rhinosinusitis using the Delphi method; development of an application for use on a mobile device, with the routine evaluation of patients with chronic rhinosinusitis. Results Based on the systematic review, the outcomes used in studies about chronic rhinosinusitis were identified, as well as guidelines recommendations, which showed discrepancies between them. These recommendations and outcomes were presented to specialists in chronic rhinosinusitis, until a consensus was reached. As a result of the Delphi method, the flowchart of the routine evaluation of patients with chronic rhinosinusitis was defined, and then was used for the development of the mobile application. Conclusion The creation of the mobile application for evaluating patients with chronic rhinosinusitis followed an adequate methodology of elaboration made by specialists in the field of chronic rhinosinusitis, standardizing the investigation of these patients. Level of evidence: Level 5.

Contributions of RNA-seq to improve in vitro embryo production (IVP).

In Vitro Embryo Production (IVP) is widely used to improve the reproductive efficiency of livestock animals, however increasing the embryo development rates and pregnancy outcomes is still a challenge for some species. Thus, the lack of biological knowledge hinders developing specie-specific IVP protocols. Therefore, the contributions of RNA-seq to generate relevant biological knowledge and improve the efficiency of IVP in livestock animals are reviewed herein.

Cutting Edge: Transcriptional Profiling Reveals Multifunctional and Cytotoxic Antiviral Responses of Zika Virus-Specific CD8+ T Cells.

Zika virus (ZIKV) constitutes an increasing public health problem. Previous studies have shown that CD8+ T cells play an important role in ZIKV-specific protective immunity. We have previously defined antigenic targets of the ZIKV-specific CD8+ T cell response in humans. In this study, we characterized the quality and phenotypes of these responses by a combined use of flow cytometry and transcriptomic methods, using PBMCs from donors deriving from different geographical locations collected in the convalescent phase of infection. We show that ZIKV-specific CD8+ T cells are characterized by a polyfunctional IFN-γ signature with upregulation of TNF-α, TNF receptors, and related activation markers, such as CD69, as well as a cytotoxic signature characterized by strong upregulation of GZMB and CRTAM. The signature is stable and not influenced by previous dengue virus exposure, geographical location, or time of sample collection postinfection. To our knowledge, this work elucidates the first in-depth characterization of human CD8+ T cells responding to ZIKV infection.

Exploring the practicing-connections hypothesis: using gesture to support coordination of ideas in understanding a complex statistical concept.

In this article, we begin to lay out a framework and approach for studying how students come to understand complex concepts in rich domains. Grounded in theories of embodied cognition, we advance the view that understanding of complex concepts requires students to practice, over time, the coordination of multiple concepts, and the connection of this system of concepts to situations in the world. Specifically, we explore the role that a teacher's gesture might play in supporting students' coordination of two concepts central to understanding in the domain of statistics: mean and standard deviation. In Study 1 we show that university students who have just taken a statistics course nevertheless have difficulty taking both mean and standard deviation into account when thinking about a statistical scenario. In Study 2 we show that presenting the same scenario with an accompanying gesture to represent variation significantly impacts students' interpretation of the scenario. Finally, in Study 3 we present evidence that instructional videos on the internet fail to leverage gesture as a means of facilitating understanding of complex concepts. Taken together, these studies illustrate an approach to translating current theories of cognition into principles that can guide instructional design.

Effect of cortisol on bovine oocyte maturation and embryo development in vitro.

Glucocorticoids (GCs) are important mediators of key cellular events. Herein, we investigated the effect of adding cortisol to the IVM medium on the acquisition of developmental competency in bovine oocytes. Cortisol (0.01, 0.1, or 1 µg/mL) had no effect on cleavage rates or cell numbers of resulting blastocysts; however, supplementation with 0.1 µg/mL during IVM increased blastocyst rates of in vitro-fertilized bovine oocytes as compared to untreated controls (41 ± 10% vs. 21 ± 1.2%, P < 0.05, respectively). This concentration was chosen to assess changes in the relative expression of potential GC target genes. Oocytes matured in the presence of cortisol and their corresponding cumulus cells did not show changes in expression for genes analyzed as compared to untreated controls. Notably, blastocysts from oocytes matured in cortisol-supplemented medium expressed higher relative levels of glucose transporter 1 (GLUT1), fatty acid synthase (FASN), and heat shock protein 70 (HSP70). This study supports a role for cortisol in the acquisition of bovine oocyte competence. This is evidenced by increased blastocyst development rates and presumably related to elevated embryonic transcripts with roles in glucose and lipid metabolism, as well as the cellular response to stress.

Effect of dexamethasone on development of in vitro-produced bovine embryos.

Studies in somatic cells have shown that glucocorticoids such as dexamethasone (DEX) may trigger or prevent apoptosis depending on the cell type in culture. Because the dysregulation of apoptosis may lower in vitro embryo production efficiency, we sought to investigate the effects of supplementing IVC medium with DEX (0.1 µg/mL) on embryo morphology, development kinetics, and apoptosis rates of in vitro-produced bovine preimplantation embryos. Embryo morphology was graded on Day 7, and development rates were assessed on Days 4 and 7 of IVC. Apoptosis was evaluated via annexin/propidium iodide staining under fluorescence microscopy where a cell labeled with annexin, propidium iodide, or both would be considered apoptotic. An embryo was counted in the apoptosis rates, if it displayed at least one such labeled cell. Although DEX supplementation did not reduce apoptosis rates, it had a positive impact on developmental kinetics and cell number both on Days 4 and 7 of embryo culture. Presumably, such effect resulted from increased cell proliferation rather than a direct inhibition of apoptosis. Further studies may evaluate the mechanisms by which glucocorticoids may affect embryo development, as DEX supplementation could become a tool to improve in vitro embryo yield in mammalian species.