In-vitro Culture with a Tilting Device in Chemically Defined Media During Meiotic Maturation and Early Development Improves the Quality of Blastocysts Derived from In-vitro Matured and Fertilized Porcine Oocytes

Under physiological conditions, mammalian oocytes and embryos appear to be stimulated not only chemically but also mechanically, such as by compression, shear stress and/or friction force in the follicle and female reproductive tract. The present study was undertaken to examine the effects of kinetic culture with a tilting device in chemically defined media during in vitro maturation (IVM) of porcine oocytes and in vitro culture (IVC) following in vitro fertilization (IVF) on the early developmental competence and quality of blastocysts. After culture in a chemically defined IVM medium, modified porcine oocyte medium (mPOM) containing gonadotropins and dibutyryl cAMP for 20 h, the mean diameter of the cumulus-oocyte complexes (COCs) was larger in the tilting culture than in the static controls, whereas the diameter of the oocytes did not differ. When culture of the COCs was continued additionally in a fresh medium without gonadotropins and dibutyryl cAMP for 24 h, the incidences of oocytes completing GVBD and developing to the metaphase-II stage did not differ between the tilting and static culture systems. Furthermore, the sperm penetration after IVF and developmental competence of the oocytes to the blastocyst stage were not different between the tilting and static systems during IVM and IVC. However, tilting culture during both IVM and IVC had a significant positive effect on the number of cells per blastocyst (P<0.05). These observations indicate that tilting culture during IVM and IVC in chemically defined media improves the quality of blastocyst, as determined by the number of cells per blastocyst, without any effects on penetrability and developmental competence

The Neutral Self-Assembling Peptide Hydrogel SPG-178 as a Topical Hemostatic Agent

Conventional self-assembling peptide hydrogels are effective as topical hemostatic agents. However, there is a possibility to harm living tissues due to their low pH. The aim of the present study was to demonstrate the efficacy of SPG-178, a neutral self-assembling peptide hydrogel, as a topical hemostatic agent. First, we measured the bleeding duration of incisions made on rat livers after application of SPG-178 (1.0% w/v), SPG-178 (1.5% w/v), RADA16 (1.0% w/v), and saline (n = 12/group). Second, we observed the bleeding surfaces by transmission electron microscopy immediately after hemostasis. Third, we measured the elastic and viscous responses (G′ and G″, respectively) of the hydrogels using a rheometer. Our results showed that bleeding duration was significantly shorter in the SPG-178 group than in the RADA16 group and that there were no significant differences in transmission electron microscopy findings between the groups. The greater the G′ value of a hydrogel, the shorter was the bleeding duration. We concluded that SPG-178 is more effective and has several advantages: it is non-biological, transparent, nonadherent, and neutral and can be sterilized by autoclaving

Systematic Understanding of Pathophysiological Mechanisms of Oxidative Stress-Related Conditions-Diabetes Mellitus, Cardiovascular Diseases, and Ischemia-Reperfusion Injury

Reactive oxygen species (ROS) plays a role in intracellular signal transduction under physiological conditions while also playing an essential role in diseases such as hypertension, ischemic heart disease, and diabetes, as well as in the process of aging. The influence of ROS has some influence on the frequent occurrence of cardiovascular diseases (CVD) in diabetic patients. In this review, we considered the pathophysiological relationship between diabetes and CVD from the perspective of ROS. In addition, considering organ damage due to ROS elevation during ischemia-reperfusion, we discussed heart and lung injuries. Furthermore, we have focused on the transient receptor potential (TRP) channels and L-type calcium channels as molecular targets for ROS in ROS-induced tissue damages and have discussed about the pathophysiological mechanism of the injury

Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress

Ischemic heart disease is one of the most common causes of death worldwide. Mitochondrial dysfunction, excessive reactive oxygen species (ROS) generation, and calcium (Ca2þ) overload are three key factors leading to myocardial death during ischemia-reperfusion (I/R) injury. Inhibition of TRPM4, a Ca2þ-activated nonselective cation channel, protects the rat heart from I/R injury, but the specific mechanism underlying this effect is unclear. In this study, we investigated the mechanism of cardioprotection against I/R injury via TRPM4 using hydrogen peroxide (H2O2), a major contributor to oxidative stress, as an I/R injury model. We knocked out the TRPM4 gene in the rat cardiomyocyte cell line H9c2 using CRISPR/Cas9. Upon H2O2 treatment, intracellular Ca2þ level and ROS production increased in wild type (WT) cells but not in TRPM4 knockout (TRPM4KO) cells. With this treatment, two indicators of mitochondrial function, mitochondrial membrane potential (DJm) and intracellular ATP levels, decreased inWT but not in TRPM4KO cells. Taken together, these findings suggest that blockade of the TRPM4 channel might protect the myocardium from oxidative stress by maintaining the mitochondrial membrane potential and intracellular ATP levels, possibly through preventing aberrant increases in intracellular Ca2þ and ROS

Development of a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells

Ischemic heart disease remains the largest cause of death worldwide. Accordingly, many researchers have sought curative options, often using laboratory animal models such as rodents. However, the physiology of the human heart differs significantly from that of the rodent heart. In this study, we developed a model of ischemic heart disease using cardiomyocytes differentiated from human induced pluripotent stem cells (hiPS-CMs). After optimizing the conditions of ischemia, including the concentration of oxygen and duration of application, we evaluated the consequent damage to hiPS-CMs. Notably, exposure to 2% oxygen, 0 mg/ml glucose, and 0% fetal bovine serum increased the percentage of nuclei stained with propidium iodide, an indicator of membrane damage, and decreased cellular viability. These conditions also decreased the contractility of hiPS-CMs. Furthermore, ischemic conditioning increased the mRNA expression of IL-8, consistent with observed conditions in the in vivo heart. Taken together, these findings suggest that our hiPS-CM-based model can provide a useful platform for human ischemic heart disease research

Spiral Trajectory Modulation of Rheotaxic Motile Human Sperm in Cylindrical Microfluidic Channels of Different Inner Diameters

We investigated the relationship between human sperm rheotaxis and motile sperm trajectories by using poly-(dimethylsiloxane) (PDMS)-based cylindrical microfluidic channels with inner diameters of 100 μm, 50 μm, and 70 μm, which corresponded to the inner diameter of the human isthmus, the length of a sperm and a diameter intermediate between the two, respectively. We counted the number of rheotaxic sperm and sperm with spiral motion. We also analyzed motile sperm trajectories. As the cylindrical channel diameter was decreased, the percentage of sperm cells exhibiting rheotaxis, the percentage of sperm cells exhibiting spiral motion, the frequency-to-diameter ratio of the sperm cells’ spiral trajectories, and the surface area of the microfluidic channel increased, while the flagellar motion at the channel wall decreased. The percentage of sperm exhibiting a spiral trajectory and the frequency-to-diameter ratio of the sperm cells’ spiral trajectories were thus affected by the channel diameter. Our findings suggest that the oviduct structure affects the swimming properties of sperm cells, guiding them from the uterus to the ampulla for egg fertilization. These results could contribute to the development of motile sperm-sorting microfluidic devices for assisted reproductive technologies

Production of TRPM4 knockout cell line using rat cardiomyocyte H9c2

The method presented in this article are related to the research article entitled as "Role of the TRPM4 channel in mitochondrial function, calcium release, and ROS generation in oxidative stress" [1]. TRPM4, a non-selective monovalent cation channel, is not only involved in the generation of the action potential in cardiomyocytes, but also thought to be a key molecule in the development of the ischemia-reperfusion injury of the brain and the heart [2-5]. However, existing pharmacological inhibitors for the TRPM4 channel have problems of non-specificity [6]. This article describes methods used for targeted genomic deletion in the rat cardiomyocyte H9c2 using the CRISPR-Cas9 genome editing system in order to suppress TRPM4 protein expression. Confocal microscopy, flow cytometry, Sanger sequencing, and western blotting are performed to confirm vector transfection and the subsequent knockout of the TRPM4 protein. These data provide information on the comprehensive analyses for knocking out the rat TRPM4 channel using CRISPR/Cas9. The analyses include confocal microscopy, flow cytometry, Sanger sequencing, and western blotting. This dataset will benefit biological and medical researchers studying the function of TRPM4-expressing cells including neurons, cardiomyocytes, and vascular endothelial cells. It is also useful to study the involvement of the TRPM4 channel in pathological processes such as cardiac arrhythmia and ischemia-reperfusion injury. The dataset can be used to guide the experiment of knocking out the TRPM4 gene and its subsequent application to the study of disease process caused by the gene

Treatment of Oxidative Stress with Exosomes in Myocardial Ischemia

A thrombus in a coronary artery causes ischemia, which eventually leads to myocardial infarction (MI) if not removed. However, removal generates reactive oxygen species (ROS), which causes ischemia-reperfusion (I/R) injury that damages the tissue and exacerbates the resulting MI. The mechanism of I/R injury is currently extensively understood. However, supplementation of exogenous antioxidants is ineffective against oxidative stress (OS). Enhancing the ability of endogenous antioxidants may be a more effective way to treat OS, and exosomes may play a role as targeted carriers. Exosomes are nanosized vesicles wrapped in biofilms which contain various complex RNAs and proteins. They are important intermediate carriers of intercellular communication and material exchange. In recent years, diagnosis and treatment with exosomes in cardiovascular diseases have gained considerable attention. Herein, we review the new findings of exosomes in the regulation of OS in coronary heart disease, discuss the possibility of exosomes as carriers for the targeted regulation of endogenous ROS generation, and compare the advantages of exosome therapy with those of stem-cell therapy. Finally, we explore several miRNAs found in exosomes against OS

Meta-Analysis-Assisted Detection of Gravity-Sensitive Genes in Human Vascular Endothelial Cells

Gravity affects the function and maintenance of organs, such as bones, muscles, and the heart. Several studies have used DNA microarrays to identify genes with altered expressions in response to gravity. However, it is technically challenging to combine the results from various microarray datasets because of their different data structures. We hypothesized that it is possible to identify common changes in gene expression from the DNA microarray datasets obtained under various conditions and methods. In this study, we grouped homologous genes to perform a meta-analysis of multiple vascular endothelial cell and skeletal muscle datasets. According to the t-distributed stochastic neighbor embedding (t-SNE) analysis, the changes in the gene expression pattern in vascular endothelial cells formed specific clusters. We also identified candidate genes in endothelial cells that responded to gravity. Further, we exposed human umbilical vein endothelial cells (HUVEC) to simulated microgravity (SMG) using a clinostat and measured the expression levels of the candidate genes. Gene expression analysis using qRT-PCR revealed that the expression level of the prostaglandin (PG) transporter gene SLCO2A1 decreased in response to microgravity, consistent with the meta-analysis of microarray datasets. Furthermore, the direction of gravity affected the expression level of SLCO2A1, buttressing the finding that its expression was affected by gravity. These results suggest that a meta-analysis of DNA microarray datasets may help identify new target genes previously overlooked in individual microarray analyses