Utilization of a serum-free primary culture of cortical neurons by using cyclodextrins in neurobiological research

神経生物学的研究における基本的分析系の確立を目的として、シクロデキストリン（CD）を用いたラット大脳皮質神経細胞の初代培養を試みた。β-およびγ-CDは、無血清培地（ダルベッコ改変MEM/ハム培地）中で胎生16および18日目ラットの神経細胞を11日以上10％胎児ウシ血清を加えた培地中と同じ程度に生存させたが、α-CDには生存維持効果が無かった。β-CDはγ-CDより安定した生存維持効果を示したが、胎生21日目ラットの神経細胞を用いた場合は有意に生存率が低下し、新生児ラットでは生存維持効果が無かった。β-CDを用いた無血清培養では10％血清培地中と比べて神経突起の伸展が悪かったが、ときに顕著な突起伸展がみられ、これはCD分子に取り込まれた生理活性物質の作用と考えられた。また、β-CDを用いた無血清培養を利用してラット脳から精製したコンドロイチン硫酸プロテオグリカン（CSPG）の作用を検討し、CSPGがグルタミン酸による神経細胞死を防止すること、弱いながら培養神経細胞の生存を維持する作用をもつことを示した。以上の結果から、この無血清培養法は神経生物学的研究において有用な分析系となりうることを指摘した

Spontaneous Bilateral Pneumothorax in a Patient with Anorexia Nervosa: The Management of Prolonged Postoperative Air Leakage

A 24-year-old Japanese female with anorexia nervosa presented to our hospital for bilateral pneumothorax, and 12-Fr thoracostomy catheters were inserted into the bilateral pleural cavities. On hospital day 9, a thoracoscopic bullectomy was performed. However, air leakage relapsed on both sides on postoperative day 1. The air leakage on the right side was particularly persistent, and we switched the drainage to a Heimlich valve. Both lungs expanded gradually and the chest tube was removed on postoperative day 19. Passive pleural drainage might be an option for prolonged air leakage after a bullectomy in patients with anorexia nervosa

Real-time TIRF observation of vinculin recruitment to stretched α-catenin by AFM

Adherens junctions (AJs) adaptively change their intensities in response to intercellular tension; therefore, they integrate tension generated by individual cells to drive multicellular dynamics, such as morphogenetic change in embryos. Under intercellular tension, α-catenin, which is a component protein of AJs, acts as a mechano-chemical transducer to recruit vinculin to promote actin remodeling. Although in vivo and in vitro studies have suggested that α-catenin-mediated mechanotransduction is a dynamic molecular process, which involves a conformational change of α-catenin under tension to expose a cryptic vinculin binding site, there are no suitable experimental methods to directly explore the process. Therefore, in this study, we developed a novel system by combining atomic force microscopy (AFM) and total internal reflection fluorescence (TIRF). In this system, α-catenin molecules (residues 276–634; the mechano-sensitive M1-M3 domain), modified on coverslips, were stretched by AFM and their recruitment of Alexa-labeled full-length vinculin molecules, dissolved in solution, were observed simultaneously, in real time, using TIRF. We applied a physiologically possible range of tensions and extensions to α-catenin and directly observed its vinculin recruitment. Our new system could be used in the fields of mechanobiology and biophysics to explore functions of proteins under tension by coupling biomechanical and biochemical information

EFFECT OF FERULIC ACID ON PIG OOCYTES

The value of laboratory and genetically-modified pigs is becoming increasingly clear; however, their in vitro development remains inefficient. Trans-ferulic acid (trans-FA) is an aromatic compound that is abundant in plant cell walls, and which exhibits antioxidant effects in vitro. Trans-FA is known to improve sperm viability and motility; however, its effects on porcine oocytes are unknown. Our aim was to investigate the effects of trans-FA supplementation during in vitro maturation on the meiotic and developmental competence of porcine oocytes. Oocytes were matured either without (control) or with trans-FA (10, 100 and 1,000 µM), fertilized, and cultured in vitro for 7 days. The maturation rate of oocytes cultured with 10 µM trans-FA (81.6%) was significantly higher than that of controls (65.0%; P<0.05). The fertilization rate of oocytes matured with 10 µM trans-FA (57.4%) was also significantly higher than that of controls (32.7%) and oocytes cultured with other concentrations (33.1% and 22.7% for 100 and 1,000 µM, respectively; P<0.05). Moreover, the blastocyst formation rate of oocytes matured with 10 µM trans-FA (6.9%) was significantly higher than that of controls (2.3%; P<0.05). Our results suggest that in vitro maturation with 10 µM trans-FA is beneficial for the in vitro production of porcine embryos and has the potential to improve production system

EMBRYONIC MOSAICISM BY MICROINJECTION

Cytoplasmic microinjection (CI) of the CRISPR/Cas9 system enabled the induction of site-specific mutations in porcine zygotes and resulting pigs. However, mosaicism is a serious problem for genetically modified pigs. In the present study, we investigated suitable timing and concentration of CRISPR/Cas9 components for introduction into oocytes/zygotes by CI, to reduce mosaicism in the resulting blastocysts. First, we introduced 20 ng/μl of Cas9 protein and guide RNA (gRNA), targeting the α-1,3-galactosyltransferase (GalT) gene in oocytes before in vitro fertilization (IVF), in zygotes after IVF, or in oocytes/zygotes before and after IVF, twice. CI treatment had no detrimental effects on blastocyst formation rates. The highest value of the rate of mutant blastocysts was observed in zygotes injected after IVF. Next, we injected Cas9 protein and gRNA into zygotes after IVF at a concentration of 20 ng/μl each (20 ng/μl group) or 100 ng/μl each (100 ng/μl group). The ratio of the number of blastocysts that carried mutations to the total number of blastocysts examined in the 100 ng/μl group was significantly higher (P < 0.05) than that in the 20 ng/μl group. Although no blastocysts from the 20 ng/μl group carried a biallelic mutation, 16.7% of blastocysts from the 100 ng/μl group carried a biallelic mutation. In conclusion, increasing the concentration of Cas9 protein and gRNA is effective in generating biallelic mutant blastocysts. To reduce mosaicism, however, further optimization of the timing of CI, and the concentration of CRISPR/Cas9 components, is needed

Model-Free Idealization: Adaptive Integrated Approach for Idealization of Ion Channel Currents (AI2)

Single-channel electrophysiological recordings provide insights into transmembrane ion permeation and channel gating mechanisms. The first step in the analysis of the recorded currents involves an "idealization" process, in which noisy raw data are classified into two discrete levels corresponding to the open and closed states of channels. This provides valuable information on the gating kinetics of ion channels. However, the idealization step is often challenging in cases of currents with poor signal-to-noise ratios (SNR) and baseline drifts, especially when the gating model of the target channel is not identified. We report herein on a highly robust model-free idealization method for achieving this goal. The algorithm, called AI2 (Adaptive Integrated Approach for the Idealization of Ion Channel Currents), is composed of Kalman filter and Gaussian Mixture Model (GMM) clustering and functions without user input. AI2 automatically determines the noise reduction setting based on the degree of separation between the open and closed levels. We validated the method on pseudo-channel-current datasets which contain either computed or experimentally recorded noise. The AI2 algorithm was then tested on actual experimental data for biological channels including gramicidin A, a voltage-gated sodium channel, and other unidentified channels. We compared the idealization results with those obtained by the conventional methods, including the 50%-threshold-crossing method

Chlorogenic and caffeic acid supplementation during sperm freezing

Chlorogenic acid (CGA) and caffeic acid (CA) are potent antioxidants that are mostly found in coffee beans. This study aimed to investigate the effects of CGA and CA supplementation during semen freezing on the quality of frozen-thawed boar spermatozoa. The antioxidants CGA and CA were added to a semen extender to achieve final concentrations of 50, 100, 200 and 400 µM. Supplementation of 100 µM CGA and CA yielded a significantly higher percentage of sperm viability (increased by 8 - 10%) and plasma membrane integrity (increased by 4 - 6%) than the control groups without the antioxidants at 0 h and 3 h after thawing (P < 0.05). At a concentration of 100 µM, CGA and CA also yielded beneficial effects on total and progressive sperm motility. Increases of CGA and CA concentrations to more than 200 µM did not enhance any sperm quality parameters. When the sperm penetrability and oocyte development by spermatozoa frozen with CGA and CA were evaluated, CGA and CA supplementations had no positive effects on the percentages of total fertilization, monospermic fertilization, cleavage and blastocyst formation. In conclusion, the supplementation of 100 µM CGA and CA during sperm freezing improved certain sperm parameters including motility, viability and plasma membrane integrity

The Relationship between Embryonic Development and the Efficiency of Target Mutations in Porcine Endogenous Retroviruses (PERVs) Pol Genes in Porcine Embryos

Porcine endogenous retrovirus (PERV) is a provirus found in the pig genome that may act as an infectious pathogen in humans who receive pig organ xenotransplantation. Inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. Therefore, the mutation of PERV pol gene in porcine embryos using genome editing may affect the embryonic development. The present study was carried out to investigate the relationship between the mutation of the PERV pol gene in porcine embryos and their development. We introduced, either alone or in combination, three different gRNAs (gRNA1, 2, and 3) into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. All three gRNAs targeted the PERV pol gene, and we assessed their effects on porcine embryonic development. Our results showed that the blastocyst formation rates of zygotes electroporated with gRNA3—alone and in combination—were significantly lower (p < 0.05) than those of zygotes electroporated with gRNA1. The mutation rates assessed by the PERV pol gene target site sequencing in individual blastocysts and pooled embryos at the 2-to-8-cell stage did not differ among the three gRNAs. However, the frequency of indel mutations in mutant embryos at the 2-to-8-cell stage trended higher in the embryos electroporated with gRNA3 alone and in combination. Embryonic development may be affected by gRNAs that induce high-frequency indel mutations.Pigs with porcine endogenous retrovirus (PERV) inactivation are preferable donor sources for xenotransplantation because the PERV may act as an infectious pathogen for humans who receive pig organ xenotransplantation. However, inactivation of the PERV pol gene in porcine cells reportedly affects cell growth. The present study clarified the relationship between the mutation of the PERV pol gene in porcine embryos and their development. Three different gRNAs targeting the PERV pol gene were introduced into porcine zygotes by genome editing using electroporation of the Cas9 protein (GEEP) system. The results demonstrated a negative relationship between the embryonic development and the efficiency of target mutations in the PERV pol gene of the porcine embryos

Effects of Tris (hydroxymethyl) aminomethane on the quality of frozen-thawed boar spermatozoa

Tris (hydroxymethyl) aminomethane (Tris) has been used as a pH regulator for buffering the pH of dilution extenders for boar semen, such as the Modena extender. The purpose of the present study was to assess the effects of Tris supplementation at different concentrations (0, 8, 24 and 72 μM) into the freezing extender on the quality and fertilising capacity of frozen-thawed boar spermatozoa. The results showed that the supplementation of 24 μM of Tris gave significantly higher percentages of sperm viability and plasma membrane integrity than those of the control group at any time point of assessment (0 h and 3 h post-thawing) (P < 0.05). However, there were no significant differences in the acrosome integrity parameter among the groups. Higher percentages of sperm motility were observed in the spermatozoa cryopreserved with 24 μM of Tris compared to the control groups when the samples were analysed 0 h after thawing (P < 0.05). However, an increase of the Tris concentration to 72 μM did not enhance the sperm motility parameters. The total numbers of fertilised oocytes and blastocysts obtained with spermatozoa frozen with 24 μM Tris were significantly higher than those of the control group without Tris (P < 0.05). In conclusion, the supplementation of 24 μM Tris into the freezing extender contributes to a better boar sperm quality and fertilising capacity after the process of freezing and thawing