Functional Roles of Charged Amino Acid Residues on the Wall of the Cytoplasmic Pore of Kir2.1

It is known that rectification of currents through the inward rectifier K+ channel (Kir) is mainly due to blockade of the outward current by cytoplasmic Mg2+ and polyamines. Analyses of the crystal structure of the cytoplasmic region of Kir2.1 have revealed the presence of both negatively (E224, D255, D259, and E299) and positively (R228 and R260) charged residues on the wall of the cytoplasmic pore of Kir2.1, but the detail is not known about the contribution of these charged residues, the positive charges in particular, to the inward rectification. We therefore analyzed the functional significance of these charged amino acids using single/double point mutants in order to better understand the structure-based mechanism underlying inward rectification of Kir2.1 currents. As a first step, we used two-electrode voltage clamp to examine inward rectification in systematically prepared mutants in which one or two negatively or positively charged amino acids were neutralized by substitution. We found that the intensity of the inward rectification tended to be determined by the net negative charge within the cytoplasmic pore. We then used inside-out excised patch clamp recording to analyze the effect of the mutations on blockade by intracellular blockers and on K+ permeation. We observed that a decrease in the net negative charge within the cytoplasmic pore reduced both the susceptibility of the channel to blockade by Mg2+ or spermine and the voltage dependence of the blockade. It also reduced K+ permeation; i.e., it decreased single channel conductance, increased open-channel noise, and strengthened the intrinsic inward rectification in the total absence of cytoplasmic blockers. Taken together, these data suggest that the negatively charged cytoplasmic pore of Kir electrostatically gathers cations such as Mg2+, spermine, and K+ so that the transmembrane pore is sufficiently filled with K+ ions, which enables strong voltage-dependent blockade with adequate outward K+ conductance

Modified cellular automaton simulation of metal additive manufacturing

Metal additive manufacturing (AM) technologies are attracting attentions not only as a fabrication process of complicated threedimensional parts but also as microstructure controlling processes. In powder bed fusion (PBF)-type AM, crystallographic texture can be controlled by scanning strategies of energy beam. To optimize microstructures, computer simulations for predicting microstructures play very important roles. In this work, we have developed simulation programs to explain the mechanism of the crystal orientation control. First, we simulated the shape of melt pool by analyzing the heat transfer using apparent heat conductivity when the penetration of laser beam through keyholes was taken into consideration because of the evaporation and accompanying convections. It was assumed that the primary crystal growth direction can be determined by the temperature gradient, and the crystals grow keeping the growth direction as generally recognized. The shapes of simulated melt pools agree well with experimental observations. The modified cellular automaton simulations successfully reproduced two typical textures with different preferential orientations along the building directions of (100) and (110) when the bidirectional scanning with and without a rotation of 90°, respectively, was accomplished between the layers.Kubo J., Koizumi Y., Ishimoto T., et al. Modified cellular automaton simulation of metal additive manufacturing. Materials Transactions 62, 864 (2021); https://doi.org/10.2320/matertrans.MT-M2021009

Voltage- and [ATP]-dependent Gating of the P2X2 ATP Receptor Channel

P2X receptors are ligand-gated cation channels activated by extracellular adenosine triphosphate (ATP). Nonetheless, P2X2 channel currents observed during the steady-state after ATP application are known to exhibit voltage dependence; there is a gradual increase in the inward current upon hyperpolarization. We used a Xenopus oocyte expression system and two-electrode voltage clamp to analyze this “activation” phase quantitatively. We characterized the conductance–voltage relationship in the presence of various [ATP], and observed that it shifted toward more depolarized potentials with increases in [ATP]. By analyzing the rate constants for the channel's transition between a closed and an open state, we showed that the gating of P2X2 is determined in a complex way that involves both membrane voltage and ATP binding. The activation phase was similarly recorded in HEK293 cells expressing P2X2 even by inside-out patch clamp after intensive perfusion, excluding a possibility that the gating is due to block/unblock by endogenous blocker(s) of oocytes. We investigated its structural basis by substituting a glycine residue (G344) in the second transmembrane (TM) helix, which may provide a kink that could mediate “gating.” We found that, instead of a gradual increase, the inward current through the G344A mutant increased instantaneously upon hyperpolarization, whereas a G344P mutant retained an activation phase that was slower than the wild type (WT). Using glycine-scanning mutagenesis in the background of G344A, we could recover the activation phase by introducing a glycine residue into the middle of second TM. These results demonstrate that the flexibility of G344 contributes to the voltage-dependent gating. Finally, we assumed a three-state model consisting of a fast ATP-binding step and a following gating step and estimated the rate constants for the latter in P2X2-WT. We then executed simulation analyses using the calculated rate constants and successfully reproduced the results observed experimentally, voltage-dependent activation that is accelerated by increases in [ATP]

The Determinant of University Spin-off Ventures that Went Public in the Short Term : A qualitative comparative analysis for management resources

We performed the qualitative comparative analysis to test which interaction among founding team characteristics and other management resources affect the short-term initial public offerings (IPOs). We used data from the Ministry of Economy, Trade and Industry (METI), 65 university-launched ventures with IPO. We found three causal pathways for achieving IPO rapidly from establishment. The first pathway is founded by experienced top management personnel and the filing of patent applications. The second pathway is a student-founded company with patent applications and university VC raised fund. And the third is a founding team of researchers and management personnel with university VC raised fund. This research contributed to imprinting theory by investigating the relationship of achieving IPO from establishment quickly and the combination of founding team and the other management resources of university-launched ventures in Japan. In addition, practical implications were presented for VCs and support organizations regarding the composition of the founding team and the nature of support for achieving IPO rapidly

Research for carbon dioxide bathing IV, Thermal effect of artificial CO(2)-bathing

1) 炭酸塩と,コ-ク酸からなる錠剤型の｢炭酸ガス浴剤｣の保温作用を健康な男子8名について,サ-モグラフィーを用いて測定した. 入浴10分後の比較で明らかに,炭酸ガス浴はよく温まった結果,表面温度が高くなっている. 2) 腰痛,四肢冷感,その他の患者24名での臨床評価の結果,患者の90%以上が手足が温まり,湯ざめしにくいことを認めた.また,患者の85%は痛みがやわらぐことを認めた. 3) 主婦664名を対象とした使用評価の結果,常時手足の冷感を訴える者の63.6%,身体の疲労感･だるさ56.5%に効果を認めていることがわかった. 4) 副作用は全く認められなかった.The artificial CO(2)-bath was prepared with a tablet (50g), made from sodium bicarbonate and succinic acid, putting simply in plain water bathtub of 150-200 litre at 40℃. Thermal effect was evaluated by means of thermography in healthy 8 men; temperature areas of 34.5℃ or greater were recorded in average compared to a plain bathing and to an artificial Na(2)SO(4)-NaHCO(3) one 10 minutes later. Clinical evaluation by questionnairings of 24 patients suffering mainly from lumbago and 4-extremity coldness revealed a long lasting peripheral warmth in 90% and the ease from pain in 85%. The artificial CO(2)-bathing was effective for women whose health were adversely affected by the cold in 63.6% and for general fatigue or dullness in 56.5% of 664 female volunteers whom the questionnairings were conducted to. No side effect was encountered in the survey

Associations between Dietary Patterns, ADRβ2 Gln27Glu and ADRβ3 Trp64Arg with Regard to Serum Triglyceride Levels : J-MICC Study

Interactions between dietary patterns and 2 β-adrenergic receptor (ADRβ) gene polymorphisms (ADRβ2 Gln27Glu and ADRβ3 Trp64Arg) were examined with regard to the effects on serum triglyceride levels. The cross-sectional study comprised 1720 men and women (aged 35–69 years) enrolled in the Japan Multi-Institutional Collaborative Cohort (J-MICC) Study. Genotyping was conducted using a multiplex polymerase chain reaction-based invader assay. We used 46 items from a validated short food frequency questionnaire and examined major dietary patterns by factor analysis. We identified four dietary patterns: healthy, Western, seafood and bread patterns. There was no significant association between any dietary pattern and serum triglyceride levels. After a separate genotype-based analysis, significant interactions between ADRβ3 Trp64Arg genotype and the bread pattern (p for interaction = 0.01) were associated with serum triglyceride levels; specifically, after adjusting for confounding factors, Arg allele carriers with the bread pattern had lower serum triglycerides (p for trend = 0.01). However, the Trp/Trp homozygous subjects with the bread pattern showed no association with serum triglycerides (p for trend = 0.55). Interactions between other dietary patterns and ADRβ polymorphisms were not significant for serum triglyceride levels. Our findings suggest that ADRβ3 polymorphism modifies the effects of the bread pattern on triglyceride levels

Distinguishing intrahepatic cholangiocarcinoma from poorly differentiated hepatocellular carcinoma using precontrast and gadoxetic acid-enhanced MRI

PURPOSEWe aimed to gain further insight in magnetic resonance imaging characteristics of mass-forming intrahepatic cholangiocarcinoma (mICC), its enhancement pattern with gadoxetic acid contrast agent, and distinction from poorly differentiated hepatocellular carcinoma (pHCC).METHODSFourteen mICC and 22 pHCC nodules were included in this study. Two observers recorded the tumor shape, intratumoral hemorrhage, fat on chemical shift imaging, signal intensity at the center of the tumor on T2-weighted image, fibrous capsule, enhancement pattern on arterial phase of dynamic study, late enhancement three minutes after contrast injection (dynamic late phase), contrast uptake on hepatobiliary phase, apparent diffusion coefficient, vascular invasion, and intrahepatic metastasis.RESULTSLate enhancement was more common in mICC (n=10, 71%) than in pHCC (n=3, 14%) (P < 0.001). A fat component was observed in 11 pHCC cases (50%) versus none of mICC cases (P = 0.002). Fibrous capsule was observed in 13 pHCC cases (59%) versus none of mICC cases (P < 0.001). On T2-weighted images a hypointense area was seen at the center of the tumor in 43% of mICC (6/14) and 9% of pHCC (2/22) cases (P = 0.018). Other parameters were not significantly different between the two types of nodules.CONCLUSIONThe absence of fat and fibrous capsule, and presence of enhancement at three minutes appear to be most characteristic for mICC and may help its differentiation from pHCC

Mendelian Randomization on hs-CRP and eGFR

Background: Inflammation is thought to be a risk factor for kidney disease. However, whether inflammatory status is either a cause or an outcome of chronic kidney disease remains controversial. We aimed to investigate the causal relationship between high-sensitivity C-reactive protein (hs-CRP) and estimated glomerular filtration rate (eGFR) using Mendelian randomization (MR) approaches. Methods: A total of 10,521 participants of the Japan Multi-institutional Collaborative Cohort Study was analyzed in this study. We used two-sample MR approaches (the inverse-variance weighted (IVW), the weighted median (WM), and the MR-Egger method) to estimate the effect of genetically determined hs-CRP on kidney function. We selected four and three hs-CRP associated single nucleotide polymorphisms (SNPs) as two instrumental variables (IV): IVCRP and IVAsian, based on SNPs previously identified in European and Asian populations. IVCRP and IVAsian explained 3.4% and 3.9% of the variation in hs-CRP, respectively. Results: Using the IVCRP, genetically determined hs-CRP was not significantly associated with eGFR in the IVW and the WM methods (estimate per 1 unit increase in ln(hs-CRP), 0.000; 95% confidence interval [CI], −0.019 to 0.020 and −0.003; 95% CI, −0.019 to 0.014, respectively). For IVAsian, we found similar results using the IVW and the WM methods (estimate, 0.005; 95% CI, −0.020 to 0.010 and −0.004; 95% CI, −0.020 to 0.012, respectively). The MR-Egger method also showed no causal relationships between hs-CRP and eGFR (IVCRP: −0.008; 95% CI, −0.058 to 0.042; IVAsian: 0.001; 95% CI, −0.036 to 0.036). Conclusion: Our two-sample MR analyses with different IVs did not support a causal effect of hs-CRP on eGFR

Association of skipping breakfast and short sleep duration with the prevalence of metabolic syndrome in the general Japanese population : Baseline data from the Japan Multi-Institutional Collaborative cohort study

The purpose of the study was to investigate sex-specific associations of skipping breakfast and short sleep duration with metabolic syndrome (MetS) and their interaction. We analyzed baseline data of 14,907 men and 14,873 women aged 35–69 years, who participated in the Japan Multi-Institutional Collaborative Cohort Study from 2005. MetS was diagnosed using a modification of the National Cholesterol Education Program Adult Treatment Panel III revised definition (NCEP-R 2005), using body mass index instead of waist circumference. Breakfast consumption was classified into two categories: ≥6 days/week (consumers) or <6 days/week (skippers). Sleep duration was classified into three categories: <6h, 6 to <8 h, and ≥8 h/day. Multivariate logistic regression analysis was performed to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) and examine the presence of interaction. In men, skipping breakfast and short sleep duration were independently associated with an increased prevalence of MetS (OR 1.26, 95%CI 1.12–1.42 and OR 1.28, 95%CI 1.12–1.45, respectively), obesity, and components of MetS. However, no significant interaction was observed between skipping breakfast and short sleep duration. In women, skipping breakfast and short sleep duration were associated with an increased prevalence of obesity, but not with MetS. These findings indicate that breakfast consumption and moderate sleep duration may be associated with a lower risk of MetS, particularly in men