Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells

Hypokalemic periodic paralysis (HypoPP) is a rare genetic disease associated with mutations in CACNA1S or SCN4A, encoding Cav1.1 or Nav1.4, respectively. Most HypoPP-associated missense changes occur at the arginine residues within the voltage-sensing domain (VSD) of these channels. It is established that such mutations destroy the hydrophobic seal separating the external water and the internal cytosolic crevices, resulting in the generation of aberrant leak currents called gating pore currents. Presently, the gating pore currents are thought to underlie HypoPP. Here, we generated HEK293T-based HypoPP-model cell lines with the Sleeping Beauty transposon system that co-express mouse inward-rectifier potassium channel (mKir2.1) and HypoPP2-associated Nav1.4 variants. Whole cell patch-clamp measurements confirmed that mKir2.1 successfully hyperpolarized the membrane potential to comparable levels to myofibers, and that some Nav1.4 variants induced notable proton-based gating pore currents. Importantly, we succeeded in fluorometrically measuring the gating pore currents in these variants using a ratiometric pH indicator, SNARF-4F. Our optical method provides a potential in vitro platform for high-throughput drug screen, not only for HypoPP but also for other channelopathies caused by VSD mutations.Kubota T., Takahashi S., Yamamoto R., et al. Optical measurement of gating pore currents in hypokalemic periodic paralysis model cells. DMM Disease Models and Mechanisms 16, A18 (2023); https://doi.org/10.1242/dmm.049704

Brain pericytes among cells constituting the blood-brain barrier are highly sensitive to tumor necrosis factor-α, releasing matrix metalloproteinase-9 and migrating in vitro

<p>Abstract</p> <p>Background</p> <p>Increased matrix metalloproteinase (MMP)-9 in the plasma and brain is associated with blood-brain barrier (BBB) disruption through proteolytic activity in neuroinflammatory diseases. MMP-9 is present in the brain microvasculature and its vicinity, where brain microvascular endothelial cells (BMECs), pericytes and astrocytes constitute the BBB. Little is known about the cellular source and role of MMP-9 at the BBB. Here, we examined the ability of pericytes to release MMP-9 and migrate in response to inflammatory mediators in comparison with BMECs and astrocytes, using primary cultures isolated from rat brains.</p> <p>Methods</p> <p>The culture supernatants were collected from primary cultures of rat brain endothelial cells, pericytes, or astrocytes. MMP-9 activities and levels in the supernatants were measured by gelatin zymography and western blot, respectively. The involvement of signaling molecules including mitogen-activated protein kinases (MAPKs) and phosphoinositide-3-kinase (PI3K)/Akt in the mediation of tumor necrosis factor (TNF)-α-induced MMP-9 release was examined using specific inhibitors. The functional activity of MMP-9 was evaluated by a cell migration assay.</p> <p>Results</p> <p>Zymographic and western blot analyses demonstrated that TNF-α stimulated pericytes to release MMP-9, and this release was much higher than from BMECs or astrocytes. Other inflammatory mediators [interleukin (IL)-1β, interferon-γ, IL-6 and lipopolysaccharide] failed to induce MMP-9 release from pericytes. TNF-α-induced MMP-9 release from pericytes was found to be mediated by MAPKs and PI3K. Scratch wound healing assay showed that in contrast to BMECs and astrocytes the extent of pericyte migration was significantly increased by TNF-α. This pericyte migration was inhibited by anti-MMP-9 antibody.</p> <p>Conclusion</p> <p>These findings suggest that pericytes are most sensitive to TNF-α in terms of MMP-9 release, and are the major source of MMP-9 at the BBB. This pericyte-derived MMP-9 initiated cellular migration of pericytes, which might be involved in pericyte loss in the damaged BBB.</p

Optimizing highly noncoplanar VMAT trajectories: the NoVo method

We introduce a new method called NoVo (Noncoplanar VMAT Optimization) to produce volumetric modulated arc therapy (VMAT) treatment plans with noncoplanar trajectories. While the use of noncoplanar beam arrangements for intensity modulated radiation therapy (IMRT), and in particular high fraction stereotactic radiosurgery (SRS), is common, noncoplanar beam trajectories for VMAT are less common as the availability of treatment machines handling these is limited. For both IMRT and VMAT, the beam angle selection problem is highly nonconvex in nature, which is why automated beam angle selection procedures have not entered mainstream clinical usage. NoVo determines a noncoplanar VMAT solution (i.e. the simultaneous trajectories of the gantry and the couch) by first computing a [Formula: see text] solution (beams from every possible direction, suitably discretized) and then eliminating beams by examing fluence contributions. Also all beam angles are scored via geometrical considerations only to find out the usefulness of the whole beam space in a very short time. A custom path finding algorithm is applied to find an optimized, continuous trajectory through the most promising beam angles using the calculated score of the beam space. Finally, using this trajectory a VMAT plan is optimized. For three clinical cases, a lung, brain, and liver case, we compare NoVo to the ideal [Formula: see text] solution, nine beam noncoplanar IMRT, coplanar VMAT, and a recently published noncoplanar VMAT algorithm. NoVo comes closest to the [Formula: see text] solution considering the lung case (brain and liver case: second), as well as improving the solution time by using geometrical considerations, followed by a time effective iterative process reducing the [Formula: see text] solution. Compared to a recently published noncoplanar VMAT algorithm, using NoVo the computation time is reduced by a factor of 2-3 (depending on the case). Compared to coplanar VMAT, NoVo reduces the objective function value by 24%, 49% and 6% for the lung, brain and liver cases, respectively

Fundamental Parameters of the Milky Way Galaxy Based on VLBI astrometry

We present analyses to determine the fundamental parameters of the Galaxy based on VLBI astrometry of 52 Galactic maser sources obtained with VERA, VLBA and EVN. We model the Galaxy's structure with a set of parameters including the Galaxy center distance R_0, the angular rotation velocity at the LSR Omega_0, mean peculiar motion of the sources with respect to Galactic rotation (U_src, V_src, W_src), rotation-curve shape index, and the V component of the Solar peculiar motions V_sun. Based on a Markov chain Monte Carlo method, we find that the Galaxy center distance is constrained at a 5% level to be R_0 = 8.05 +/- 0.45 kpc, where the error bar includes both statistical and systematic errors. We also find that the two components of the source peculiar motion U_src and W_src are fairly small compared to the Galactic rotation velocity, being U_src = 1.0 +/- 1.5 km/s and W_src = -1.4 +/- 1.2 km/s. Also, the rotation curve shape is found to be basically flat between Galacto-centric radii of 4 and 13 kpc. On the other hand, we find a linear relation between V_src and V_sun as V_src = V_sun -19 (+/- 2) km/s, suggesting that the value of V_src is fully dependent on the adopted value of V_sun. Regarding the rotation speed in the vicinity of the Sun, we also find a strong correlation between Omega_0 and V_sun. We find that the angular velocity of the Sun, Omega_sun, which is defined as Omega_sun = Omega_0 + V_sun/R_0, can be well constrained with the best estimate of Omega_sun = 31.09 +/- 0.78 km/s/kpc. This corresponds to Theta_0 = 238 +/- 14 km/s if one adopts the above value of R_0 and recent determination of V_sun ~ 12 km/s.Comment: 14 pages, 6 figures, PASJ in pres

Laser-driven multi-MeV high-purity proton acceleration via anisotropic ambipolar expansion of micron-scale hydrogen clusters

強力なレーザーを使ってエネルギーがそろった純度100％の陽子ビーム発生に成功 --レーザー駆動陽子ビーム加速器の実現へ向けて大きく前進--. 京都大学プレスリリース. 2022-10-13.Multi-MeV high-purity proton acceleration by using a hydrogen cluster target irradiated with repetitive, relativistic intensity laser pulses has been demonstrated. Statistical analysis of hundreds of data sets highlights the existence of markedly high energy protons produced from the laser-irradiated clusters with micron-scale diameters. The spatial distribution of the accelerated protons is found to be anisotropic, where the higher energy protons are preferentially accelerated along the laser propagation direction due to the relativistic effect. These features are supported by three-dimensional (3D) particle-in-cell (PIC) simulations, which show that directional, higher energy protons are generated via the anisotropic ambipolar expansion of the micron-scale clusters. The number of protons accelerating along the laser propagation direction is found to be as high as 1.6 ±0.3 × 10⁹/MeV/sr/shot with an energy of 2.8 ±1.9 MeV, indicating that laser-driven proton acceleration using the micron-scale hydrogen clusters is promising as a compact, repetitive, multi-MeV high-purity proton source for various applications

Discriminative detection of laser-accelerated multi-MeV carbon ions utilizing solid state nuclear track detectors

A new diagnosis method for the discriminative detection of laser‐accelerated multi‐MeV carbon ions from background oxygen ions utilizing solid‐state nuclear track detectors (SSNTDs) is proposed. The idea is to combine two kinds of SSNTDs having different track registration sensitivities: Bisphenol A polycarbonate detects carbon and the heavier ions, and polyethylene terephthalate detects oxygen and the heavier ions. The method is calibrated with mono‐energetic carbon and oxygen ion beams from the heavy ion accelerator. Based on the calibration data, the method is applied to identify carbon ions accelerated from multilayered graphene targets irradiated by a high‐power laser, where the generation of high‐energy high‐purity carbon ions is expected. It is found that 93 ± 1% of the accelerated heavy ions with energies larger than 14 MeV are carbons. The results thus obtained support that carbon‐rich heavy ion acceleration is achieved

Adiponectin receptor-1 expression is associated with good prognosis in gastric cancer

<p>Abstract</p> <p>Background</p> <p>Adiponectin is inversely related to BMI, positively correlates with insulin sensitivity, and has anti-atherogenic effects. In recent years, adiponectin has been well studied in the field of oncology. Adiponectin has been shown to have antiproliferative effects on gastric cancer, and adiponectin expression is inversely correlated with clinical staging of the disease. However, no studies have reported the correlation between serum adiponectin and receptor expression with disease progression.</p> <p>Methods</p> <p>In this study, we evaluated expression levels of 2 adiponectin receptors--AdipoR1 and AdipoR2--and attempted to correlate their expression with prognosis in gastric cancer patients. AdipoR1 and AdipoR2 expression in gastric cancer cell lines (MKN45, TMK-1, NUGC3, and NUGC4) was evaluated by western blotting analysis, and the antiproliferative potential of adiponectin was examined in vitro. Serum adiponectin levels were evaluated in 100 gastric cancer patients, and the expression of AdipoR1 and AdipoR2 was assessed by immunohistochemical staining.</p> <p>Results</p> <p>MKN45 and NUGC3 expressed higher levels of AdipoR1 compared to NUGC4, even though there was no significance in AdipoR2 expression. The antiproliferative effect of adiponectin was confirmed in MKN45 and NUGC3 at 10 μg/ml. No significant associations were observed between serum adiponectin levels and clinicopathological characteristics, but lymphatic metastasis and peritoneal dissemination were significantly higher in the negative AdipoR1 immunostaining group (24/32, <it>p </it>= 0.013 and 9/32, <it>p </it>= 0.042, respectively) compared to the positive AdipoR1 group (lymphatic metastasis, 33/68; peritoneal dissemination, 8/68). On the other hand, AdipoR2 expression was only associated with histopathological type (<it>p </it>= 0.001). In survival analysis, the AdipoR1 positive staining group had significantly longer survival rates than the negative staining group (<it>p </it>= 0.01). However, multivariate analysis indicated that AdipoR1 was not an independent prognostic factor on patient's survival on gastric cancer.</p> <p>Conclusions</p> <p>In gastric cancer, adiponectin has the possibility to be involved in cell growth suppression via AdipoR1. The presence of AdipoR1 could be a novel anticancer therapeutic target in gastric cancer.</p

Farnesyl pyrophosphate regulates adipocyte functions as an endogenous PPARγ agonist

The cholesterol biosynthetic pathway produces not only sterols but also non-sterol mevalonate metabolites involved in isoprenoid synthesis. Mevalonate metabolites affect transcriptional and post-transcriptional events that in turn affect various biological processes including energy metabolism. In the present study, we examine whether mevalonate metabolites activate PPARγ (peroxisome-proliferator-activated receptor γ), a ligand-dependent transcription factor playing a central role in adipocyte differentiation. In the luciferase reporter assay using both GAL4 chimaera and full-length PPARγ systems, a mevalonate metabolite, FPP (farnesyl pyrophosphate), which is the precursor of almost all isoprenoids and is positioned at branch points leading to the synthesis of other longer-chain isoprenoids, activated PPARγ in a dose-dependent manner. FPP induced the in vitro binding of a co-activator, SRC-1 (steroid receptor co-activator-1), to GST (glutathione transferase)–PPARγ. Direct binding of FPP to PPARγ was also indicated by docking simulation studies. Moreover, the addition of FPP up-regulated the mRNA expression levels of PPARγ target genes during adipocyte differentiation induction. In the presence of lovastatin, an HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) reductase inhibitor, both intracellular FPP levels and PPARγ-target gene expressions were decreased. In contrast, the increase in intracellular FPP level after the addition of zaragozic acid, a squalene synthase inhibitor, induced PPARγ-target gene expression. The addition of FPP and zaragozic acid promotes lipid accumulation during adipocyte differentiation. These findings indicated that FPP might function as an endogenous PPARγ agonist and regulate gene expression in adipocytes