In Vitro Support of Primary Leukemia Cells

No in vitro method for culturing Primary Leukemia Cells exists. Thus, study of Primary Leukemia biology requires immunodeficient mouse models, which is costly and time consuming. Identifying a method to grow and maintain Primary Leukemia Cells in vitro would allow biological and molecular assays to facilitate bench to bedside translation

Implantable pneumatically actuated microsystem for renal pressure-mediated transfection in mice.

In vivo transfection is an important technique used in biological research and drug therapy development. Previously, we developed a renal pressure-mediated transfection method performed by pressing a kidney after an intravenous injection of naked nucleic acids. Although this is a useful method because of its safety and wide range of applications, an innovative approach for performing this method without repeatedly cutting open the abdomen is required. In this study, we developed an implantable microsystem fabricated by Micro-Electro-Mechanical Systems (MEMS) technologies for renal pressure-mediated transfection. The system consists of a polydimethylsiloxane pneumatic balloon actuator (PBA) used as an actuator to press the target kidney. The PBA of the implanted microsystem can be actuated without opening the abdomen by applying air pressure from outside the body to the pressure-supplying port via a needle. We successfully performed renal pressure-mediated transfection using the newly developed system when the implanted system was activated at 60kPa for 10s. This is the first report of an implantable MEMS-based microsystem that demonstrates in vivo transfection to a kidney using naked plasmid DNA

Inhibition of ATR protein kinase activity by schisandrin B in DNA damage response

ATM and ATR protein kinases play a crucial role in cellular DNA damage responses. The inhibition of ATM and ATR can lead to the abolition of the function of cell cycle checkpoints. In this regard, it is expected that checkpoint inhibitors can serve as sensitizing agents for anti-cancer chemo/radiotherapy. Although several ATM inhibitors have been reported, there are no ATR-specific inhibitors currently available. Here, we report the inhibitory effect of schisandrin B (SchB), an active ingredient of Fructus schisandrae, on ATR activity in DNA damage response. SchB treatment significantly decreased the viability of A549 adenocarcinoma cells after UV exposure. Importantly, SchB treatment inhibited both the phosphorylation levels of ATM and ATR substrates, as well as the activity of the G2/M checkpoint in UV-exposed cells. The protein kinase activity of immunoaffinity-purified ATR was dose-dependently decreased by SchB in vitro (IC50: 7.25 μM), but the inhibitory effect was not observed in ATM, Chk1, PI3K, DNA-PK, and mTOR. The extent of UV-induced phosphorylation of p53 and Chk1 was markedly reduced by SchB in ATM-deficient but not siATR-treated cells. Taken together, our demonstration of the ability of SchB to inhibit ATR protein kinase activity following DNA damage in cells has clinical implications in anti-cancer therapy

Regulation of the unfolded protein response via S-nitrosylation of sensors of endoplasmic reticulum stress

Protein S-nitrosylation modulates important cellular processes, including neurotransmission, vasodilation, proliferation, and apoptosis in various cell types. We have previously reported that protein disulfide isomerase (PDI) is S-nitrosylated in brains of patients with sporadic neurodegenerative diseases. This modification inhibits PDI enzymatic activity and consequently leads to the accumulation of unfolded/misfolded proteins in the endoplasmic reticulum (ER) lumen. Here, we describe S-nitrosylation of additional ER pathways that affect the unfolded protein response (UPR) in cell-based models of Parkinson's disease (PD). We demonstrate that nitric oxide (NO) can S-nitrosylate the ER stress sensors IRE1α and PERK. While S-nitrosylation of IRE1α inhibited its ribonuclease activity, S-nitrosylation of PERK activated its kinase activity and downstream phosphorylation/inactivation or eIF2α. Site-directed mutagenesis of IRE1α(Cys931) prevented S-nitrosylation and inhibition of its ribonuclease activity, indicating that Cys931 is the predominant site of S-nitrosylation. Importantly, cells overexpressing mutant IRE1α(C931S) were resistant to NO-induced damage. Our findings show that nitrosative stress leads to dysfunctional ER stress signaling, thus contributing to neuronal cell death

Serum amyloid alpha 1-2 are not required for liver inflammation in the 4T1 murine breast cancer model

がんに起因して起こる宿主の肝臓の急性期応答と炎症 --血清アミロイドαは乳がんモデルにおける肝臓の炎症の原因ではない--. 京都大学プレスリリース. 2023-02-06.Cancers induce the production of acute phase proteins such as serum amyloid alpha (SAA) in the liver and cause inflammation in various host organs. Despite the well-known coincidence of acute phase response and inflammation, the direct roles of SAA proteins in inflammation in the cancer context remains incompletely characterized, particularly in vivo. Here, we investigate the in vivo significance of SAA proteins in liver inflammation in the 4T1 murine breast cancer model. 4T1 cancers elevate the expression of SAA1 and SAA2, the two major murine acute phase proteins in the liver. The elevation of Saa1-2 correlates with the up-regulation of immune cell-related genes including neutrophil markers. To examine this correlation in detail, we generate mice that lack Saa1-2 and investigate immune-cell phenotypes. RNA-seq experiments reveal that deletion of Saa1-2 does not strongly affect 4T1-induced activation of immune cell-related genes in the liver. Flow cytometry experiments demonstrate the dispensable roles of SAA1-2 in cancer-dependent neutrophil infiltration to the liver. Consistently, 4T1-induced gene expression changes in bone marrow do not require Saa1-2. This study clarifies the negligible contribution of SAA1-2 proteins in liver inflammation in the 4T1 breast cancer model

Demonstration of periodic nanostructure formation with less ablation by double-pulse laser irradiation on titanium

By pairing femtosecond laser pulses (duration ∼40 fs and central wavelength ∼810 nm) at an appropriate time interval, a laser-induced periodic surface structure (LIPSS) is formed with much less ablation than one formed with a single pulse. On a titanium plate, a pair of laser pulses with fluences of 70 and 140 mJ/cm² and a rather large time interval (>10 ps) creates a LIPSS with an interspace of 600 nm, the same as that formed by a single pulse of 210 mJ/cm², while the double pulse ablates only 4 nm, a quarter of the ablation depth of a single pulse

Simple noise reduction for DWI

Our purpose in this study was to reduce the noise in order to improve the SNR of Dw images with high b-value by using two correction schemes. This study was performed with use of phantoms made from water and sucrose at different concentrations, which were 10, 30, and 50 weight percent (wt%). In noise reduction for Dw imaging of the phantoms, we compared two correction schemes that are based on the Rician distribution and the Gaussian distribution. The highest error values for each concentration with use of the Rician distribution scheme were 7.3 % for 10 wt%, 2.4 % for 30 wt%, and 0.1 % for 50 wt%. The highest error values for each concentration with use of the Gaussian distribution scheme were 20.3 % for 10 wt%, 11.6 % for 30 wt%, and 3.4 % for 50 wt%. In Dw imaging, the noise reduction makes it possible to apply the correction scheme of Rician distribution