Genome Editing Using Cas9 Ribonucleoprotein Is Effective for Introducing PDGFRA Variant in Cultured Human Glioblastoma Cell Lines

Many variants of uncertain significance (VUS) have been detected in clinical cancer cases using next-generation sequencing-based cancer gene panel analysis. One strategy for the elucidation of VUS is the functional analysis of cultured cancer cell lines that harbor targeted gene variants using genome editing. Genome editing is a powerful tool for creating desired gene alterations in cultured cancer cell lines. However, the efficiency of genome editing varies substantially among cell lines of interest. We performed comparative studies to determine the optimal editing conditions for the introduction of platelet-derived growth factor receptor alpha (PDGFRA) variants in human glioblastoma multiforme (GBM) cell lines. After monitoring the copy numbers of PDGFRA and the expression level of the PDGFRα protein, four GBM cell lines (U-251 MG, KNS-42, SF126, and YKG-1 cells) were selected for the study. To compare the editing efficiency in these GBM cell lines, the modes of clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) delivery (plasmid vs. ribonucleoprotein (RNP)), methods of transfection (lipofection vs. electroporation), and usefulness of cell sorting were then evaluated. Herein, we demonstrated that electroporation-mediated transfer of Cas9 with single-guide RNA (Cas9 RNP complex) could sufficiently edit a target nucleotide substitution, irrespective of cell sorting. As the Cas9 RNP complex method showed a higher editing efficiency than the Cas9 plasmid lipofection method, it was the optimal method for single-nucleotide editing in human GBM cell lines under our experimental conditions.     

Effects of polyglycerol esters of fatty acids and ethylene‐vinyl acetate co‐polymer on crystallization behavior of biodiesel

Biodiesel fuels (BDF) have many problems in the cold due to their crystallization properties. In particular, precipitation of large crystals of high‐melting fractions in BDF at low temperatures remarkably changes cold flow property of BDF and, thereby, it increases the values of cold filter plugging point. In this study, we evaluated polyglycerol esters of fatty acids (PGE) and ethylene‐vinyl acetate co‐polymer (EVA) as chemical additives to improve the cold flow property of palm oil‐based FAME (PFME). The results of solid fat content measurement indicate that the simultaneous addition of PGE and EVA showed synergistic effects on suppression of crystallization of PFME, however such effect was not observed when EVA was used alone. DSC thermograms indicated that the PGE additives not only decreased the crystallization temperature but also kinetically suppressed the crystal growth. Polarized light microscopy showed that the simultaneous addition of PGE and EVA led to the formation of considerably small and fine‐dispersed crystals of PFME. These results indicate that combined effects of PGE and EVA caused the formation of fine‐dispersed PFME crystals, which could improve the viscous properties of palm oil‐based BDF at relatively cold temperatures.     

Lysine‐241 Has a Role in Coupling 2OG Turnover with Substrate Oxidation During KDM4‐Catalysed Histone Demethylation

The JmjC histone lysyl demethylases (KDMs) play important roles in modulating histone methylation states and have the potential to be regulated by oxygen availability. Lys241 of the KDM4 subfamily is proposed to be important in oxygen binding by KDM4A. We report evidence that, although Lys241 is unlikely to be directly involved in oxygen binding, it has an important role in coupling 2‐oxoglutarate cosubstrate oxidation with lysine demethylase activity. The results suggest that compounds promoting the uncoupling of substrate oxidation are of interest as JmjC‐KDM inhibitors.     

Sweep‐out flow of film condensation on vertical plates with horizontal grooves

We have investigated fluid flow characteristics of film‐wise condensation on vertical plates with horizontal periodic grooves. Condensate stays at the edge of the grooves due to the surface tension. The condensate starts to flow, however, when the balance between the surface tension and the condensate's own weight is broken. It is found that the condensate flows downward successively and periodically from the top part of the plate as a group. In addition, we have obtained the relation between the frequencies of the periodic flow and the degree of sub‐cooling for two different pitches of the grooves. © 2009 Wiley Periodicals, Inc. Heat Trans Asian Res; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/htj.20249     

A Novel Approach to the Polaronic Metallic State of Cuprate Superconductors and the d-Wave Pairing Mechanism

The present novel approach consists of two stages: in the first stage the many-electron states of a CuO₆ octahedron or a CuO₅ pyramid are calculated by the first-principles variational method, by taking into account the local distortions of a CuO₆ octahedron or a CuO₅ pyramid. In the second stage a metallic state is constructed in the presence of the local AF ordering constructed by the localized spins. In this metallic state the local distortions are treated in the mean field approximation, so that a pseudopolaronic effect is taken into account. Based on this approach, the hole-concentration dependence of T _c and the isotope effect are calculated for LSCO, and compared with experimental results. This approach leads to d-wave pairing mechanism.     

Endothelin A-receptor blockade worsens endotoxin-induced hepatic microcirculatory changes and necrosis

BACKGROUND & AIMS: Endothelin 1 is considered to be an important regulator of sinusoidal blood flow and increases during endotoxemia. The purpose of this study was to investigate the role of endothelin 1 in hepatic microcirculation, oxygen transport, and liver injury during endotoxemia. METHODS: Male Sprague-Dawley rats were continuously infused with 2.5 mL/h of saline, 0.8 mg . kg-1 . h-1 of lipopolysaccharide (LPS), 3 mg . kg-1 . h-1 of BQ-485, an endothelin A-receptor antagonist, or LPS plus BQ-485 for 7 hours. RESULTS: BQ-485 infusion had no significant effect on hepatic microcirculation and liver injury. LPS increased the plasma levels of aspartate aminotransferase (AST) and total bilirubin and decreased the hepatic adenosine triphosphate (ATP) level and bile flow rate. LPS + BQ-485 infusion further increased the plasma levels of AST and total bilirubin and decreased the bile flow rate and the hepatic ATP level. Dual-spot microspectroscopy revealed mild decreases in sinusoidal erythrocyte velocity and oxygen transport in the LPS group and profound decreases in these parameters in the LPS + BQ-485 group. Histological examinations revealed massive necrotic changes in the pericentral regions of the LPS + BQ-485 group. CONCLUSIONS: These results suggest that blockade of endothelin A receptors disturbs hepatic microcirculation and oxygen transport and aggravates the necrotic injury induced by endotoxin.     

Bacillus cereus septicemia in a patient with severe aplastic anemia

A 78-year-old female was admitted with complaints of malaise and fatigue in the legs. The patient was diagnosed as severe aplastic anemia and treatment was started with metenolone and steroid pulse therapy. Administration of antibiotics and granulocyte-colony stimulating factor which led to a resolution of the high fever. About four months after admission, the patient developed vomiting and abdominal pain with a spiking fever. The next day after suddenly losing consciousness, she died. B. cereus was isolated from blood cultures. Autopsy specimens of the liver, cardiac muscle and lung showed changes due to B. cereus. This pathogen is widely distributed in nature. We should not overlook B. cereus as a contamination, but rather should consider it a potential pathogen in immunocompromised hosts, when it is isolated from blood cultures.     

Arrhythmogenic right ventricular cardiomyopathy underlies syndrome of right bundle branch block, ST-segment elevation, and sudden death

In all experimental mammals tested (rats, dogs, primates), intramuscular injections of the oil-soluble antimalarial artemisinin derivatives artemether and arteether have produced an unusual pattern of selective damage to brain stem centers predominantly involved in auditory processing and vestibular reflexes. Artesunate, the most widely used of these compounds, is a water soluble hemisuccinate derivative given parenterally either by intravenous or intramuscular injection. The neurotoxic potential of parenteral artesunate and artemether was compared in a murine model. Adult Swiss albino mice were assigned randomly to 28-day regimens of intramuscular artemether or artesunate in doses ranging from 30 to 100 mg/kg/day. At 30 mg/kg/day, no abnormalities were detected with either drug. At 50 mg/kg/day, abnormalities were observed in six of 12 artemether recipients and two of 12 artesunate recipients. These were reversible in all but one (artemether) mouse. At 100 mg/kg/day, eight of 36 artemether recipients, two of 36 artesunate recipients, and one of 18 control mice died. All but four surviving mice in the artemether group (86%) showed obvious and usually irreversible abnormalities of balance and equilibrium, whereas only four artesunate recipients (11%) exhibited abnormalities, and these were reversible in each case (P < 0.001). At this dose the relative risk (95% confidence interval) for death or disability was 5.3 (2.6-11.2) for artemether recipients. Intramuscular artemether is significantly more neurotoxic than intramuscular artesunate in this murine model.     

Survival by Mac-1-mediated adherence and anoikis in phorbol ester-treated HL-60 cells

During the exposure of human myelocytic leukemia HL-60 cells to phorbol diester, nonadherent cells die by apoptosis, but adherent cells survive and growth-arrest at G1 phase of the cell cycle. Here we have shown that the adherent cells rapidly died by apoptosis after forced detachment (anoikis), indicating that phorbol diester induced apoptosis by default. Dimethylsphingosine induced apoptosis in the adherent cells, and sphingosine-1-phosphate rescued the detached cells from apoptosis. Sphingosine kinase activity in adherent cells was higher than that in nonadherent cells and was decreased by forced detachment. It is likely that the phorbol diester-induced apoptosis and the adhesion-mediated survival are modulated by sphingosine and sphingosine-1-phosphate, respectively. The adherent cells were reverted and reproliferated when allowed to spontaneously detach from plastic surfaces by removal of phorbol diester. This result suggests that after removal of phorbol diester, the commitment signal of apoptosis by default is lost faster than the survival signal by adherence.