Rapid and efficient generation of GFP-knocked-in Drosophila by the CRISPR-Cas9-mediated genome editing.

The CRISPR-Cas9 technology has been a powerful means to manipulate the genome in a wide range of organisms. A series of GFP knocked-in (GFPKI ) Drosophila strains have been generated through CRISPR-Cas9-induced double strand breaks coupled with homology-directed repairs in the presence of donor plasmids. They visualized specific cell types or intracellular structures in both fixed and live specimen. We provide a rapid and efficient strategy to identify KI lines. This method requires neither co-integration of a selection marker nor prior establishment of sgRNA-expressing transgenic lines. The injection of the mixture of a sgRNA/Cas9 expression plasmid and a donor plasmid into cleavage stage embryos efficiently generated multiple independent KI lines. A PCR-based selection allows to identify KI fly lines at the F1 generation (approximately 4 weeks after injection). These GFPKI strains have been deposited in the Kyoto Drosophila stock center, and made freely available to researchers at non-profit organizations. Thus, they will be useful resources for Drosophila research.

Early therapeutic effects of adaptive servo-ventilation on cardiac sympathetic nervous function in patients with heart failure evaluated using a combination of 11C-HED PET and 123I-MIBG SPECT.

RATIONALE: Adaptive servo-ventilation (ASV), a novel respiratory support therapy for sleep disorders, may improve cardiac function in heart failure (HF). However, the reasons that ASV improves cardiac function have not been fully studied especially in sympathetic nervous function (SNF). The purpose of the present study was to investigate the effects of ASV therapy on cardiac SNF in patients with HF. METHODS: We evaluated ASV therapeutic effects before and 6 months after ASV therapy in 9 HF patients [57.3 ± 17.3 years old, left ventricular ejection fraction (LVEF) 36.1 ± 16.7%]. We performed echocardiography, polysomnography, biomarkers, 11C-hydroxyephedrine (HED) PET as a presynaptic function marker and planar 123I-metaiodobenzylguanidine (MIBG) to evaluate washout rate. RESULTS: ASV therapy reduced apnea-hypopnea index (AHI) and improved plasma brain natriuretic peptide (BNP) concentration. In 123I-MIBG imaging, the early heart/mediastinum (H/M) ratio increased after ASV therapy (2.19 ± 0.58 to 2.40 ± 0.67; P = 0.045). Washout rate did not change (23.8 ± 7.3% to 23.8 ± 8.8%; P = 0.122). Global 11C-HED retention index (RI) improved from 0.068 ± 0.033/s to 0.075 ± 0.034/s (P = 0.029). CONCLUSIONS: ASV reduced AHI and improved BNP. ASV might initially improve presynaptic cardiac sympathetic nervous function in HF patients after 6 months of treatment.

Value of Virtual Touch Quantification Elastography for Assessing Liver Congestion in Patients With Heart Failure.

BACKGROUND: Heart failure (HF) causes organ congestion, which is thought to increase organ stiffness. The virtual touch quantification (VTQ) method can be used to assess liver stiffness in patients with chronic liver diseases. This study aimed to measure liver and kidney stiffness using VTQ and to determine its value for assessing organ congestion in patients with HF. METHODS AND RESULTS: This study included 10 normal subjects and 38 HF patients (age 52.3±16.7 years, left ventricular ejection fraction 27.0±9.4%, plasma B-type natriuretic peptide [BNP] 1,297.3±1,155.1 pg/ml). We investigated the relationships between clinical characteristics and hemodynamics and liver and kidney stiffness, and assessed the effects of medical treatment on these measurements. Liver stiffness was significantly higher in HF patients (1.17±0.13 m/s vs. 2.03±0.91 m/s, P=0.004) compared with normal subjects, but kidney stiffness was similar in both groups. Central venous pressure (CVP) (P=0.021) and BNP (P=0.025) were independent predictive factors for increased liver stiffness in HF patients. Liver stiffness decreased significantly from 2.37±1.09 to 1.27±0.33 m/s (P<0.001) after treatment. Changes in liver stiffness in HF patients significantly correlated with changes in CVP (R=0.636, P=0.014) and cardiac index (R=-0.557, P=0.039) according to univariate analysis, and with changes in CVP in multivariate analysis. CONCLUSIONS: Liver stiffness measured by noninvasive VTQ methods can be used to assess liver congestion and therapeutic effects in patients with HF. (Circ J 2016; 80: 1187-1195).

Higher Hemoglobin A1c After Discharge Is an Independent Predictor of Adverse Outcomes in Patients With Acute Coronary Syndrome　- Findings From the PACIFIC Registry.

BACKGROUND: Optimal medical therapy (OMT) and the management of coronary risk factors are necessary for secondary prevention of major adverse cardiac and cerebrovascular events (MACCE) in post-acute coronary syndrome (ACS) patients. However, the effect of post-discharge patient adherence has not been investigated in Japanese patients. METHODS AND RESULTS: The Prevention of AtherothrombotiC Incidents Following Ischemic Coronary Attack (PACIFIC) registry was a multicenter, prospective observational study of 3,597 patients with ACS. Death or MACCE occurred in 229 patients between hospitalization and up to 1 year after discharge. Among 2,587 patients, the association between OMT adherence and risk factor control at 1 year and MACCE occurring between 1 and 2 years after discharge was assessed. OMT was defined as the use of antiplatelet agents, angiotensin-converting enzyme inhibitors, ß-blockers, and statins. Risk factor targets were: low-density lipoprotein-cholesterol <100 mg/dl, HbA1c <7.0%, non-smoking status, blood pressure <130/80 mmHg, and 18.5≤body mass index≤24.9 kg/m(2). The incidence of MACCE was 1.8% and associated with female sex (P=0.020), age ≥75 years (P=0.004), HbA1c ≥7.0% (P=0.004), LV ejection fraction <35% (P<0.001), estimated glomerular filtration rate <60 ml/min (P=0.008), and history of cerebral infarction (P=0.003). In multivariate analysis, lower post-discharge HbA1c was strongly associated with a lower risk of MACCE after ACS (P=0.004). CONCLUSIONS: Hyperglycemia after discharge is a crucial target for the prevention of MACCE in post-ACS patients. (Circ J 2016; 80: 1607-1614).

Adaptive Servo-Ventilation Has More Favorable Acute Effects on Hemodynamics Than Continuous Positive Airway Pressure in Patients With Heart Failure.

Adaptive servo-ventilation (ASV) has been attracting attention as a novel respiratory support therapy for heart failure (HF). However, the acute hemodynamic effects have not been compared between ASV and continuous positive airway pressure (CPAP) in HF patients.We studied 12 consecutive patients with stable chronic HF. Hemodynamic measurement was performed by right heart catheterization before and after CPAP 5 cmH2O, CPAP 10 cmH2O, and ASV for 15 minutes each.Heart rate, blood pressure, pulmonary capillary wedge pressure (PCWP), and stroke volume index (SVI) were not changed by any intervention. Right atrial pressure significantly increased after CPAP 10 cmH2O (3.6 ± 3.3 to 6.7 ± 1.6 mmHg, P = 0.005) and ASV (4.1 ± 2.6 to 6.8 ± 1.5 mmHg, P = 0.026). Cardiac index was significantly decreased by CPAP 10 cmH2O (2.3 ± 0.4 to 1.9 ± 0.3 L/minute/m(2), P = 0.048), but was not changed by ASV (2.3 ± 0.4 to 2.0 ± 0.3 L/ minute/m(2), P = 0.299). There was a significant positive correlation between baseline PCWP and % of baseline SVI by CPAP 10 cmH2O (r = 0.705, P < 0.001) and ASV (r = 0.750, P < 0.001). ASV and CPAP 10 cmH2O had significantly greater slopes of this correlation than CPAP 5 cmH2O, suggesting that patients with higher PCWP had a greater increase in SVI by ASV and CPAP 10 cmH2O. The relationship between baseline PCWP and % of baseline SVI by ASV was shifted upwards compared to CPAP 10 cmH2O. Furthermore, based on the results of a questionnaire, patients accepted CPAP 5 cmH2O and ASV more favorably compared to CPAP 10 cmH2O.ASV had more beneficial effects on acute hemodynamics and acceptance than CPAP in HF patients.

Cardiac magnetic resonance performs better in the detection of functionally significant coronary artery stenosis compared to single-photon emission computed tomography and dobutamine stress echocardiography.

BACKGROUND: Fractional flow reserve (FFR) measured on catheterization is now widely used for the diagnosis of functional myocardial ischemia in patients with coronary artery disease (CAD). FFR, however, is invasive and carries potential procedural complications. Therefore, the aim of this study was to compare the diagnostic capability in functionally significant stenosis identified on FFR, between cardiac magnetic resonance myocardial perfusion imaging (CMR-MPI), single-photon emission computed tomography MPI (SPECT-MPI), and dobutamine stress echocardiography (DSE) in patients with CAD. METHODS AND RESULTS: A total of 25 patients who had at least 1 angiographic stenosis ≥50% on coronary angiography was studied. CMR-MPI, SPECT-MPI and DSE were done before FFR measurement. FFR was measured in all 3 major epicardial coronary arteries. Out of 71 vascular territories excluding 4 territories due to inadequate imaging, 29 (41%) had FFR <0.80. The sensitivity of CMR-MPI was significantly higher than that of SPECT-MPI and DSE (P=0.02 and P=0.001, respectively). The area under the receiver operating characteristic curve (AUC) for CMR-MPI (AUC, 0.92) was significantly greater than for SPECT-MPI (AUC, 0.73; P=0.006) and DSE (AUC, 0.69; P<0.001). CONCLUSIONS: CMR-MPI performed well in the detection of functionally significant stenosis defined according to FFR, and had the highest diagnostic sensitivity among the 3 modalities tested in patients with CAD.

Impact of the haplotype CYP3A4*16B harboring the Thr185Ser substitution on paclitaxel metabolism in Japanese patients with cancer.

OBJECTIVE: Paclitaxel is one of the most important anticancer drugs for the treatment of various tumors such as non-small cell lung cancer. We investigated the association between CYP3A4 haplotypes and pharmacokinetic parameters of paclitaxel metabolism. METHODS: This study enrolled 235 Japanese patients with cancer who were receiving paclitaxel. These patients were screened for CYP3A4 gene polymorphisms by either direct sequencing or pyrosequencing. Plasma concentrations of paclitaxel and its 3 metabolites were determined by HPLC in 229 patients. RESULTS: Median values of paclitaxel clearance, normalized for body surface area, were lower in the high-dose group (>or=175 mg/m2, n = 199) than in the low-dose group (

Clinical implication of reverse redistribution on 99mTc-sestamibi images for evaluating ischemic heart disease.

OBJECTIVE: The purpose of this study was to clarify the usefulness of 99mTc-sestamibi (MIBI) delayed imaging in the assessment of the severity of myocardial ischemia in patients with coronary artery stenosis. METHODS: Forty-three angina pectoris with coronary stenosis of greater than 75% were enrolled in this study. Myocardial perfusion SPECT images were obtained 1 and 6 hours after an intravenous injection of MIBI at rest. Stress myocardial perfusion SPECT images were also acquired after the injection of MIBI. And myocardial fatty acid metabolism images were obtained 30 minutes after the injection of BMIPP at rest. Myocardial perfusion SPECT images were divided into 20 segments which were semiquantitatively assessed according to a 4-level defect score scale: score 0 (normal) to score 3 (severely); then the extent score (ES) and severity score (SS) were calculated. RESULTS: The sensitivity for myocardial ischemia showed the highest rate at 88.3% with MIBI delayed SPECT. According to the coronary angiography findings, MIBI stress SPECT and MIBI delayed SPECT detected the severity and extent of ischemia with more sensitivity than MIBI early SPECT in 12 patients (group A) with stenosis of more than 75% but less than 90% (p < 0.01). Even though MIBI stress SPECT detected the severity and extent of ischemia in 31 patients (group B) with stenosis of more than 90% but less than 100%, there was no significant difference between MIBI stress SPECT and MIBI delayed SPECT. BMIPP SPECT revealed significant differences between group A and group B regarding the severity of myocardial ischemia. MIBI reverse redistribution was observed in 33 patients and no significant difference existed between groups A and B. CONCLUSIONS: Myocardial washout of MIBI was frequently observed in patients with angina pectoris and the detection accuracy for ischemia was high. MIBI imaging is considered useful for assessment not only of myocardial perfusion but also mitochondrial function. The imagings with BMIPP and delayed MIBI could serve to determine the severity of myocardial ischemia more accurately.

CYP2C8 haplotype structures and their influence on pharmacokinetics of paclitaxel in a Japanese population.

OBJECTIVE: CYP2C8 is known to metabolize various drugs including an anticancer drug paclitaxel. Although large interindividual differences in CYP2C8 enzymatic activity and several nonsynonymous variations were reported, neither haplotype structures nor their associations with pharmacokinetic parameters of paclitaxel were reported. METHODS: Haplotype structures of the CYP2C8 gene were inferred by an expectation-maximization based program using 40 genetic variations detected in 437 Japanese patients, which included cancer patients. Associations of the haplotypes and paclitaxel pharmacokinetic parameters were analyzed for 199 paclitaxel-administered cancer patients. RESULTS: Relatively strong linkage disequilibriums were observed throughout the CYP2C8 gene. We estimated 40 haplotypes without an amino-acid change and nine haplotypes with amino acid changes. The 40 haplotypes were classified into six groups based on network analysis. The patients with heterozygous *IG group haplotypes harboring several intronic variations showed a 2.5-fold higher median area under concentration-time curve of C3'-p-hydroxy-paclitaxel and a 1.6-fold higher median value of C3'-p-hydroxy-paclitaxel/paclitaxel area under concentration-time curve ratio than patients bearing no *IG group haplotypes (P<0.001 for both comparisons by Mann-Whitney U-test). No statistically significant differences, however, were observed between patients with and without the *IG group (haplotypes) in clearance and area under concentration-time curve of paclitaxel, area under concentration-time curve of 6alpha-hydroxy-paclitaxel and 6alpha-, C3'-p-dihydroxy-paclitaxel, and area under concentration-time curve ratio of 6alpha-hydroxy-paclitaxel/paclitaxel. CONCLUSION: CYP2C8*IG group haplotypes were associated with increased area under concentration-time curve of C3'-p-hydroxy-paclitaxel and area under concentration-time curve ratio of C3'-p-hydroxy-paclitaxel/paclitaxel. Thus, *IG group haplotypes might be associated with reduced CYP2C8 activity, possibly through its reduced protein levels.

Effect of experimental renal failure on the pharmacokinetics of losartan in rats.

The purpose of this investigation was to determine whether the pharmacokinetics of the angiotensin II receptor antagonist losartan is altered in renal failure. Male Wistar rats were pretreated with uranyl nitrate or subjected to bilateral ureteral ligation to produce acute renal failure (ARF). Saline-injected and sham-operated rats, respectively, served as controls. Uranyl nitrate-treated rats showed significantly higher serum concentrations of losartan after oral administration and the area under the serum concentration-time curve (AUC(0-24)) of losartan increased about 3-fold compared to control rats. The systemic clearance of losartan significantly decreased from 410 +/- 254ml/h/kg in control to 177 +/- 112ml/h/kg in uranyl nitrate-treated rats. In order to investigate the mechanisms of reduced clearance of losartan associated with ARF, a hepatic microsome fraction was prepared from normal and ARF rats. No significant difference was found in the metabolism of losartan by hepatic microsomes prepared from ARF and control rats. In addition, the metabolic activity of microsomes was examined in the presence of uremic rat serum. The unbound clearance of losartan and the unbound clearance associated with the formation of EXP3174 in the presence of uremic serum were significantly lower than those in the presence of control serum. Furthermore, the metabolism of losartan was inhibited by indoxyl sulfate, a uremic toxin, in an uncompetitive manner. These results suggest that ARF is associated with reduced clearance of losartan due to the inhibition of hepatic metabolism by accumulated uremic toxin(s).