Optimization of RK2-based gene introduction system for Bacillus subtilis.

The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double crossover recombinants. The optimized conjugation method described here allowed facility and efficient gene introduction into B. subtilis from E. coli.

Impact of the sampling rate of dynamic myocardial computed tomography perfusion on the quantitative assessment of myocardial blood flow.

BACKGROUND: The relationship between shot-to-shot sampling rates for dynamic myocardial computed tomography perfusion (CTP) and robustness of CTP-derived myocardial blood flow (CT-MBF) is debatable. We retrospectively investigated the influence of a reduced sampling rate for dynamic CTP on CT-MBF computation and diagnostic performance for detecting myocardial perfusion abnormalities. METHODS: Pharmacological stress dynamic whole-heart CTP was performed in 120 patients suspected with coronary artery disease. Dynamic CTP data were obtained for 30 continuous heartbeats during the R-peak to R-peak (1RR) interval on electrocardiography. Three additional datasets were created with sub-sampling acquisitions every 2, 3, and 4 heartbeats from the original dataset as interval times of 2RR, 3RR, and 4RR, respectively. CT-MBF was calculated using deconvolution analysis and determined as the mean value of the whole heart (global CT-MBF) and using the 16-segment model (segmental CT-MBF). The diagnostic performance of segmental CT-MBF for detecting perfusion abnormalities was compared to that of cardiac magnetic resonance imaging as the gold standard in 32 of 120 patients. These results were compared among the four CTP datasets. RESULTS: Global CT-MBFs for 1RR, 2RR, 3RR, and 4RR sampling were 1.57 ±â€¯0.34, 1.54 ±â€¯0.36, 1.51 ±â€¯0.37, and 1.41 ±â€¯0.33 mL/g/min, respectively. Areas under the receiver operating characteristic curves of segmental CT-MBF for 1RR, 2RR, 3RR, and 4RR sampling were 0.84, 0.83, 0.79, and 0.76, respectively (1RR versus [vs.] 2RR, non-significant; 1RR vs. 3RR or 4RR, p < 0.05). CONCLUSION: CT-MBF with 2RR sampling has similar performance with regard to quantification and detecting myocardial perfusion abnormalities as that with 1RR sampling.

Late iodine enhancement computed tomography with image subtraction for assessment of myocardial infarction.

OBJECTIVE: To evaluate the feasibility of image subtraction in late iodine enhancement CT (LIE-CT) for assessment of myocardial infarction (MI). METHODS: A comprehensive cardiac CT protocol and late gadolinium enhancement MRI (LGE-MRI) was used to assess coronary artery disease in 27 patients. LIE-CT was performed after stress CT perfusion (CTP) and CT angiography. Subtraction LIE-CT was created by subtracting the mask volume of the left ventricle (LV) cavity from the original LIE-CT using CTP dataset. The %MI volume was quantified as the ratio of LIE to entire LV volume, and transmural extent (TME) of LIE was classified as 0%, 1-24%, 25-49%, 50-74% or 75-100%. These results were compared with LGE-MRI using the Spearman rank test, Bland-Altman method and chi-square test. RESULTS: One hundred twenty-five (29%) of 432 segments were positive on LGE-MRI. Correlation coefficients for original and subtraction LIE-CT to LGE-MRI were 0.79 and 0.85 for %MI volume. Concordances of the 5-point grading scale between original and subtraction LIE-CT with LGE-MRI were 75% and 84% for TME; concordance was significantly improved using the subtraction technique (p <0.05). CONCLUSION: Subtraction LIE-CT allowed more accurate assessment of MI extent than the original LIE-CT. KEY POINTS: • Subtraction LIE-CT allows for accurate assessment of the extent of myocardial infarction. • Subtraction LIE-CT shows a close correlation with LGE-MRI in %MI volume. • Subtraction LIE-CT has significantly higher concordance with TME assessment than original LIE-CT.

Two-Phase Contrast Injection Protocol for Pediatric Cardiac Computed Tomography in Children with Congenital Heart Disease.

To assess a two-phase contrast injection protocol for contrast enhancement during cardiac computed tomography (CT) in children with congenital heart disease. Forty-three children (20 boys, 23 girls) of median age 13 months (range 3 days-8.3 years) and weighing ≤ 20 kg who underwent cardiac CT using a two-phase contrast injection protocol at our institution were retrospectively identified. High-pitch spiral third-generation dual-source cardiac CT (tube voltage 70 kV) was performed with a fixed delay of 60 s after contrast injection in the order of 10 mgI/kg/s (30 s), 15 mgI/kg/s (20 s), and a saline chaser (10 s). Attenuation in the inferior vena cava (IVC), superior vena cava (SVC), right atrium (RA), right ventricle (RV), pulmonary artery (PA), left atrium (LA), left ventricle (LV), and descending aorta (AO) was compared using the Steel-Dwass and Fisher's exact tests. The median (interquartile range) attenuation in the IVC, SVC, RA, RV, PA, LA, LV, and AO was 285 (264-347) Hounsfield units (HU), 416 (370-445) HU, 368 (320-388) HU, 373 (322-417) HU, 397 (330-432) HU, 425 (373-469) HU, 435 (385-468) HU, and 437 (392-491) HU, respectively (p < 0.05, IVC vs. the other anatomic sites). There was no significant difference in diagnostic success rate for attenuation > 250 HU between the IVC (41 children, 95.3%) and the other sites (43 children, 100%). A two-phase contrast injection protocol is useful for effective contrast enhancement in pediatric cardiac CT.

Peak enhancement ratio of myocardium to aorta for identification of myocardial ischemia using dynamic myocardial computed tomography perfusion imaging.

BACKGROUND: This study aimed to evaluate the feasibility of peak enhancement (PE) ratio of myocardium to aorta (PER) derived from stress dynamic computed tomography myocardial perfusion imaging (CTP) for the detection of myocardial ischemia assessed by magnetic resonance (MR) imaging. METHODS: Forty-four patients who underwent stress dynamic CTP and MR imaging were retrospectively evaluated. From the time-attenuation curve, myocardial PE, PER, and myocardial blood flow (MBF) were calculated on a segment-based analysis. The correlation between myocardial and aortic PE was assessed by Spearman's correlation, and the differences in myocardial PE and PER between normal and ischemic myocardium were assessed by the Mann-Whitney U-test. The diagnostic accuracies of myocardial PE, PER, and MBF for detecting myocardial ischemia were compared by receiver operating characteristic analysis. RESULTS: Of 704 segments, 258 segments (37%) were diagnosed as myocardial ischemia with MR imaging. Myocardial and aortic PE were significantly correlated in both normal and ischemic segments (r=0.76 and 0.58; p<0.05, in each). The myocardial PE and PER of ischemic segments were significantly lower than those of normal segments (p<0.05, in each). Sensitivity and specificity were 61% [95% confidence interval (CI), 55-70%] and 83% (95% CI, 73-87%) for myocardial PE, 78% (67-88%) and 82% (95% CI, 70-91%) for PER, and 81% (95% CI, 73-87%) and 85% (95% CI, 79-92%) for MBF. There was a significantly larger area under the curve for PER (0.87; 95% CI, 0.84-0.90) and MBF (0.88; 95%CI, 0.85-0.91), compared to myocardial PE (0.75; 95% CI, 0.70-0.79) (p<0.05, in each). There was no significant difference in area under the curve between PER and MBF. CONCLUSIONS: The semi-quantitative parameter of PER showed a high diagnostic accuracy for the detection of myocardial ischemia, comparable to that of MBF.

Impact of knowledge-based iterative model reconstruction on myocardial late iodine enhancement in computed tomography and comparison with cardiac magnetic resonance.

We evaluated the image quality and diagnostic performance of late iodine enhancement computed tomography (LIE-CT) with knowledge-based iterative model reconstruction (IMR) for the detection of myocardial infarction (MI) in comparison with late gadolinium enhancement magnetic resonance imaging (LGE-MRI). The study investigated 35 patients who underwent a comprehensive cardiac CT protocol and LGE-MRI for the assessment of coronary artery disease. The CT protocol consisted of stress dynamic myocardial CT perfusion, coronary CT angiography (CTA) and LIE-CT using 256-slice CT. LIE-CT scans were acquired 5 min after CTA without additional contrast medium and reconstructed with filtered back projection (FBP), a hybrid iterative reconstruction (HIR), and IMR. The signal-to-noise ratio (SNR) and contrast-to-noise ratio (CNR) were assessed. Sensitivity and specificity of LIE-CT for detecting MI were assessed according to the 16-segment model. Image quality scores, and diagnostic performance were compared among LIE-CT with FBP, HIR and IMR. Among the 35 patients, 139 of 560 segments showed MI in LGE-MRI. On LIE-CT with FBP, HIR, and IMR, the median SNRs were 2.1, 2.9, and 6.1; and the median CNRs were 1.7, 2.2, and 4.7, respectively. Sensitivity and specificity were 56 and 93% for FBP, 62 and 91% for HIR, and 80 and 91% for IMR. LIE-CT with IMR showed the highest image quality and sensitivity (p < 0.05). The use of IMR enables significant improvement of image quality and diagnostic performance of LIE-CT for detecting MI in comparison with FBP and HIR.

Synthesis of Recombinant Mouse Crystallin Proteins and in Vitro Measurement of Their Refractivity.

The eye lens is an organ that focuses light onto the retina and is reported to have a high refractive index in vertebrates. An analysis of refractivity was conducted using recombinant mouse Crystallin proteins produced in Escherichia coli (E. coli) compared with bovine serum albumin (BSA) and other commercially available proteins. Not only did we measure the refractivity but for one of the crystallins, Cryba1, we also confirmed that it responds uniquely to its environmental conditions. The crystallin showed high refractivity, as expected, and we confirmed that the electrical charge of the Cryba1 molecule influences its refractivity.

Three-dimensional maximum principal strain using cardiac computed tomography for identification of myocardial infarction.

OBJECTIVES: To evaluate the feasibility of three-dimensional (3D) maximum principal strain (MP-strain) derived from cardiac computed tomography (CT) for detecting myocardial infarction (MI). METHODS: Forty-three patients who underwent cardiac CT and magnetic resonance imaging (MRI) were retrospectively selected. Using the voxel tracking of motion coherence algorithm, the peak CT MP-strain was measured using the 16-segment model. With the trans-mural extent of late gadolinium enhancement (LGE) and the distance from MI, all segments were classified into four groups (infarcted, border, adjacent, and remote segments); infarcted and border segments were defined as MI with LGE positive. Diagnostic performance of MP-strain for detecting MI was compared with per cent systolic wall thickening (%SWT) assessed by MRI using receiver-operating characteristic curve analysis at a segment level. RESULTS: Of 672 segments excluding16 segments influenced by artefacts, 193 were diagnosed as MI. Sensitivity and specificity of peak MP-strain to identify MI were 81 % [95 % confidence interval (95 % CI): 74-88 %] and 86 % (81-92 %) compared with %SWT: 76 % (60-95 %) and 68 % (48-84 %), respectively. The area under the curve of peak MP-strain was superior to %SWT [0.90 (0.87-0.93) vs. 0.80 (0.76-0.83), p < 0.05]. CONCLUSIONS: CT MP-strain has a potential to provide incremental value to coronary CT angiography for detecting MI. KEY POINTS: • CT MP-strain allows for three-dimensional assessment of regional cardiac function. • CT-MP strain has high diagnostic accuracy for detecting myocardial infarction. • CT-MP strain may assist in tissue characterisation of myocardium assessed by LGE-MRI. • CT-MP strain provides incremental values to coronary CTA for detecting myocardial infarction.

Optimal Scan Time for Single-Phase Myocardial Computed Tomography Perfusion to Detect Myocardial Ischemia　- Derivation Cohort From Dynamic Myocardial Computed Tomography Perfusion.

BACKGROUND: Single-phase myocardial computed tomography perfusion (CTP) is useful for detecting myocardial ischemia, but determining the optimal scan time is difficult. The present study evaluated this by analyzing dynamic CTP data.Methods and Results:We retrospectively selected 32 patients, all of whom had undergone stress dynamic CTP and magnetic resonance myocardial perfusion imaging (MR-MPI). Myocardial ischemia was assessed by MR-MPI using the 16-segment model. Whole-heart dynamic CTP data were acquired for 30 consecutive heartbeats without spatial or temporal gaps using a wide-detector CT, and redistributed into 11 series of single-phase CTP acquired from -2 s to 8 s from the time of maximal enhancement (Tmax) in the ascending aorta. Single-phase CTP images were visually assessed at the segment level, and diagnostic performance of single-phase CTP images for detecting myocardial ischemia was compared with dynamic CTP. Of 512 segments, 177 segments (35%) were diagnosed as ischemic by MR-MPI. The diagnostic accuracy of single-phase CTP acquired at 2-6 s from Tmax in the ascending aorta (median 86%, range 84-87%) was comparable to that of dynamic CTP. CONCLUSIONS: The optimal scan time for detecting myocardial ischemia with single-phase CTP was at 2-6 s from Tmax in the ascending aorta. (Circ J 2016; 80: 2506-2512).

Engineered yeast whole-cell biocatalyst for direct degradation of alginate from macroalgae and production of non-commercialized useful monosaccharide from alginate.

Alginate is a major component of brown macroalgae. In macroalgae, an endolytic alginate lyase first degrades alginate into oligosaccharides. These oligosaccharides are further broken down into monosaccharides by an exolytic alginate lyase. In this study, genes encoding various alginate lyases derived from alginate-assimilating marine bacterium Saccharophagus degradans were isolated, and their enzymes were displayed using the yeast cell surface display system. Alg7A-, Alg7D-, and Alg18J-displaying yeasts showed endolytic alginate lyase activity. On the other hand, Alg7K-displaying yeast showed exolytic alginate lyase activity. Alg7A, Alg7D, Alg7K, and Alg18J, when displayed on yeast cell surface, demonstrated both polyguluronate lyase and polymannuronate lyase activities. Additionally, polyguluronic acid could be much easily degraded by Alg7A, Alg7K, and Alg7D than polymannuronic acid. In contrast, polymannuronic acid could be much easily degraded by Alg18J than polyguluronic acid. We further constructed yeasts co-displaying endolytic and exolytic alginate lyases. Degradation efficiency by the co-displaying yeasts were significantly higher than single alginate lyase-displaying yeasts. Alg7A/Alg7K co-displaying yeast had maximum alginate degrading activity, with production of 1.98 g/L of reducing sugars in a 60-min reaction. This system developed, along with our findings, will contribute to the efficient utilization and production of useful and non-commercialized monosaccharides from alginate by Saccharomyces cerevisiae.