Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level

Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca^[2+]-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca^[2+]-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects

Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety

AbstractA mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the 3 GalCers, the mAb reacted with two GalCers which contained α-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid

The structural changes in crystalline cellulose and effects on enzymatic digestibility

The enzymatic hydrolysis of cellulose I achieves almost complete digestion when sufficient enzyme loading as much as 20 mg/g-substrate is applied. However, the yield of digestion reaches the limit when the enzyme dosage is decreased to 2 mg/g-substrate. Therefore, we have performed three pretreatments such as mercerization, dissolution into phosphoric acid and EDA treatment. Transformation into cellulose II hydrate by mercerization and dissolution into phosphoric acid were not sufficient because substrate changed to highly crystalline structure during saccharification. On the other hand, in the case of crystalline conversion of cellulose I to IIII by EDA, almost perfect digestion was achieved even in enzyme loading as small as 0.5 mg/g-substrate, furthermore, hydrolyzed residue was typical cellulose I. The structural analysis of substrate after saccharification provides an insight into relationships between cellulose crystalline property and cellulase toward better enzymatic digestion

Rosa davurica Pall., a useful Rosa species for functional rose hip production with high content of antioxidants and multiple antioxidant activities in hydrophilic extract

Fruits of the genus Rosa plants are called rose hips. The common hips of R. canina are well known as a rich source of antioxidants like ascorbic acid and polyphenols. To investigate availability in the hips in Rosa spp., wild Rosa hips originating from East Asia, i.e. R. acicularis, R. davurica, R. multiflora and R. rugosa were evaluated in terms of the content of antioxidants and antioxidant ability in the hydrophilic extracts. The hips from R. glauca originating from south Europe and its interspecific hybrids ('Kitaayaka' and 'Consared'), and purchased R. canina hips were also examined. In addition to the colorimetric detections of DPPH and ORAC, ESR-ST methods were employed for evaluating antioxidant ability, which can determine scavenging activities against naturally-occurring ROS i.e. superoxide anion radical (center dot O2-), hydroxyl radical (HO center dot), alkoxyl radical (RO center dot) and singlet oxygen (1O2), individually. The hips of R. davurica and 'Consared' showed quite high values in both the total content of ASA plus DHA (40.8-103.1 g/kg DW) and total polyphenols (119.2-161.5 g quercetin eq./kg DW) regardless of the years collected. They also had high antioxidant activities against each radical compared to other rose hips, and thus their antioxidant ability seems multiple. Both ASA and polyphenols could scavenge radicals of ROO center dot and 1O2, since significant correlations (P< 0.05) were confirmed. However, polyphenols might have greater contribution to the antioxidant activities, because the correlation coefficients were higher in total polyphenols than ASA. R. davurica can be one of the useful genetic resources for breeding cultivars which will bear antioxidant-rich rose hips, since 'Consared' is a progeny of R. davurica x glauca

Association between Intraoperative Early Warning Score and Mortality and In-Hospital Stay in Lower Gastrointestinal Spontaneous Perforation

Background. Early warning scores (EWSs) can be easily calculated from physiological indices; however, the extent to which intraoperative EWSs and the corresponding changes are associated with patient prognosis is unknown. In this study, we investigated whether EWS and the corresponding time-related changes are associated with patient outcomes during the anesthetic management of lower gastrointestinal perforation. Methods. This was a single-center, retrospective cohort study conducted at a tertiary emergency care center. Adult patients who underwent surgery for spontaneous lower gastrointestinal perforations between September 1, 2012, and December 31, 2019, were included. The National Early Warning Score (NEWS) and Modified Early Warning Score (MEWS) were calculated based on the intraoperative physiological indices, and the associations with in-hospital death and length of hospital stay were investigated. Results. A total of 101 patients were analyzed. The median age was 70 years, and there were 11 cases of in-hospital death (mortality rate: 10.9%). There was a significant association between the intraoperative maximum NEWS and in-hospital death (odds ratio (OR): 1.60, 95% confidence interval (CI): 1.10–2.32, p=0.013) and change from initial to maximum NEWS (OR: 1.60, 95% CI: 1.07–2.40, p=0.023) in the crude analysis. However, when adjustments were made for confounding factors, no statistically significant associations were found. Other intraoperative EWS values and changes were not significantly associated with the investigated outcomes. The preoperative sepsis-related organ failure assessment score and the intraoperative base excess value were significantly associated with in-hospital death. Conclusions. No clear association was observed between EWSs and corresponding changes and in-hospital death in cases of lower gastrointestinal perforation. The preoperative sepsis-related organ failure assessment score and intraoperative base excess value were significantly associated with in-hospital death