High-Throughput Metabolic Profiling of Diverse Green Coffea arabica Beans Identified Tryptophan as a Universal Discrimination Factor for Immature Beans

The maturity of green coffee beans is the most influential determinant of the quality and flavor of the resultant coffee beverage. However, the chemical compounds that can be used to discriminate the maturity of the beans remain uncharacterized. We herein analyzed four distinct stages of maturity (immature, semi-mature, mature and overripe) of nine different varieties of green Coffea arabica beans hand-harvested from a single experimental field in Hawaii. After developing a high-throughput experimental system for sample preparation and liquid chromatography-mass spectrometry (LC-MS) measurement, we applied metabolic profiling, integrated with chemometric techniques, to explore the relationship between the metabolome and maturity of the sample in a non-biased way. For the multivariate statistical analyses, a partial least square (PLS) regression model was successfully created, which allowed us to accurately predict the maturity of the beans based on the metabolomic information. As a result, tryptophan was identified to be the best contributor to the regression model; the relative MS intensity of tryptophan was higher in immature beans than in those after the semi-mature stages in all arabica varieties investigated, demonstrating a universal discrimination factor for diverse arabica beans. Therefore, typtophan, either alone or together with other metabolites, may be utilized for traders as an assessment standard when purchasing qualified trading green arabica bean products. Furthermore, our results suggest that the tryptophan metabolism may be tightly linked to the development of coffee cherries and/or beans.     

Identification and physiological evaluation of the components from Citrus fruits as potential drugs for anti-corpulence and anticancer

On the basis of monitoring the prevention of accumulation of lipid droplets in mouse 3T3-L1 preadipocyte cells and inhibition of the proliferation of human colon cancer HT-29 cells, effective anti-corpulence and anticancer compounds were isolated from the peel of Citrus fruits. These bioactive components were identified as polymethoxyflavones and coumarin derivatives by spectroscopic analyses. 5-Hydroxy-6,7,8,3′,4′-pentamethoxyflavone had the greatest anti-corpulence effects and 3,5,6,7,8,3′,5′-heptamethoxyflavone had the greatest anticancer effects. Furthermore, distributions of those bioactive components in the peel of 10 species of Citrus fruits were demonstrated by HPLC analyses.     

Discovery of novel 2-(4-aryl-2-methylpiperazin-1-yl)-pyrimidin-4-ones as glycogen synthase kinase-3β inhibitors

We herein describe the results of further evolution of glycogen synthase kinase (GSK)-3β inhibitors from our promising compounds containing a 3-methylmorpholine moiety. Transformation of the morpholine moiety into a piperazine moiety resulted in potent GSK-3β inhibitors. SAR studies focused on the nitrogen atom of the piperazine moiety revealed that a phenyl group afforded potent inhibitory activity toward GSK-3β. Docking studies indicated that the phenyl group on the piperazine nitrogen atom and the methyl group on the piperazine make cation-π and CH-π interactions with GSK-3β respectively. 4-Methoxyphenyl analogue 29 showed most potent inhibitory activity toward GSK-3β with good in vitro and in vivo pharmacokinetic profiles, and 29 demonstrated a significant decrease in tau phosphorylation after oral administration in mice.     

Imaging-cytometry revealed spatial heterogeneities of marker expression in undifferentiated human pluripotent stem cells

Human pluripotent stem cells (hPSCs) provide a good model system for studying human development and are expected as a source for both cell-based medical and pharmaceutical research application. However, stable maintenance of undifferentiated hPSCs is yet challenging, and thus routine characterization is required. Flow-cytometry is one of the popular quantitative characterization tools for hPSCs, but it has drawback of spatial information loss of the cells in the culture. Here, we have applied a two-dimensional imaging cytometry that examines undifferentiated state of hPSCs to analyze localization and morphological information of immunopositive cells in the culture. The whole images of cells in a culture vessel were acquired and analyzed by an image analyzer, IN Cell Analyzer 2000, and determined staining intensity of the cells with their positional information. We have compared the expression of five hPSC-markers in four hPSC lines using the two-dimensional imaging cytometry and flow cytometry. The results showed that immunopositive ratios analyzed by the imaging cytometry had good correlation with those by the flow cytometry. Furthermore, the imaging cytometry revealed spatially heterogenic expression of hPSC-markers in undifferentiated hPSCs. Imaging cytometry is capable of reflecting minute aberrance without losing spatial and morphological information of the cells. It would be a powerful, useful, and time-efficient tool for characterizing hPSC colonies.     

Aboveground biomass increments over 26 years (1993–2019) in an old-growth cool-temperate forest in northern Japan

Assessing long-term changes in the biomass of old-growth forests with consideration of climate effects is essential for understanding forest ecosystem functions under a changing climate. Long-term biomass changes are the result of accumulated short-term changes, which can be affected by endogenous processes such as gap filling in small-scale canopy openings. Here, we used 26 years (1993–2019) of repeated tree census data in an old-growth, cool-temperate, mixed deciduous forest that contains three topographic units (riparian, denuded slope, and terrace) in northern Japan to document decadal changes in aboveground biomass (AGB) and their processes in relation to endogenous processes and climatic factors. AGB increased steadily over the 26 years in all topographic units, but different tree species contributed to the increase among the topographic units. AGB gain within each topographic unit exceeded AGB loss via tree mortality in most of the measurement periods despite substantial temporal variation in AGB loss. At the local scale, variations in AGB gain were partially explained by compensating growth of trees around canopy gaps. Climate affected the local-scale AGB gain: the gain was larger in the measurement periods with higher mean air temperature during the current summer but smaller in those with higher mean air temperature during the previous autumn, synchronously in all topographic units. The influences of decadal summer and autumn warming on AGB growth appeared to be counteracting, suggesting that the observed steady AGB increase in KRRF is not fully explained by the warming. Future studies should consider global and regional environmental factors such as elevated CO₂ concentrations and nitrogen deposition, and include cool-temperate forests with a broader temperature range to improve our understanding on biomass accumulation in this type of forests under climate change.

     

Appropriate preoperative membranous urethral length predicts recovery of urinary continence after robot-assisted laparoscopic prostatectomy

Purpose

We investigated that preoperative membranous urethral length (MUL) would be associated with the recovery of urinary continence after robot-assisted laparoscopic prostatectomy (RALP).

Patients and methods

We studied 204 patients who underwent RALP between May 2013 and March 2016. All patients underwent pelvic magnetic resonance imaging (MRI) preoperatively to measure MUL. Urinary continence was defined as the use of one pad or less (safety pad). The 204 patients were divided into two groups: continence group, those who achieved recovery of continence at 3, 6, and 12?months after RALP, and incontinence group, those who did not. We retrospectively analyzed the patients in terms of preoperative clinical factors including age, body mass index (BMI), estimated prostate volume, neurovascular bundle salvage, history of preoperative hormonal therapy, and MUL.

Results

The safety pad use rate was 69.6%, 86.9%, and 91.1% at 3, 6, and 12?months, respectively. On univariate and multivariate analyses, MUL were significant factors in every term of recovery of urinary continence in both groups. According to the receiver operating characteristic (ROC) curve analysis, the preoperative MUL that could best predict early recovery of urinary continence at 3 months after RALP was 12?mm.

Conclusions

We suggest that preoperative MUL >?12?mm would be a predictor of early recovery of urinary continence after RALP.

     

Human OS-9, a Lectin Required for Glycoprotein Endoplasmic Reticulum-associated Degradation, Recognizes Mannose-trimmed N-Glycans

In the endoplasmic reticulum (ER), lectins and processing enzymes are involved in quality control of newly synthesized proteins for productive folding as well as in the ER-associated degradation (ERAD) of misfolded proteins. ER quality control requires the recognition and modification of the N-linked oligosaccharides attached to glycoproteins. Mannose trimming from the N-glycans plays an important role in targeting of misfolded glycoproteins for ERAD. Recently, two mammalian lectins, OS-9 and XTP3-B, which contain mannose 6-phosphate receptor homology domains, were reported to be involved in ER quality control. Here, we examined the requirement for human OS-9 (hOS-9) lectin activity in degradation of the glycosylated ERAD substrate NHK, a genetic variant of α1-antitrypsin. Using frontal affinity chromatography, we demonstrated that the recombinant hOS-9 mannose 6-phosphate receptor homology domain specifically binds N-glycans lacking the terminal mannose from the C branch in vitro. To examine the specificity of OS-9 recognition of N-glycans in vivo, we modified the oligosaccharide structures on NHK by overexpressing ER α1,2-mannosidase I or EDEM3 and examined the effect of these modifications on NHK degradation in combination with small interfering RNA-mediated knockdown of hOS-9. The ability of hOS-9 to enhance glycoprotein ERAD depended on the N-glycan structures on NHK, consistent with the frontal affinity chromatography results. Thus, we propose a model for mannose trimming and the requirement for hOS-9 lectin activity in glycoprotein ERAD in which N-glycans lacking the terminal mannose from the C branch are recognized by hOS-9 and targeted for degradation.Recognition and sorting of improperly folded proteins is essential to cell survival, and hence, an elaborate quality control system is found in cells. ER⁴ quality control is well characterized with respect to the N-linked oligosaccharides regulating the folding and degradation of newly synthesized proteins in the ER (1). Immediately after polypeptides enter the ER, Glc₃Man₉GlcNAc₂ (G3M9) precursor oligosaccharides are covalently attached and subsequently processed. Terminally misfolded proteins are removed from the ER by the ERAD machinery (1–4). Aberrant conformers are recognized, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (5, 6). Processing of mannose residues from the N-linked oligosaccharides acts as a timer for the recognition of misfolded glycoproteins in the ER lumen (1, 7). ER α1,2-mannosidase I (ER ManI) in mammals and ER α-mannosidase in yeast preferentially trim mannose residues from the middle branch of N-glycans, generating the Man₈GlcNAc₂ (M8) isomer B (M8B) (8). In mammals, further mannose processing is required as a signal for degradation (1, 9, 10), whereas the presence of M8B is sufficient to signal degradation in yeast (11). The postulated lectin EDEMs in mammals, their yeast homolog Htm1p/Mnl1p, and the yeast MRH domain-containing lectin Yos9p have all been proposed to recognize glycoproteins targeted for degradation (12).The role of Yos9p in glycoprotein ERAD was identified using a genetic screen in Saccharomyces cerevisiae (13). Yos9p, a homolog of hOS-9, contains an MRH domain (14) and functions as a lectin. Yos9p recognizes substrates of the ERAD-lumenal pathway (15–17), generating a large ER membrane complex containing the Hrd1p-Hrd3p ubiquitin ligase core complex (18–20). The M8B and Man₅GlcNAc₂ (M5) N-glycans are predicted to function as ligands for Yos9p (17). Bipartite recognition of both glycan and polypeptide by Yos9p has also been reported (15).Recent studies revealed that two mammalian MRH domain-containing lectins, OS-9 and XTP3-B, are ER luminal proteins involved in ER quality control and form a large complex containing the HRD1-SEL1L ubiquitin-ligase in the ER membrane (21–24). The components of the complex are similar to yeast, suggesting evolutionary conservation, although the molecular mechanisms underlying the role of OS-9 and XTP3-B remain elusive. Studies using lectin mutants have suggested that the MRH domains are required not for binding to ERAD substrates but for interactions with SEL1L (21), which has multiple N-glycans (25, 26). Additionally, lectin activity appears to be dispensable for hOS-9 binding to misfolded glycoproteins (21, 24). Thus, to understand the role of hOS-9 in the ER quality control pathway, the specific carbohydrate structures recognized by the hOS-9 MRH domain need to be identified, and the requirement of the lectin domain in substrate recognition needs to be determined.In the present study we demonstrate that the lectin activity of hOS-9 is required for enhancement of glycoprotein ERAD. We identified the N-glycan structures recognized by the recombinant hOS-9 MRH domain in vitro by frontal affinity chromatography (FAC). Using a model ERAD substrate, NHK (27), we show that the ability of hOS-9 to enhance ERAD in vivo depends on the oligosaccharides present on NHK, consistent with the FAC results.     

Organ and detriment-weighted dose rate coefficients for exposure to radionuclide-contaminated soil considering body morphometries that differ from reference conditions: adults and children

Radiation and Environmental Biophysics - The system of protection established by the International Commission on Radiological Protection (ICRP) provides a robust framework for ionizing radiation...