Comprehensive Geriatric Assessment Using the Yoitoko Check-Up, a Novel Health Check-Up Providing Positive Feedback to Older Adults: A Before-After Study.

PURPOSE: The Yoitoko check-up, a novel health check-up providing positive feedback, has been developed to promote health among older adults, and consists of several comprehensive geriatric assessment items. This report aimed to describe the details of the Yoitoko check-up and to explore the future possibility of the check-up by evaluating the participants' short-term behavioral changes in terms of comprehensive functioning, using a before-after study design. PATIENTS AND METHODS: Four Yoitoko check-ups were conducted, at 3-month intervals, between December 2018 and September 2019. Study participants aged ≥65 years included those who had undergone ≥2 Yoitoko check-ups. The results of each visit after the second check-ups were retrospectively compared with those of the baseline, and the mean changes and the odds ratios were calculated using a paired t-test or a McNemar test, respectively. RESULTS: Of 84 participants, the results of 16 (19.0%) participants were analyzed. The mean (standard deviation) age was 75.3 (4.7) years. The mean Tokyo Metropolitan Institute of Gerontology Index of Competence score, a measure of high-level functional capacity, increased 0.9 (95% confidence interval; range, 0.2-1.5) points between the first and second visits. CONCLUSION: We developed the Yoitoko check-up and introduced the details of it. Our study findings suggested that the Yoitoko check-up may further motivate older adults to improve their health and promote positive behavioral changes. Future studies are needed to evaluate the effectiveness of this novel assessment method.

The NADPH oxidase NOX4 promotes the directed migration of endothelial cells by stabilizing vascular endothelial growth factor receptor 2 protein.

Directed migration of endothelial cells (ECs) is an important process during both physiological and pathological angiogenesis. The binding of vascular endothelial growth factor (VEGF) to VEGF receptor-2 (VEGFR-2) on the EC surface is necessary for directed migration of these cells. Here, we used TAXIScan, an optically accessible real-time horizontal cell dynamics assay approach, and demonstrate that reactive oxygen species (ROS)-producing NADPH oxidase 4 (NOX4), which is abundantly expressed in ECs, mediates VEGF/VEGFR-2-dependent directed migration. We noted that a continuous supply of endoplasmic reticulum (ER)-retained VEGFR-2 to the plasma membrane is required to maintain VEGFR-2 at the cell surface. siRNA-mediated NOX4 silencing decreased the ER-retained form of VEGFR-2, resulting in decreased cell surface expression levels of the receptor. We also found that ER-localized NOX4 interacts with ER-retained VEGFR-2 and thereby stabilizes this ER-retained form at the protein level in the ER. We conclude that NOX4 contributes to the directed migration of ECs by maintaining VEGFR-2 levels at their surface.

Role of chemotherapy in 5000 patients with head and neck cancer treated by curative surgery: A subgroup analysis of the meta-analysis of chemotherapy in head and neck cancer.

OBJECTIVE: To evaluate the effect of chemotherapy added to a surgical locoregional treatment (LRT) for patients with locally advanced head and neck squamous cell carcinoma (HNSCC). MATERIALS AND METHODS: We studied the sub-group of trials with surgical LRT included in the meta-analysis on chemotherapy in head and neck cancer (MACH-NC). Data from published and unpublished randomized trials comparing the addition of chemotherapy to LRT in HNSCC patients were sought using electronic database searching for the period 1965-2000, hand searching and by contacting experts in the field. Trials with less than 60 patients, or preoperative radiotherapy or where the type of LRT could not be individually determined were excluded. All individual patient data were checked for internal consistency, compared with published reports, and validated with trialists. Data were pooled using a fixed-effect model. Heterogeneity was assessed using Cochrane test and I2 statistic. RESULTS: Twenty-four trials were eligible (5000 patients). Chemotherapy improved overall survival (HR = 0.92 [95%CI: 0.85-0.99] p = 0.02). There was a significant interaction between treatment effect and timing of chemotherapy (p = 0.08 at pre-specified threshold of 0.10) with a greater effect for concomitant chemotherapy (HR = 0.79, 95%CI: 0.69-0.92). The benefit of chemotherapy was greater in women (HRwomen = 0.63, 95%CI: 0.50-0.80) compared to men (HRmen = 0.96, 95%CI: 0.89-1.04; p for interaction = 0.001). CONCLUSIONS: This analysis confirmed the benefit of concomitant chemotherapy added to surgical LRT. The role of induction therapy as yet to be determined as it did not improve OS. Women may benefit more than men from chemotherapy.

Production of Human ß-Actin Using a Bacterial Expression System with a Cold Shock Vector.

Actin is one of the most abundant proteins in the cytoplasm of eukaryotic cells and plays important roles in a variety of cellular functions. However, it has been difficult to produce actin in substantial amounts using bacterial expression systems. In this article, a new method is described for the production of recombinant actin in bacterial cells. Human ß-actin (His-tagged) can be expressed using a cold shock vector, pCold, in a bacterial expression system and then separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant ß-actin shows normal polymerization ability compared with commercially available ß-actin purified from human platelets. This article also describes the preparation of mutant actin(G168R). This purified mutant exhibits impaired polymerization ability. The system and procedures described here will provide a useful method for the production of actin isoforms and their mutants. © 2018 by John Wiley & Sons, Inc.

Floral structure and development in Nartheciaceae (Dioscoreales), with special reference to ovary position and septal nectaries.

We present a comparative study of the floral structure and development of Nartheciaceae, a small dioscorealean family consisting of five genera (Aletris, Lophiola, Metanarthecium, Narthecium, and Nietneria). A noticeable diversity existed in nine floral characters. Analyses of their respective character states in the light of a phylogenetic context revealed that the flowers of Nartheciaceae, whose plesiomorphies occur in Aletris and Metanarthecium, have evolved toward in all or part of Lophiola, Narthecium, and Nietneria: (1) loss of a perianth tube; (2) stamen insertion at the perianth base; (3) congenital carpel fusion; (4) loss of the septal nectaries; (5) unilocular style; (6) unfused lateral carpellary margins in the style; (7) flower with the median outer tepal on the abaxial side; (8) flower with moniliform hairs; and (9) flower with weak monosymmetry. We further found that, as the flowers developed, the ovary shifted its position from inferior to superior. As a whole, their structure changes suggest that the Nartheciaceae flowers have evolved in close association with pollination and seed dispersal. By considering inferior ovaries and the presence of septal nectaries as plesiomorphies of Nartheciaceae, we discussed evolution of the ovary position and septal nectaries in all the monocots.

Phosphorylation of Ser-525 in ßPix impairs Nox1-activating ability in Caco-2 cells.

ßPix activates Nox1, an O2--generating NADPH oxidase, through Rac activation. In this study, we found that S525E mutation of ßPix eliminated its Nox1-activating ability in transfected Caco-2 cells. Unexpectedly, affinity for Rac was not diminished but rather enhanced by S525E mutation, and guanine nucleotide exchange factor (GEF) activity was not altered. The N-terminal fragment (amino acids 1-400) showed similar Rac-binding and GEF activity to wild-type ßPix. In contrast, the C-terminal fragment (amino acids 408-646) had higher Rac-binding activity, particularly for Rac-GTP, than wild-type ßPix, and showed no GEF activity. These data suggest that a second Rac-binding site within the C-terminal region is opened by phosphorylation of Ser-525. The site may bind not only Rac-GDP but also Rac-GTP released from the N-terminal catalytic region, which interrupts Rac-GTP translocation to the membrane where Nox1 resides. If one considers that S340E mutation enhances Nox1 activation (Kaito et al., 2014), the present study suggests that ßPix can also play an inhibitory role in O2- production, depending on the sites of phosphorylation.

Evolutionary timescale of monocots determined by the fossilized birth-death model using a large number of fossil records.

Although the phylogenetic relationships between monocot orders are sufficiently understood, a timescale of their evolution is needed. Several studies on molecular clock dating are available, but their results have been biased by their calibration schemes. Recently, the fossilized birth-death model, a type of Bayesian dating method, was proposed, and it does not require prior calibration and allows the use all available fossils. Using this model, we conducted divergence-time estimations of monocots to explore their evolutionary timeline without calibration bias. This is the first application of this model to seed plants. The dataset contained the matK and rbcL chloroplast genes of 118 monocot genera covering all extant orders. We employed information from 247 monocot fossils, which exceeded previous dating analyses that used a maximum of 12 monocot fossils. The crown group of monocots was dated to approximately the Late Jurassic-Early Cretaceous periods, and most extant monocot orders were estimated to diverge throughout the Early Cretaceous. Our results overlapped with the divergence time of insect lineages, such as beetles and flies, suggesting an association with pollinators in early monocot evolution. In addition, we proposed three new orders based on divergence time: Orchidales separated from Asparagales and Tofieldiales and Arales separated from Aslimatales.

Good manufacturing practices production of a purification-free oral cholera vaccine expressed in transgenic rice plants.

KEY MESSAGE: The first Good Manufacturing Practices production of a purification-free rice-based oral cholera vaccine (MucoRice-CTB) from transgenic plants in a closed cultivation system yielded a product meeting regulatory requirements. Despite our knowledge of their advantages, plant-based vaccines remain unavailable for human use in both developing and industrialized countries. A leading, practical obstacle to their widespread use is producing plant-based vaccines that meet governmental regulatory requirements. Here, we report the first production according to current Good Manufacturing Practices of a rice-based vaccine, the cholera vaccine MucoRice-CTB, at an academic institution. To this end, we established specifications and methods for the master seed bank (MSB) of MucoRice-CTB, which was previously generated as a selection-marker-free line, evaluated its propagation, and given that the stored seeds must be renewed periodically. The production of MucoRice-CTB incorporated a closed hydroponic system for cultivating the transgenic plants, to minimize variations in expression and quality during vaccine manufacture. This type of molecular farming factory can be operated year-round, generating three harvests annually, and is cost- and production-effective. Rice was polished to a ratio of 95 % and then powdered to produce the MucoRice-CTB drug substance, and the identity, potency, and safety of the MucoRice-CTB product met pre-established release requirements. The formulation of MucoRice-CTB made by fine-powdering of drug substance and packaged in an aluminum pouch is being evaluated in a physician-initiated phase I study.

An improved superoxide-generating nanodevice for oxidative stress studies in cultured cells.

The effects of reactive oxygen species on cells have attracted great attention from both physiological and pathological aspects. Superoxide (O2-) is the primary reactive oxygen species formed in animals. We previously developed an O2--generating nanodevice consisting of NADPH oxidase 2 (Nox2) and modulated activating factors. However, the device was subsequently found to be unstable in a standard culture medium. Here we improved the device in stability by cross-linking. This new nanodevice, Device II, had a half-life of 3 h at 37 °C in the medium. Device II induced cell death in 80% of HEK293 cells after 24 h of incubation. Superoxide dismutase alone did not diminish the effect of the device, but eliminated the effect when used together with catalase, confirming that the cell death was caused by H2O2 derived from O2-. Flow cytometric analyses revealed that Device II induced caspase-3 activation in HEK293 cells, suggesting that the cell death proceeded largely through apoptosis.

Induction of apoptosis in Caco-2 cells by exogenously added O2⁻ produced by a nanodevice.

The effects of reactive oxygen species on cells have attracted considerable attention in relation to oxidative stress and related disorders. Superoxide (O2(-)) is the primary reactive oxygen species formed in animals as a byproduct or purposeful product of enzymes. We recently established an O2(-)-generating nanodevice that produces O2(-) continuously even in culture medium, by improving an original nanodevice. The new nanodevice, named Device II, efficiently induced cell death in Caco-2 cells in a time- and dose-dependent manner. Catalase largely recovered the cell viability, while superoxide dismutase rather lowered the viability. Flow cytometric and fluorescence microscopic analyses revealed that phosphatidylserine was exposed on the cells and that caspase-3 was activated in the cells after treatment with Device II. These findings indicated that exogenously added O2(-) caused apoptosis in Caco-2 cells through its derivative H2O2.

Nox1 activation by ßPix and the role of Ser-340 phosphorylation.

Rac is an activating factor for Nox1, an O2(-)-generating NADPH oxidase, expressed in the colon and other tissues. Rac requires a GDP-GTP exchange factor for activation. Nox1 activation by ßPix has been demonstrated in cell lines. We examined the effects of ßPix and its phosphomimetic mutant on endogenous Nox1 in Caco-2 cells transfected with Noxo1 and Noxa1. ßPix expression enhanced O2(-) production in resting cells and cells stimulated with EGF or phorbol ester. ßPix(S340E) further enhanced O2(-) production, while ßPix(S340A) eliminated the ßPix effect. ßPix(S340E), but not ßPix(S340A), had higher affinity and GEF activity for Rac than wild-type ßPix. These results suggest that ßPix phosphorylation at Ser-340 upregulates Nox1 through Rac activation, confirming Rac as a trigger for acute Nox1-dependent ROS production.

New flavonol glycosides from the leaves of Triantha japonica and Tofieldia nuda.

Two new flavonol glycosides were isolated from the leaves of Triantha japonica, together with eight known flavonols, kaempferol 3-O-sophoroside, kaempferol 3-O-sambubioside, kaempferol 3-O-glucosyl-(1 --> 2)-[glucosyl-(1 --> 6)-glucoside], quercetin 3-O-sophoroside, quercetin 3-O-sambubioside, isorhamnetin 3-O-glucoside, isorhamnetin 3-O-sophoroside and isorhamnetin 3-O-sambubioside. The new compounds were identified as kaempferol 3-O-beta-xylopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-glucopyranoside] (1) and isorhamnetin 3-O-beta-xylopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-glucopyranoside] (3) by UV, LC-MS, acid hydrolysis, and 1H and 13C NMR spectroscopy. Another two new flavonol glycosides were isolated from theleaves of Tofieldia nuda, and identified as kaempferol 3-O-beta-glucopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-galactopyranoside] (4) and quercetin 3-O-beta-glucopyranosyl-(1 --> 2)-[beta-glucopyranosyl-(1 --> 6)-beta-galactopyranoside] (5). Though the genera Triantha and Tofieldia were treated as Tofieldia sensu lato, they were recently divided into two genera. It was shown by this survey that their flavonoid composition were also different to each other.

C-terminal truncation of Noxa1 greatly enhances its ability to activate Nox2 in a pure reconstitution system.

Noxa1 activates Nox2 together with Noxo1 and Rac in a pure reconstitution system, but the resulting activity is considerably lower than that induced by p67(phox) and p47(phox). In this study, we found that C-terminal-truncated forms of Noxa1 exhibited higher activities than full-length Noxa1. Of the truncations examined, Noxa1(1-225) showed the highest ability for activation. Kinetic studies revealed that Noxa1(1-225) had a threefold higher Vmax value than full-length Noxa1 with a similar EC50 value. The affinities of Noxo1 and RacQ61L were not much altered by the truncation. Conversely, the affinity of FAD for the Nox2 complex was enhanced after the truncation. In the absence of Noxo1, Noxa1(1-225) showed much higher activity with a lower EC50 than full-length Noxa1. Noxa1(1-225) showed comparable activity to that of p67(phox) with either Noxo1 or p47(phox), although the stability was lower than that with p67(phox) and p47(phox). These findings indicate that the role of the C-terminal half of Noxa1 is autoinhibition. The data suggest a two-step autoinhibition mechanism, comprising self-masking to interrupt the binding to the oxidase, and holding of the activation domain in a suboptimal position to the oxidase. This study reveals that when both types of inhibition are released, Noxa1 achieves high-level superoxide production.

Noxa1 as a moderate activator of Nox2-based NADPH oxidase.

Noxa1 was discovered as an activating factor for Nox1, an O(2)(-)-generating enzyme. Subsequent studies have shown that Noxa1 is colocalized with Nox2 in several cell types, including vascular cells. Nox2 activation by Noxa1 has been examined in reconstituted model cells. However, little is known about the kinetic properties of Noxa1 in Nox2 activation. In the present study, we used purified cyt.b(558) (Nox2 plus p22(phox)), Rac(Q61L), and Noxo1 to examine the ability of Noxa1 to activate Nox2. In the pure reconstitution system, Noxa1 activated Nox2 with lower efficiency than p67(phox), a canonical activator of Nox2. The EC(50) value of Noxa1 was considerably higher than that of p67(phox). The V(max) value with Noxa1 and Noxo1 was one-third of that with p67(phox) and p47(phox). The EC(50) value of Noxo1 or Rac(Q61L) was also higher when Noxa1 was used. The affinity of FAD for the oxidase and the stability of the active complex were remarkably low when Noxa1 and Noxo1 were used compared with p67(phox) and p47(phox). The stability was not improved by fusion of Noxa1 with Rac(Q61L). These findings show that Noxa1 has quite different kinetic properties from p67(phox) and suggest that Noxa1 may function as a moderate activator of Nox2.

Production of human ß-actin and a mutant using a bacterial expression system with a cold shock vector.

Actin is the most abundant protein in the cytoplasm of most eukaryotic cells and is involved in a variety of cellular functions. It has been difficult to produce actin in bacterial expression systems in good yields. In this study, we developed a new simple method for the production of recombinant actin in Escherichia coli cells. Human ß-actin was successfully expressed using a cold shock vector, pCold, in the bacterial expression system. The expressed ß-actin (hexahistidine-tagged) was separated with a Ni-chelating resin, followed by a polymerization/depolymerization cycle or column chromatography with the Ni-chelating resin. The purified recombinant ß-actin showed a normal polymerization ability compared with ß-actin purified from human platelets. We produced a recombinant mutant actin with a Gly-168Arg mutation in the system and confirmed that it exhibited an impaired polymerization ability. The system developed in this study will provide a useful method for the production of actin isoforms and their mutants.

Activation of NADPH oxidase 1 in tumour colon epithelial cells.

In the plasma membrane fraction from Caco-2 human colon carcinoma cells, active Nox1 (NADPH oxidase 1) endogenously co-localizes with its regulatory components p22(phox), NOXO1, NOXA1 and Rac1. NADPH-specific superoxide generating activity was reduced by 80% in the presence of either a flavoenzyme inhibitor DPI (diphenyleneiodonium) or NADP(+). The plasma membranes from PMA-stimulated cells showed an increased amount of Rac1 (19.6 pmol/mg), as compared with the membranes from unstimulated Caco-2 cells (15.1 pmol/mg), but other components did not change before and after the stimulation by PMA. Spectrophotometric analysis found approx. 36 pmol of FAD and 43 pmol of haem per mg of membrane and the turnover of superoxide generation in a cell-free system consisting of the membrane and FAD was 10 mol/s per mol of haem. When the constitutively active form of Rac, Rac1(Q61L) or GTP-bound Rac1 was added exogenously to the membrane, O(2)(-)-producing activity was enhanced up to 1.5-fold above the basal level, but GDP-loaded Rac1 did not affect superoxide-generating kinetics. A fusion protein [NOXA1N-Rac1(Q61L)] between truncated NOXA1(1-211) and Rac1-(Q61L) exhibited a 6-fold increase of the basal Nox1 activity, but NOXO1N(1-292) [C-terminal truncated NOXO1(1-292)] alone showed little effect on the activity. The activated forms of Rac1 and NOXA1 are essentially involved in Nox1 activation and their interactions might be responsible for regulating the O(2)(-)-producing activity in Caco-2 cells.

p40phox as an alternative organizer to p47phox in Nox2 activation: a new mechanism involving an interaction with p22phox.

p40(phox) activated phagocyte NADPH oxidase without p47(phox) in a cell-free system consisting of p67(phox), Rac and cytochrome b(558) relipidated with phosphatidylinositol 3-phosphate. The activation reached to 70% of that by p47(phox). Addition of p47(phox) slightly increased the activation, but not additively. p40(phox) improved the efficiency of p67(phox) in the activation. The C-terminus-truncated p67(phox), p40(phox)(D289A), p40(phox)(R58A), or p40(phox)(W207R) showed an impaired activation. A peptide corresponding to the p22(phox) Pro-rich region suppressed the activation, and far-western blotting revealed its interaction with p40(phox) SH3 domain. Thus, p40(phox) can substitute for p47(phox) in the activation, interacting with p22(phox) and p67(phox) through their specific regions.

Identification of an actin-binding site in p47phox an organizer protein of NADPH oxidase.

Actin has been reported to enhance the superoxide-generating activity of neutrophil NADPH oxidase in a cell-free system and to interact with p47phox, a regulatory subunit of the oxidase. In the present study, we searched for an actin-binding site in p47phox by far-western blotting and blot-binding assays using truncated forms of p47phox. The amino-acid sequence 319-337 was identified as an actin-binding site, and a synthetic peptide of this sequence bound to actin. The sequence shows no homology to other known actin-binding motifs. It is located in the autoinhibitory region of p47phox and includes Ser-328, a phosphorylation site essential for unmasking. Although a phosphorylation-mimetic p47phox mutant bound to actin with a lower affinity than the wild type, the same mutant interacted with filamentous actin more efficiently than the wild type. A mutant peptide p47phox (319-337, Ser328Glu) bound to filamentous actin more tightly than to monomer actin. These results suggest that p47phox moves to cortical actin when it becomes unmasked in the cells.

A new type of O(2)(-)-generating tool for oxidative stress studies by remodeling neutrophil NADPH oxidase.

The effects of reactive oxygen species on cells have attracted much attention in relation to redox regulation and oxidative stress-related diseases. Superoxide (O(2)(-)) is the reactive oxygen species primarily formed in biological systems. However, no convenient O(2)(-)-generating device has been available for use in cell or tissue culture. The neutrophil NADPH oxidase, a professional enzyme for killing bacteria, has a high ability to produce O(2)(-). However, the cell-free activation process requires several protein factors and an anionic amphiphile, and moreover, the activation is transient. To utilize the enzyme as an O(2)(-) generator, we improved the cell-free activation method by remodeling regulatory components, optimizing lipid composition, and modifying the mixing conditions. We established a new method to produce an active enzyme that is stable, efficient, and preservable. As an application, we examined the effect of the device on cultured HEK293 cells and observed that it caused cell death. This system has several advantages over the xanthine oxidase system often used. The new device will be useful for studies of oxidative stress and related diseases.