Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold-acclimated Arabidopsis.

Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea during the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.

Comparison between the 0- and 30-s balloon dilation time in percutaneous transluminal angioplasty for restenosed arteriovenous fistula among hemodialysis patients: a multicenter, prospective, randomized trial (CARP study).

BACKGROUND: This study aims to compare patency rates of the 0- and 30-s (sec) balloon dilation time in hemodialysis (HD) patients with restenosis after percutaneous transluminal angioplasty (PTA). METHODS: The patients who underwent PTA within 6 months for failed arteriovenous fistula at the forearm were randomly assigned the 0-s or 30-s dilation time group. Effect of dilation time on the 3- and 6-month patency rates after PTA was examined. RESULTS: Fifty patients were enrolled in this study. The 3-month patency rate in the 30-s dilation group was better than that in the 0-s dilation group (P = 0.0050), while the 6-month patency rates did not show a significant difference between the two groups (P = 0.28). Cox's proportional hazard model revealed that 30-s of inflation time (hazard ratio 0.027; P = 0.0072), diameter of the proximal (hazard ratio 0.32; P = 0.031), and dilation pressure (hazard ratio 0.63; P = 0.014) were associated with better 3-month patency. Dilation pressure between previous and present PTA did not differ in the 0-s (P = 0.15) and 30-s dilation groups (P = 0.16). The 6-month patency rate of the present PTA in the 30-s dilation group was higher than that of the previous PTA (P = 0.015). The visual analog scale did not differ between the two groups (P = 0.51). CONCLUSION: The presenting data suggest that 30-s dilation potentially results in a better 3-month patency rate than 0-s dilation in HD patients with restenosis after PTA.

Hybrid and Conventional Endoscopic Submucosal Dissection for Early Gastric Neoplasms: A Multi-Center Randomized Controlled Trial.

BACKGROUND & AIMS: Hybrid endoscopic submucosal dissection (H-ESD), which incorporates endoscopic submucosal dissection (ESD) with endoscopic mucosal resection, has been developed to make ESD technically easier. This study aimed to determine if H-ESD is superior to conventional ESD (C-ESD) for small early gastric neoplasms (EGNs). METHODS: We conducted a multi-center, prospective, open-label, randomized controlled trial to compare the treatment outcomes of H-ESD and C-ESD (Hybrid-G Trial). Patients with differentiated type intramucosal EGN ≤20 mm in diameter and without ulceration were randomly assigned (1:1) to groups that underwent H-ESD or C-ESD. A single multi-functional snare, SOUTEN (ST1850-20, Kaneka, Medix, Tokyo, Japan), was used for H-ESD. The primary outcome was procedure time. Secondary outcomes included mucosal incision time, time and speed of submucosal dissection, curability, and endoscopic procedural adverse events. RESULTS: A total of 39 and 40 patients underwent H-ESD and C-ESD, respectively. The procedure time of H-ESD was significantly shorter than that of C-ESD (33.16 min vs 62.46 min; H-ESD/C-ESD ratio: 0.53; 95% confidence interval, 0.41-0.69; P < .0001). There was no significant difference in mucosal incision time between the 2 groups; the time and speed of submucosal dissection of H-ESD were significantly shorter than those of C-ESD. No difference was observed between the 2 groups in other outcomes. CONCLUSIONS: H-ESD has significantly shorter procedure time than C-ESD, with high and comparable curability and safety for both H-ESD and C-ESD. H-ESD can be a good option for the endoscopic treatment of small EGNs. (UMIN Clinical Trials Registry, Numbers: UMIN000041244).

A new fluorescence labeling method for molecular analysis of double-stranded DNA.

In this study, a double-stranded DNA (dsDNA) fluorescent labeling method was developed using the fusion proteins of fluorescent protein (FP), and 7 kDa DNA-binding family members including Sso7d from Sulfolobus solfataricus, Aho7c from Acidianus hospitalis, ATSV7 from Acidianus tailed spindle virus and Sto7 from Sulfolobus tokodaii. Using this fluorescent DNA labeling method, we succeeded in single-molecule imaging of bacteriophage λDNA molecules stretched on glass surfaces. The fluorescence of the λDNA with FP fusion proteins decayed 2.4- to 6.4-fold slower than that of the typical intercalating method with SYTOX Green (SxG). In addition, the dynamic behaviors of FP-fused Aho7c-λDNA were relaxed and stretched with and without buffer flow, respectively, in microflow channels and were similar to that with typical intercalating dye, such as YOYO-1 and SxG. this fluorescent DNA labeling method. This fluorescent DNA labeling method can solve the problem of rapid fluorescence decay due to the intercalating dyes and therefore can be expected as an alternative to compound-based fluorescent dye. Thus, this study establishes FP fusion proteins as useful fluorescent DNA probes at the single-molecule level.

Activity of anticoagulant proteins on the polymer-coated and heparin-coated membranes in an extracorporeal circulation circuit.

INTRODUCTION: We performed in vitro experiments using whole human blood without anticoagulants to clarify the activity of anticoagulant proteins on membranes coated with acrylate-copolymer (ACP) with a hydrophilic blood-contacting layer compared to those coated by immobilizing heparin (IHP) in extracorporeal circulation. METHODS: Whole human blood from healthy volunteers was recirculated in two types of experimental circuits with an ACP-coated reservoir and tubes and an ACP-coated or IHP-coated membrane. To compare the fluctuation of anticoagulant proteins, the circuit pressure at the inlet and outlet of the membrane was measured every 5 min; antithrombin antigen (ATQ), antithrombin activity, protein-C quantitation (PCQ), protein-C activity, protein-S free antigen (PSQ), and protein-S activity were measured at 0, 30, 60, 120, and 180 min in each experiment (n = 5). RESULTS: The time taken to achieve high circuit pressure (> 300 mmHg) at the inlet of the membrane was significantly shorter in the ACP-coated membrane circuit (28 ± 2.7 min) than in the IHP-coated membrane circuit (54 ± 24 min); however, the ATQ, PCQ, and PSQ at 180 min of recirculation were significantly higher in the former than in the latter (all p < .05). CONCLUSIONS: ACP-coated membranes can prevent the consumption of anticoagulant proteins but cannot delay circuit thrombogenicity compared to IHP-coated membranes. Considering patient care during the post-extracorporeal circulation period, the use of ACP coating, which can preserve anticoagulant protein, is better in extracorporeal circulation circuits.

Comparison of complement consumption and platelet accumulation between membrane oxygenators coated with a polymer or heparin.

INTRODUCTION: The membrane oxygenator in extracorporeal circulation circuits is coated with acrylate-copolymer (ACP) or immobilized heparin (IHP) to enhance hemocompatibility. To evaluate the relative features of both coatings, we compared blood components circulated in the circuits with ACP-and IHP-coated membranes in vitro using whole human blood. METHODS: Whole human blood was heparinized and circulated in two experimental circuits with an ACP-coated reservoir, tubes, and an ACP- or IHP-coated membrane. Platelet (PLT) counts and the amount of total protein (TP), complement component 3 (C3), and complement component 4 (C4) were measured at 0, 8, 16, 24, and 32 h in each experiment (n = 5). RESULTS: The PLT count at 0-h circulation was lower in the IHP-coated than in the ACP-coated circuits (p = 0.034); however, no significant difference was observed at other time points. Reduction in TP at 8-h and 16-h circulation and in C3 at 32-h circulation was lesser in the ACP-coated than in the IHP-coated circuits (p = 0.004, 0.034, and 0.027, respectively); reduction in TP and C3 at other time points and C4 at each time point was not significantly different. There were significant interactions between coating type and circulation duration in the PLT, TP, and C3 transitions (p = 0.008, 0.020, and 0.043, respectively). CONCLUSIONS: Our findings suggest that ACP-coated membranes can prevent the initial drop in PLT count and C3 consumption over 32 h, whereas IHP-coated membranes could not prevent this drop in extracorporeal circulation. Therefore, ACP-coated membranes are suitable for short- and long-term extracorporeal life support.

Loss of function of Hog1 improves glycerol assimilation in Saccharomyces cerevisiae.

We previously isolated a mutant of Saccharomyces cerevisiae strain 85_9 whose glycerol assimilation was improved through adaptive laboratory evolution. To investigate the mechanism for this improved glycerol assimilation, genome resequencing of the 85_9 strain was performed, and the mutations in the open reading frame of HOG1, SIR3, SSB2, and KGD2 genes were found. Among these, a frameshift mutation in the HOG1 open reading frame was responsible for the improved glycerol assimilation ability of the 85_9 strain. Moreover, the HOG1 gene disruption improved glycerol assimilation. As HOG1 encodes a mitogen-activated protein kinase (MAPK), which is responsible for the signal transduction cascade in response to osmotic stress, namely the high osmolarity glycerol (HOG) pathway, we investigated the effect of the disruption of PBS2 gene encoding MAPK kinase for Hog1 MAPK on glycerol assimilation, revealing that PBS2 disruption can increase glycerol assimilation. These results indicate that loss of function of Hog1 improves glycerol assimilation in S. cerevisiae. However, single disruption of the SSK2, SSK22 and STE11 genes encoding protein kinases responsible for Pbs2 phosphorylation in the HOG pathway did not increase glycerol assimilation, while their triple disruption partially improved glycerol assimilation in S. cerevisiae. In addition, the HOG1 frameshift mutation did not improve glycerol assimilation in the STL1-overexpressing RIM15 disruptant strain, which was previously constructed with high glycerol assimilation ability. Furthermore, the effectiveness of the HOG1 disruptant as a bioproduction host was validated, indicating that the HOG1 CYB2 double disruptant can produce L-lactic acid from glycerol.

A newly developed solution for the preservation of short-chain fatty acids, bile acids, and microbiota in fecal specimens.

Recent studies have revealed that the gut microbiome affects various health conditions via its metabolites, including short-chain fatty acids (SCFAs) and bile acids (BAs). In the analysis of these, appropriate collection, handling, and storage of fecal specimens are required, and convenient specimen handling processes will facilitate their investigation. Here, we developed a novel preservation solution, "Metabolokeeper®", to stabilize fecal microbiota, organic acids including SCFAs, and BAs at room temperature. In the present study, we collected fecal samples from 20 healthy adult volunteers and stored them at room temperature with Metabolokeeper® and at -80°C without preservatives for up to four weeks to evaluate the usefulness of the novel preservative solution. We found that microbiome profiles and short chain fatty acid contents were stably maintained at room temperature with Metabolokeeper® for 28 days, while the bile acids were stably maintained for 7 days under the same conditions. We conclude that this convenient procedure to obtain a fecal sample for collecting the gut microbiome and gut metabolites can contribute to a better understanding of the health effects of fecal metabolites produced by the gut microbiome.

Effects of Diabetes and Voluntary Exercise on IgA Concentration and Polymeric Immunoglobulin Receptor Expression in the Submandibular Gland of Rats.

Background and Objectives: Patients with diabetes are more susceptible to upper respiratory tract infections (URTIs) because they are easily infected. Salivary IgA (sali-IgA) levels play a major role in transmitting URTIs. Sali-IgA levels are determined by salivary gland IgA production and polymeric immunoglobulin receptor (poly-IgR) expression. However, it is unknown whether salivary gland IgA production and poly-IgR expression are decreased in patients with diabetes. While exercise is reported to increase or decrease the sali-IgA levels, it is unclear how exercise affects the salivary glands of patients with diabetes. This study aimed to determine the effects of diabetes and voluntary exercise on IgA production and poly-IgR expression in the salivary glands of diabetic rats. Materials and Methods: Ten spontaneously diabetic Otsuka Long-Evans Tokushima Fatty (OLETF) rats (eight-week-old) were divided into two groups of five rats each: a non-exercise group (OLETF-C) and a voluntary wheel-running group (OLETF-E). Five Long-Evans Tokushima Otsuka (LETO) rats without diabetes were bred under the same conditions as the OLETF-C. Sixteen weeks after the study began, the submandibular glands (SGs) were collected and analyzed for IgA and poly-IgR expression levels. Results: IgA concentrations and poly-IgR expression levels in SGs were lower in OLETF-C and OLETF-E than in LETO (p < 0.05). These values did not differ between the OLETF-C and OLETF-E. Conclusions: Diabetes decreases IgA production and poly-IgR expression in the salivary glands of rats. Moreover, voluntary exercise increases sali-IgA levels but does not increase IgA production and poly-IgR expression in the salivary glands of diabetic rats. Increasing IgA production and poly-IgR expression in the salivary glands, which is reduced in diabetes, might require slightly higher-intensity exercise than voluntary exercise under the supervision of a doctor.

Reconstitution of Circulating Mucosal-Associated Invariant T Cells after Allogeneic Hematopoietic Cell Transplantation: Its Association with the Riboflavin Synthetic Pathway of Gut Microbiota in Cord Blood Transplant Recipients.

Mucosal-associated invariant T (MAIT) cells are a type of innate lymphocyte and recognize riboflavin (vitamin B2) synthesis products presented by MHC-related protein 1. We investigated long-term reconstitution of MAIT cells and its association with chronic graft-versus-host disease (cGVHD) in a cross-sectional cohort of 173 adult patients after allogeneic hematopoietic cell transplantation. According to donor source, the number of MAIT cells significantly correlated with time after cord blood transplantation (CBT) but not with time after bone marrow transplantation or peripheral blood stem cell transplantation. The number of MAIT cells was significantly lower in patients with cGVHD compared with patients without cGVHD. We also examined the association between MAIT cell reconstitution and gut microbiota as evaluated by 16S ribosomal sequencing of stool samples 1 mo post-CBT in 27 adult patients undergoing CBT. The diversity of gut microbiota was positively correlated with better MAIT cell reconstitution after CBT. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States analysis indicated that amounts of ribB and ribA genes were significantly higher in the microbiomes of patients with subsequent MAIT cell reconstitution after CBT. In conclusion, long-term MAIT cell reconstitution is dependent on the type of donor source. Our data also unveiled an important role for the interaction of circulating MAIT cells with gut microbiota in humans.

Impact of Stellate Ganglion Block on Tissue Blood Flow/Oxygenation and Postoperative Mandibular Nerve Hypoesthesia: A Cohort Study.

PURPOSE: Although a stellate ganglion block (SGB) increases tissue blood flow in the mandibular region, the change in tissue oxygenation after SGB and therapeutic effect of SGB for postoperative mandibular nerve hypoesthesia remain to be established. The study aim was to measure the change in tissue oxygenation in the mandibular region after SGB. METHODS: To determine the variation in tissue oxygenation in the mandibular region, the tissue oxygen index (TOI; percentage of oxygenated hemoglobin in the total hemoglobin) was measured at the skin near the mental foramen bilaterally, at the primary site of unilateral SGB, achieved using 6 mL of 1% lidocaine hydrochloride, for the treatment of bilateral postoperative mandibular nerve injury. The primary outcome of this study is the temporal variation in TOI after SGB (0.5, 1, 5, 10, 15, 20, and 25 minutes after SGB), and the control group in this study is the TOI at the end of SGB injection (0 minute). All data are expressed as the mean ± standard deviation and 95% confidence interval (CI). Repeated-measures analysis of variance with Dunnett's test was used to determine parametric statistical significance. A P-value <.05 was considered statistically significant. RESULTS: Thirteen patients were enrolled in this study. On both the blocked and contralateral side, the TOI was significantly increased compared to that before SGB (ΔTOI at 15 minute after SGB, 5.87 ± 2.89%, P < .001, 95% CI: 4.122 to 7.617% in the blocked side, 1.88 ± 2.73%, P = .005, 95% CI: 1.877 to 2.725% in the contralateral side). CONCLUSIONS: Unilateral SGB using 6 mL of 1% lidocaine hydrochloride results in an increase in tissue oxygenation in the mandibular region. Based on these findings, we hypothesize that a series of SGBs may contribute to a more rapid recovery of postoperative trigeminal nerve injury.

Gingival blood flow before, during, and after clenching, measured by laser Doppler blood flowmeter: A pilot study.

INTRODUCTION: This study aimed to investigate the effects of the strong occlusal force on the hemodynamics of gingival microcirculation. METHODS: Eleven adult volunteers with healthy periodontium and normal occlusion participated in this study. Using a noncontact laser Doppler flowmeter placed at the attached gingiva and the interdental papilla of the maxillary first premolar, changes in gingival blood flow (GBF) were examined during and after clenching. RESULTS: When the strong occlusal pressure was applied on the maxillary first premolar by clenching, GBF in the attached gingiva on the buccal side decreased significantly compared with the resting GBF, with medians of 2.3 mL/min/100 g and 5.4 mL/min/100 g, respectively (P <0.05). After the release of the maximum clenching, GBF recovered immediately and transiently increased to a median of 2.4 mL/min/100 g, showing a significant difference to the resting GBF (P <0.05). In contrast, in the interdental papilla, no significant change in GBF was found by clenching. CONCLUSIONS: Ischemia of the buccal attached gingiva associated with strong clenching may be due to compression of the vascular network of the periodontal membrane. Through reactive hyperemia resulting from the release of clenching, it is possible not only that blood flow will be restored to the tissue but that the tissue itself may be damaged by the reperfusion. During active orthodontic treatment, it is suggested that occlusal management to prevent occlusal trauma is important to avoid detrimental effects on periodontal tissues.

DNA Manipulation and Single-Molecule Imaging.

DNA replication, repair, and recombination in the cell play a significant role in the regulation of the inheritance, maintenance, and transfer of genetic information. To elucidate the biomolecular mechanism in the cell, some molecular models of DNA replication, repair, and recombination have been proposed. These biological studies have been conducted using bulk assays, such as gel electrophoresis. Because in bulk assays, several millions of biomolecules are subjected to analysis, the results of the biological analysis only reveal the average behavior of a large number of biomolecules. Therefore, revealing the elementary biological processes of a protein acting on DNA (e.g., the binding of protein to DNA, DNA synthesis, the pause of DNA synthesis, and the release of protein from DNA) is difficult. Single-molecule imaging allows the analysis of the dynamic behaviors of individual biomolecules that are hidden during bulk experiments. Thus, the methods for single-molecule imaging have provided new insights into almost all of the aspects of the elementary processes of DNA replication, repair, and recombination. However, in an aqueous solution, DNA molecules are in a randomly coiled state. Thus, the manipulation of the physical form of the single DNA molecules is important. In this review, we provide an overview of the unique studies on DNA manipulation and single-molecule imaging to analyze the dynamic interaction between DNA and protein.

Impact of Intestinal Microbiota on Reconstitution of Circulating Monocyte, Dendritic Cell, and Natural Killer Cell Subsets in Adults Undergoing Single-Unit Cord Blood Transplantation.

The intestinal microbiota plays a fundamental role in the development of host innate immune cells, such as monocytes, dendritic cells (DCs), and natural killer (NK) cells. We examined the association between intestinal microbiota and subsequent immune reconstitution of circulating monocyte, DC, and NK cell subsets in 38 adult patients undergoing single-unit cord blood transplantation (CBT). A higher diversity of intestinal microbiota at 1 month was significantly associated with higher counts of plasmacytoid DCs at 7 months after CBT, as measured by the Chao1 index. Principal coordinate analysis of unweighted UniFrac distances showed significant differences between higher and lower classical monocyte reconstitution at 7 months post-CBT. The families Neisseriaceae, Burkholderiaceae, Propionibacteriaceae, and Coriobacteriaceae were increased in higher classical monocyte reconstitution at 7 months post-CBT, whereas the family Bacteroidaceae was increased in lower classical monocyte reconstitution at 7 months post-CBT. These data show that intestinal microbiota composition affects immune reconstitution of classical monocyte and plasmacytoid DCs following single-unit CBT.

Asymmetric Total Synthesis of Fawcettimine-Type Lycopodium Alkaloid, Lycopoclavamine-A.

An asymmetric total synthesis of lycopoclavamine-A (1), a structurally unique fawcettimine-type Lycopodium alkaloid, was achieved via a stereoselective Pauson-Khand reaction and a stereoselective conjugate addition to construct a quaternary carbon center at C-12.

Two cases of unilateral cone-rod dysfunction with negative electroretinograms.

PURPOSE: To report the findings in two patients with unilateral cone-rod dysfunction with the a-wave larger than the b-wave, i.e., negative-type, full-field electroretinogram (ERG). METHODS: Standard ophthalmological examinations were performed including the medical history, measurements of the best-corrected visual acuity and intraocular pressures, slit-lamp biomicroscopy, ophthalmoscopy, spectral-domain optical coherence tomography, fundus autofluorescence, fluorescein angiography, and perimetry. ERG examinations were carried out with the ISCEV standard. Immunoblot analysis using the patient's sera was performed to determine the presence of the recoverin1 antibody. RESULTS: The common findings in these two patients were: unilateral, male sex, sudden onset of photophobia or a reduction in the vision at an advanced age, preserved visual acuity, no complaint of night blindness, normal fundus appearance, negative-type dark-adapted 3.0 ERGs with reduced a-wave amplitudes, absent light-adapted 3.0 ERGs, and very reduced but recordable dark-adapted 0.01 ERGs. In addition, the multifocal ERGs in all areas except that in a hexagonal area within a 2.5° radius of the fovea were very reduced. Patients with similar findings have been reported earlier, but the subnormal a-wave of the dark-adapted 3.0 ERGs and extensive morphological alterations of the retina in the posterior pole in the OCT images were different from those of the reported patients. The OCT images showed an indistinct interdigitation zone and discontinuous ellipsoid zone. Anti-recoverin antibodies were not detected. CONCLUSIONS: Negative ERGs with severely reduced cone and rod components suggest that both the cone and rod bipolar cell visual pathways may be disturbed. Slightly decreased a-wave suggests minor abnormality of photoreceptors. It is important to determine whether these patients represent a new clinical entity or a phenotypic variation of an already described retinal disorder.

Fungemia due to Fusarium solani under low-dose liposomal amphotericin B in a patient after cord blood transplantation.

The introduction of the prophylactic use of antifungal drugs caused the increased occurrence of invasive fungal infections due to previously rare molds, such as fusariosis, after allogeneic hematopoietic stem cell transplantation. We herein report the case of a patient with diffuse large B-cell lymphoma who developed fungemia due to Fusarium solani during liposormal amphotericin B on day 25 after cord blood transplantation (CBT). Because Fusarium species might differ in virulence and drug susceptibility, the sequencing of the internal transcribed spacer region of the ribosomal RNA gene accurately identified Fusarium solani to be the cause of fungemia at the species level. This case highlights Fusarium solani as the cause of fungemia in a patient under liposormal amphotericin B treatment after CBT.

Off-axis virtual-image display and camera by holographic mirror and blur compensation.

We propose off-axis virtual-image display and camera systems, which integrate a vertically-standing holographic off-axis mirror, blur-compensation optical systems, and digital imaging devices. In the system, the holographic mirror is used for an off-axis reflector, which realizes an upright and thin screen for virtual-image formation. By combining it with a display unit, an off-axis virtual-image display is realized, where the virtual image can be seen behind the upright holographic mirror. Simultaneously, by combining it with a camera unit, an off-axis camera is implemented, which realizes frontal shooting of objects by a camera placed at an off-axis position. Since both the off-axis display and the camera can be implemented by a single holographic mirror, it can be applied to a two-way visual-telecommunication system with a thin screen, which implements eye contact and the observer--image distance. A problem with the proposed system is image blur, which is caused by the chromatic dispersion of the holographic mirror. To solve this, we designed optical blur-compensation systems using a diffractive optical element and a diffuser or a lens. Experimental results verify the concept of the proposed systems with clarifying the effect of designed blur-compensation methods.

A New Method for Immobilization of His-Tagged Proteins with the Application of Low-Frequency AC Electric Field.

Continued advancement of protein array, bioelectrode, and biosensor technologies is necessary to develop methods for higher amount and highly oriented immobilization activity of proteins. In pursuit of these goals, we developed a new immobilization method by combining electrostatic transport and subsequent molecular diffusion of protein molecules. Our developed immobilization method is based on a model that transports proteins toward the substrate surface due to steep concentration gradient generated by low-frequency AC electric field. The immobilization of the maximum amounts can be obtained by the application of the AC voltage of 80 Vpp, 20 Hz both for His-tagged Green Fluorescent Protein (GFP) and Discosoma sp. Red Fluorescent Protein (DsRed), used as model proteins. The amounts of the immobilized His-tagged GFP and DsRed were approximately seven-fold higher than that in the absence of the application of low-frequency AC electric field. Furthermore, the positively and negatively charged His-tagged GFP at acidic and alkaline pH were immobilized by applying of low-frequency AC electric field, whereas the non-charged His-tagged GFP at the pH corresponding to its isoelectric point (pI) was not immobilized. Therefore, unless the pH is equal to pI, the immobilization of electrically charged proteins was strongly enhanced through electrostatic transport and subsequent molecular diffusion.

Cinacalcet hydrochloride relieves hypercalcemia in Japanese patients with parathyroid cancer and intractable primary hyperparathyroidism.

Pharmacological treatment of hypercalcemia is essential for patients with parathyroid carcinoma and intractable primary hyperparathyroidism (PHPT). Use of the calcimimetic cinacalcet hydrochloride (cinacalcet) is an option to treat such patients. We investigated the efficacy and safety of cinacalcet in Japanese patients with parathyroid carcinoma and intractable PHPT. Five Japanese patients with parathyroid carcinoma and two with intractable PHPT were enrolled in an open-label, single-arm study consisting of titration and maintenance phases. Cinacalcet doses were titrated until the albumin-corrected serum calcium concentration decreased to 10.0 mg/dL or less or until dose escalation was considered not necessary or feasible. Serum calcium concentration at the baseline was 12.1 ± 1.3 mg/dL (mean ± standard deviation; range 10.4-14.6 mg/dL) and decreased to 10.1 ± 1.6 mg/dL (range 8.6-13.3 mg/dL) at the end of the titration phase with cinacalcet at a dosage of up to 75 mg three times a day. At the end of the titration phase, at least a 1 mg/dL reduction in serum calcium concentration from the baseline was observed in five patients (three with carcinoma and two with PHPT), and it decreased to the normocalcemic range in five patients (three with carcinoma and two with PHPT). Common adverse events were nausea and vomiting. One patient discontinued participation in the study because of an adverse event, liver disorder. Cinacalcet effectively relieved hypercalcemia in 60% of the Japanese patients with parathyroid carcinoma and might be effective in those with intractable PHPT. The drug might be tolerable and safe at a dosage of at most 75 mg three times a day.