Highly Efficient Multi-Step Oxidation Bioanode Using Microfluidic Channels.

With the rapid decline of fossil fuels, various types of biofuel cells (BFCs) are being developed as an alternative energy source. BFCs based on multi-enzyme cascade reactions are utilized to extract more electrons from substrates. Thus, more power density is obtained from a single molucule of substrate. In the present study, a bioanode that could extract six electrons from a single molecule of L-proline via a three-enzyme cascade reaction was developed and investigated for its possible use in BFCs. These enzymes were immobilized on the electrode to ensure highly efficient electron transfer. Then, oriented immobilization of enzymes was achieved using two types of self-assembled monolayers (SAMs). In addition, a microfluidic system was incorporated to achieve efficient electron transfer. The microfluidic system, in which the electrodes were arranged in a tooth-shaped comb, allowed for substrates to be supplied continuously to the cascade, which resulted in smooth electron transfer. Finally, we developed a high-performance bioanode which resulted in the accumulation of higher current density compared to that of a gold disc electrode (205.8 µA cm-2: approximately 187 times higher). This presents an opportunity for using the bioanode to develop high-performance BFCs in the future.

Falsely elevated plasma ACTH levels measured by the Elecsys assay related to heterophilic antibody in a case of secondary adrenocortical insufficiency.

A 49-year-old woman with membranous nephropathy was referred to our hospital during the tapering of oral prednisolone, because of suspicion of primary adrenal insufficiency based on a plasma ACTH level of 399.1 pg/mL in the Elecsys assay and a serum cortisol level of 3.1 µg/dL. A rapid ACTH stimulation test revealed a suboptimal response, whereas a prolonged ACTH simulation test showed a sufficient increase in her urinary free cortisol. Also, big ACTH was not detected by gel exclusion chromatography. Therefore, we speculated that ACTH levels were falsely elevated due to some interference substances. Pretreatment of her plasma with either polyethylene glycol precipitation or a heterophilic blocking tube substantially reduced her ACTH values. When either the Immulite ACTH II or the TOSOH II ACTH was tried instead of the Elecsys ACTH, her plasma ACTH values turned out to be lower and appropriate for her clinical status. These results indicated that heterophilic antibodies interfered only with the Elecsys ACTH assay presumably by bridging the capture and tracer antibodies. To our knowledge, this is the first case in which the Elecsys ACTH assay yielded falsely elevated results. Regardless of the measurement system used, if there is a discordance between assay results and clinical findings, it should be considered to adopt additional procedures and/or another assay.

Identification of Mitosis-Specific Phosphorylation in Mitotic Chromosome-Associated Proteins.

During mitosis, phosphorylation of chromosome-associated proteins is a key regulatory mechanism. Mass spectrometry has been successfully applied to determine the complete protein composition of mitotic chromosomes, but not to identify post-translational modifications. Here, we quantitatively compared the phosphoproteome of isolated mitotic chromosomes with that of chromosomes in nonsynchronized cells. We identified 4274 total phosphorylation sites and 350 mitosis-specific phosphorylation sites in mitotic chromosome-associated proteins. Significant mitosis-specific phosphorylation in centromere/kinetochore proteins was detected, although the chromosomal association of these proteins did not change throughout the cell cycle. This mitosis-specific phosphorylation might play a key role in regulation of mitosis. Further analysis revealed strong dependency of phosphorylation dynamics on kinase consensus patterns, thus linking the identified phosphorylation sites to known key mitotic kinases. Remarkably, chromosomal axial proteins such as non-SMC subunits of condensin, TopoIIα, and Kif4A, together with the chromosomal periphery protein Ki67 involved in the establishment of the mitotic chromosomal structure, demonstrated high phosphorylation during mitosis. These findings suggest a novel mechanism for regulation of chromosome restructuring in mitosis via protein phosphorylation. Our study generated a large quantitative database on protein phosphorylation in mitotic and nonmitotic chromosomes, thus providing insights into the dynamics of chromatin protein phosphorylation at mitosis onset.

Insulin resistance in nonobese Japanese women with polycystic ovary syndrome is associated with poorer glucose tolerance, delayed insulin secretion, and enhanced insulin response.

Purpose: To determine the prevalence of insulin resistance (IR) and impaired glucose tolerance (IGT) in PCOS patients, the optimal screening method, and to compare our findings between nonobese and obese Japanese women with PCOS. Methods: Ninety-eight PCOS patients were included in this research from 2006 to 2013. Glucose tolerance test (OGTT) was performed. Serum glucose and insulin concentration were assayed before and 30, 60, and 120 min after taking 75 g of glucose. Results: All examined metabolic parameters were significantly favorable in the nonobese subjects, below 25 kg/m2. HOMA-IR, fasting insulin, glucose120, and insulin120 showed strong correlations with BMI. A total of 1.4 % of nonobese women had IR based on fasting insulin or HOMA-IR. However, 15.5 % (11/71) of nonobese women had IR as determined by a continuous increase of serum insulin level in OGTT. In comparison, the prevalence of IR among the obese women ranged from 41 to 59 %. AUCglucose, glucose60, glucose120, and insulin120 in nonobese women with a continuous insulin increase were higher than those without such a continuous increase. Conclusions: All examined metabolic parameters were significantly correlated with BMI. As the presence of a continuous increase of insulin level reflects to some degree poorer glucose tolerance, delayed insulin secretion, and enhanced insulin response compared with non-continuous insulin increase, OGTT might not been excluded to determine IR and IGT for nonobese women with PCOS.

Comparison of the efficacy of gefitinib in patients with non-small cell lung cancer according to the type of epidermal growth factor receptor mutation.

BACKGROUND: Exon 19 deletion and L858R point mutation of the epidermal growth factor receptor (EGFR) are the most commonly encountered EGFR mutations in non-small cell lung cancer (NSCLC), and predict higher clinical outcomes following treatment with gefitinib. The objective of this study was to evaluate the differential clinical outcomes of gefitinib in patients with NSCLC according to the type of active EGFR mutation, i.e. exon 19 deletion or L858R point mutation. METHODS: We identified patients with advanced NSCLC harboring the exon 19 deletion or the L858R point mutation of EGFR who were on gefitinib treatment. The clinical outcomes were evaluated. RESULTS: Of the 124 patients with NSCLC harboring active EGFR mutations, the overall response rate, progression-free survival and overall survival were 60.5%, 11.3 and 27.3 months, respectively, and did not differ significantly between patients with the exon 19 deletion (61.8%, 11.3 and 32.2 months, respectively) and those with the L858R point mutation (58.9%, 9.0 and 27.7 months, respectively). CONCLUSION: It may be considered that there is no difference in the clinical efficacy of gefitinib between NSCLC patients who harbor the exon 19 deletion and those with the L858R point mutation.

[Comparison of ultrasound-guided and blindly placed radial artery catheterization].

BACKGROUND: The purpose of this study was to evaluate whether ultrasound (US)-guided radial artery catheterization decreases the number of failed attempts. METHODS: A total of 36 patients requiring an arterial catheter were enrolled. Patients were divided into 3 groups: 1) a blind palpation technique group, 2) an US-guided short-axis group, and 3) an US-guided long-axis group. The vascular access was performed by one anesthesiologist. The number of attempts at catheter placement was compared within the groups. RESULTS: There were no significant differences among the groups in number of attemps (P = 0.268); however, it seems that the deviation of attempts was small in the US groups. CONCLUSIONS: Because the artery and catheter are actually visualized by US, it seems, that US-guided radial artery catheterization is a safe and educational technique.

[Efficacy of chemotherapy with carboplatin-paclitaxel plus bevacizumab for previously treated patients with advanced non-small cell carcinoma].

BACKGROUND: There have been few reports on the efficacy or safety of combination therapy with carboplatin-paclitaxel plus bevacizumab(TC+Bev)in previously treated patients with non-small cell lung carcinoma(NSCLC). PURPOSE: The objective of this study was to assess the efficacy and safety of TC+Bev as second-line therapy and beyond for previously treated NSCLC patients. METHODS: A total of 17 patients previously treated with NSCLC at the Kitasato University Hospital between April 2010 and February 2012 were enrolled in this retrospective study. On day 1 all patients received a 200mg/m2 dose of paclitaxel by intravenous infusion over a 3-h period, followed by infusion of carboplatin at a dose corresponding to an AUC of 6.0 min mg/mL, and then by Bev at a dose of 15mg/m2. The treatment course was repeated every three or four weeks. Patients continued to receive Bev monotherapy every 3 weeks thereafter until evidence of disease progression or unacceptable toxicity developed. RESULTS: Median age: 60 years old(range 39-74 years old); gender: male/female, 6/11; PS 0-1/≥2, 17/0; clinical stage:III B/IV postoperative recurrence, 0/16/1; EGFR mutation status: positive/negative/unknown, 7/9/1; histological type: adenocarcinoma in all patients; median number of prior regimens: 3. 4(1-6); median number of cycles(induction phase): 3(1-6). The objective response rate and disease control rate were 17. 6% and 70. 6%, respectively, and the median progression-free survival time was 4. 7 months. There were no treatment-related deaths, and the toxicities of the treatment regimen were acceptable. CONCLUSION: TC+Bev therapy exhibits activity in previously treated NSCLC patients and has acceptable toxicity. Further study is warranted to confirm our results.

[Pemetrexed as second-line treatment and beyond for elderly patients with advanced non-small-cell lung cancer].

BACKGROUND: In Japan, the standard first-line therapy for elderly patients with advanced non-small lung cancer(NSCLC)is docetaxel(DOC)monotherapy. However, there is very limited information about second-line and beyond chemotherapy regimens for elderly patients with advanced NSCLC. Pemetrexed(PEM)monotherapy has been recognized as a standard regimen for advanced NSCLC in second-line settings, just as DOC monotherapy has been. PURPOSE: The objective of this study was to examine the efficacy and safety of PEM as second-line therapy and beyond for elderly patients. METHODS: The records of previously -treated elderly patients with advanced NSCLC, who had been treated with PEM as second-line therapy and beyond between July 2009 and December 2010, were retrospectively reviewed. RESULTS: median age: 73 years old(range 70-79 years old); gender: male/female, 11/8; PS 0-1/≥2, 19/0; clinical stage: III B/IV/postoperative recurrence, 4/10/5; pathology: adeno/LCNEC/other, 17/1/1 patient. The objective response-rate and disease control-rate were 15. 8% and 57. 9%, respectively. Median progression-free survival time was 3. 2 months. There were no treatment-related deaths, and most of the toxicities of the treatment regimen were mild and acceptable. CONCLUSION: PEM monotherapy exhibits activity in previously treated elderly NSCLC patients and has an acceptably low toxicity. Further study is warranted to confirm our results.

Molecular principles of recruitment and dynamics of guest proteins in liquid droplets.

Despite the continuous discovery of host and guest proteins in membraneless organelles, complex host-guest interactions hinder the understanding of the molecular grammar governing liquid-liquid phase separation. In this study, we characterized the localization and dynamic properties of guest proteins in liquid droplets using single-molecule fluorescence microscopy. Eighteen guest proteins of different sizes, structures, and oligomeric states were examined in host p53 liquid droplets. Recruitment did not significantly depend on the structural properties of the guest proteins, but was moderately correlated with their length, total charge, and number of R and Y residues. In contrast, the diffusion of disordered guest proteins was comparable to that of host p53, whereas that of folded proteins varied widely. Molecular dynamics simulations suggest that folded proteins diffuse within the voids of the liquid droplet while interacting weakly with neighboring host proteins, whereas disordered proteins adapt their structures to form tight interactions with the host proteins. Our study provides insights into the key molecular principles of the localization and dynamics of guest proteins in liquid droplets.

Comprehensive identification of translation start sites by tetracycline-inhibited ribosome profiling.

Tetracycline-inhibited ribosome profiling (TetRP) provides a powerful new experimental tool for comprehensive genome-wide identification of translation initiation sites in bacteria. We validated TetRP by confirming the translation start sites of protein-coding genes in accordance with the 2006 version of Escherichia coli K-12 annotation record (GenBank U000962) and found ∼150 new start sites within 60 nucleotides of the annotated site. This analysis revealed 72 per cent of the genes whose initiation site annotations were changed from the 2006 GenBank record to the newer 2014 annotation record (GenBank U000963), indicating a high sensitivity. Also, results from reporter fusion and proteomics of N-terminally enriched peptides showed high specificity of the TetRP results. In addition, we discovered over 300 translation start sites within non-coding, intergenic regions of the genome, using a threshold that retains ∼2,000 known coding genes. While some appear to correspond to pseudogenes, others may encode small peptides or have previously unforeseen roles. In summary, we showed that ribosome profiling upon translation inhibition by tetracycline offers a simple, reliable and comprehensive experimental tool for precise annotation of translation start sites of expressed genes in bacteria.

Phase I and pharmacokinetic study of erlotinib administered in combination with amrubicin in patients with previously treated, advanced non-small cell lung cancer.

OBJECTIVES: We conducted a phase I trial of erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, combined with amrubicin, a topoisomerase II inhibitor. The aim was to determine the maximum tolerated dose, the dose-limiting toxicities (DLTs), and the pharmacokinetics of this combination in patients with non-small cell lung cancer who had received previous chemotherapy. METHODS: A total of 9 patients with stage IV disease were treated at 3-week intervals with erlotinib once daily on days 1 through 21 plus a 5-minute intravenous injection of amrubicin on days 1 through 3. RESULTS: The dose levels evaluated were erlotinib (mg/body)/amrubicin (mg/m): 100/30 (n=3), 100/35 (n=3), and 150/30 (n=3). The maximum tolerated dose of erlotinib and amrubicin was 100 mg/body and 35 mg/m because 2 of the 3 patients experienced DLTs during the first cycle of treatment at the third dose level of 150 mg/body and 30 mg/m. Cessation of erlotinib administration for 8 days because of grade 3 leukopenia and grade 3 skin infection (erysipelas) were the DLTs. No drug-drug interactions between erlotinib and amrubicin were observed in this study. The overall response rate was 33%, including 3 partial responses, in the 9 patients. The median progression-free survival for all patients was quite long, 11.3 months, and the median overall survival has not yet been reached. CONCLUSIONS: Combined erlotinib plus amrubicin therapy seems to be highly effective, with acceptable toxicity, against non-small cell lung cancer. The recommended dose for phase II studies was erlotinib 100 mg once daily on days 1 through 21, and amrubicin 35 mg/m on days 1 through 3 administered every 21 days.

Canine herpesvirus ORF2 is a membrane protein modified by N-linked glycosylation.

Canine herpesvirus (CHV) ORF2, located downstream of the glycoprotein C (gC) gene, has homologues with some of the alphaherpesviruses. To characterize CHV OFR2, a recombinant CHV carrying a LacZ gene in the ORF2 locus, and recombinant vaccinia virus expressing ORF2 protein were constructed. Northern blot analysis revealed ORF2 and a gamma2 class late gene, and its protein product was detectable in CHV-infected cells reacted with ORF2 protein antiserum. Tunicamycin and N-glycosidase F treatment revealed that the ORF2 protein was modified by N-linked glycosylation. Fractionation and immune fluorescence analyses of the CHV-infected cells showed the ORF2 as a membrane protein transportable to the surface of infected cells. In vitro, the ORF2 protein did not affect viral replication and cell-to-cell viral spreading. Present findings represent the first evidence pointing to the CHV ORF2 as a membrane protein modified by an N-linked glycosylation.

Evaluation of gefitinib efficacy according to body surface area in patients with non-small cell lung cancer harboring an EGFR mutation.

BACKGROUND: Exon 19 deletions and L858R point mutation are the most commonly encountered active epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC), and they predict greater efficacy of gefitinib therapy. The objective of this study was to evaluate whether body surface area (BSA) affects the efficacy of gefitinib in patients with NSCLC harboring an active EGFR mutation. METHODS: We reviewed the medical records of consecutive patients with advanced NSCLC harboring an active EGFR mutation who received gefitinib monotherapy. The median BSA value was used as the cutoff value to evaluate the impact of BSA on the efficacy of gefitinib. RESULTS: The median BSA of the 103 NSCLC patients harboring an active EGFR mutation was 1.45 m(2). The overall response rate, progression-free survival (PFS), and median survival time (MST) were 65.0 %, 11.3 months, and 26.2 months, respectively. There were no significant differences in clinical outcomes between the high-BSA group (BSA ≥ 1.45 m(2)) and low-BSA group (BSA < 1.45 m(2)), i.e., the response rates was 60.0 % and 69.8 %, respectively (P = 0.20), and their MST was 24.7 and 26.2 months, respectively (P = 0.78). Although BSA was predictive of PFS between high-BSA group and low-BSA group in the univariate analysis (9.0 and 12.2 months, P = 0.04), the multivariate analysis identified only performance status and smoking status as independent predictors of PFS. CONCLUSIONS: The efficacy of gefitinib in patients with NSCLC harboring an EGFR mutation does not differ according to their BSA.

Successful chemotherapy with Carboplatin and s-1 for thymic carcinoma: a case report.

Thymic carcinoma is a rare but aggressive neoplasm. Although there is no clearly optimal first- or second-line chemotherapy regimen for thymic carcinoma, platinum-based chemotherapy has repeatedly been shown to be of benefit to patients with advanced thymic carcinoma. Some case reports have described S-1 as a novel agent with good activity against advanced thymic carcinoma. A 74-year-old female was diagnosed with thymic carcinoma complicated by pleural dissemination and pericardial effusion of carcinomatosa. She was treated with carboplatin on day 1 plus S-1 on days 1-14 in cycles repeated every 3 or 4 weeks. Four cycles of this regimen were administered, and a partial response was confirmed. There were no severe hematological or nonhematological toxicities, and no dose reduction was necessary. To our knowledge, this is the first report to demonstrate the efficacy of combination chemotherapy consisting of carboplatin and S-1 against thymic carcinoma.