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Control of the formation of liquid crystalline Ia 3̄ d gyroid phases and their nanostructures is critical to advance materials chemistry based on the structural feature of three-dimensional helical networks. Here, we present that introducing methyl side-group(s) and slight non-symmetry into aryloyl-hydrazine-based molecules is unexpectedly crucial for their formation and can be a new design strategy through tuning intermolecular interactions: the two chemical modifications in the core portion of the chain-core-chain type molecules effectively lower and extend the Ia 3̄ d phase temperature ranges with the increased twist angle between neighboring molecules along the network. The detailed analyses of the aggregation structure revealed the change in the core assembly mode from the double-layered core mode of the mother molecule (without methyl groups) to the single-layered core mode. Such changes are explained in terms of modified intermolecular interactions in those phases employing quantum chemical calculations.

Background: The applicability of ultra-hypofractionated (ultra-HF) whole-breast irradiation (WBI) remains unknown in Japanese women. This study aimed to evaluate the safety and efficacy of this approach among Japanese women and report the results of an interim analysis performed to assess acute adverse events (AEs) and determine whether it was safe to continue this study., Methods: We enrolled Japanese women with invasive breast cancer or ductal carcinoma in situ who had undergone breast-conserving surgery, were aged ≥ 40 years, had pathological stages of Tis-T3 N0-N1, and had negative surgical margins. Ultra-HF-WBI was delivered at 26 Gy in five fractions over one week. When the number of enrolled patients reached 28, patient registration was paused for three months. The endpoint of the interim analysis was the proportion of acute AEs of grade ≥ 2 (Common Terminology Criteria for Adverse Events v5.0) within three months., Results: Of the 28 patients enrolled from seven institutes, 26 received ultra-HF-WBI, and 2 were excluded due to postoperative infections. No AEs of grade ≥ 3 occurred. One patient (4%) experienced grade 2 radiation dermatitis, and 18 (69%) had grade 1 radiation dermatitis. The other acute grade 1 AEs experienced were skin hyperpigmentation (n = 10, 38%); breast pain (n = 4, 15%); superficial soft tissue fibrosis (n = 3, 12%); and fatigue (n = 1, 4%). No other acute AEs of grade ≥ 2 were detected., Conclusions: Acute AEs following ultra-HF-WBI were within acceptable limits among Japanese women, indicating that the continuation of the study was appropriate., (© 2024. The Author(s).)

A chemodivergent synthesis of polycyclic aromatic diarylamines and carbazoles was established by employing thermally or photochemically controlled processes using KO t Bu/1,10-phenanthroline. The synthetic processes involved the dephosphinylation of 9-amino-10-(phosphinyl)phenanthrenes, which were obtained through a regioselective palladium-catalyzed direct [4 + 2] benzannulation of phosphinyl ynamines with 2-iodobiphenyls. When the dephosphinylation was conducted under heating conditions (∼100 °C), it proceeded to yield 9-aminophenanthrene. However, when the reaction was performed under the illumination of purple light (LEDs, λ max = ca. 390 nm), KO t Bu/1,10-phenanthroline promoted single-electron-transfer-triggered dephosphinylation followed by cyclization, producing the corresponding π-expanded carbazoles. We successfully synthesized a highly π-expanded dicarbazole through a dual dephosphinylative cyclization. Additionally, we present the optical properties of a series of amino compounds produced through the dephosphinylative processes.

Background: Few studies have reported reliable prognostic factors for immune checkpoint inhibitors (ICIs) in renal cell carcinoma (RCC). Therefore, we investigated prognostic factors in patients treated with ICIs for unresectable or metastatic RCC., Methods: We included 43 patients who received ICI treatment for RCC between January 2018 and October 2021. Blood samples were drawn before treatment, and 73 soluble factors in the plasma were analyzed using a bead-based multiplex assay. We examined factors associated with progression-free survival (PFS), overall survival (OS), and immune-related adverse events (irAE) using the Chi-squared test, Kaplan-Meier method, and the COX proportional hazards model., Results: Patients exhibited a median PFS and OS of 212 and 783 days, respectively. Significant differences in both PFS and OS were observed for MMP1 (PFS, p < 0.001; OS, p = 0.003), IL-1β (PFS, p = 0.021; OS, p = 0.008), sTNFR-1 (PFS, p = 0.017; OS, p = 0.005), and IL-6 (PFS, p = 0.004; OS, p < 0.001). Multivariate analysis revealed significant differences in PFS for MMP1 (hazard ratio [HR] 5.305, 95% confidence interval [CI], 1.648-17.082; p = 0.005) and OS for IL-6 (HR 23.876, 95% CI, 3.426-166.386; p = 0.001). Moreover, 26 patients experienced irAE, leading to ICI discontinuation or withdrawal. MMP1 was significantly associated with irAE (p = 0.039)., Conclusion: MMP1 may be associated with severe irAE, and MMP1, IL-1β, sTNFR-1, and IL-6 could serve as prognostic factors in unresectable or metastatic RCC treated with ICIs. MMP1 and IL-6 were independent predictors of PFS and OS, respectively. Thus, inhibiting these soluble factors may be promising for enhancing antitumor responses in patients with RCC treated with ICIs., (© 2024. The Author(s).)

A 35-year-old man was diagnosed with stage IIIC non-seminoma with paralysis of the lower half of his body due to 8th thoracic spine metastasis. The patient received bleomycin, etoposide, and cisplatin (BEP) therapy. On day 4 of the second course of BEP, the patient developed a fever and was diagnosed with coronavirus disease (COVID-19). COVID-19 was suspected to worsen because of cancer and chemotherapy-induced immunosuppression. However, the benefits of continuing BEP therapy outweighed these risks. After obtaining fully informed consent, BEP therapy was continued from day 5, while sotrovimab (anti-COVID-19 drug) was administered. The second course of BEP was completed without worsening severe COVID-19 or bleomycin-induced lung injury. The patient completed four courses of BEP, with normalization of tumor markers, partial response on imaging, and improvement in lower body paralysis. In this case, we successfully treated a patient with testicular germ cell tumor with chemotherapy while having COVID-19 without treatment delay. During the COVID-19 pandemic, concomitant chemotherapy and COVID-19 treatment are warranted because delaying treatment will decrease the efficacy of highly curative diseases such as germ cell tumors., Competing Interests: The authors have no conflicts of interest to declare., (© 2023 The Author(s). Published by S. Karger AG, Basel.)

Purpose: The UK-FAST-Forward study showed that ultra-hypofractionated whole-breast irradiation (ultra-HF-WBI) involving five fractions of 26 Gy radiation over 1 week was not inferior to HF-WBI. However, it is not used in Japan due to safety concerns. In April 2022, we commenced a multi-institutional, single-arm, phase II trial. Our aim is to confirm the safety of ultra-HF-WBI after breast-conserving surgery (BCS) for breast cancer in Japanese women., Method: We plan to enroll 98 patients from 13 institutions. The primary endpoint is the proportion of late adverse events of grades ≥2 within 3 years., Discussion: We believe that this highly promising clinical study can positively impact the Japanese guidelines for breast cancer treatment. The results will help us decide whether or not ultra-HF-WBI can be used as a more convenient alternative to WBI., Registration Number and Date: This trial was registered in the UMIN Clinical Trials Registry (UMIN000047080) on March 4, 2022., (© The Author(s) 2022. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

Background: The American Society of Surgery and American Society for Surgical Infections issued guidelines for surgical site infections (SSIs) in December 2016. These guidelines recommend a purse-string suture (PSS) for stoma closure as it facilitates granulation and enables open wound drainage. This study investigated the effect of using negative pressure wound therapy (NPWT) along with standard PSS and aimed to determine the optimal period of NPWT use., Methods: The patients were divided into three groups as follows: Group A, postoperative wound management alone with gauze exchange as the representative of conventional PSS; Group B, the performed management was similar to that of Group A plus NPWT for 1 week; and Group C, the performed management was similar to that of Group A plus NPWT for 2 weeks. Regarding objective measures, the wound reduction rate was the primary outcome, and the incidence of SSIs, length of hospital stay, and wound healing duration were the secondary outcomes., Results: In total, 30 patients (male: 18, female: 12) were enrolled. The average age was 63 (range: 43-84) years. The wound reduction rate was significantly higher in Group B than in Group A on postoperative days (PODs) 7 (66.1 vs. 48.4%, p = 0.049) and 10 (78.6 vs. 58.2%, p = 0.011), whereas no significant difference was observed on POD 14. Compared with Group A, Group C (POD 7: 65.9%, POD 10: 69.2%) showed an increase in the wound reduction rate on POD 7, although the difference was not significant (p = 0.075). SSIs were observed in Groups B (n = 2) and C (n = 2) (20%) but not in Group A (0%)., Conclusions: The most effective duration of NPWT use for ileostomy closure with PSS in terms of the maximum wound reduction rate was from PODs 3 to 10. However, NPWT did not shorten the wound healing duration. NPWT may reduce the wound size but should be used with precautions for SSIs. The small sample size (30 cases), the use of only one type of NPWT system, and the fact that wound assessment was subjective and not blinded were the limitations of this study. Further studies are needed to confirm our findings., Trial Registration: UMIN Clinical Trials Registry; UMIN000032174 (10/04/2018)., (© 2021. The Author(s).)

Recent progress in research on glucagon and α-cells highlights their pathophysiological roles in diabetes. We previously showed that plasma glucagon levels measured by newly developed enzyme-linked immunosorbent assay were dysregulated in patients with type 1 diabetes with respect to plasma glucose levels, suggesting dysregulated secretion. In the current study, the annual change in plasma glucagon levels was assessed in these same patients. We found that the plasma glucagon levels in the 66 Japanese patients involved in the study were significantly correlated between both years. In addition, they were significantly associated with serum blood urea nitrogen levels in a multivariate linear regression analysis, as reported in our previous study. The statistical correlation in glucagon levels between annual checkups and the sustained significant correlation between glucagon and blood urea nitrogen suggest a constant dysregulation of glucagon in association with altered amino acid metabolism in patients with type 1 diabetes., (© 2019 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Background: The main purposes of metastatic breast cancer (MBC) treatment are to prolong survival and maintain health-related quality of life (HRQOL). Compliance with the HRQOL assessment can be poor, particularly among patients who receive long-term treatment. One possible solution to overcoming this problem is to engage in real-time home monitoring by having patients report outcomes on their personal electronic devices. The objective of this study was to investigate compliance with HRQOL monitoring from home among MBC patients using the Computer-Based Health Evaluation System (CHES) to collect patient data., Methods: Sixteen MBC patients who received outpatient chemotherapy or endocrine therapy, both with and without targeted therapy, were recruited. One eligibility criterion was the availability of a personal electronic device with access to the Internet. Patients were asked to enter HRQOL ratings from their personal electronic devices via CHES once every week for 12 weeks. The European Organization for Research and Treatment of Cancer (EORTC) Quality of Life Questionnaire-Core 30 (QLQ C30) was used to evaluate HRQOL. The outcome examined was the questionnaire collection rate., Results: Six patients (37.5%) were treated with chemotherapy only, one (6.2%) with endocrine therapy only, three (18.8%) with a combination of chemotherapy and targeted therapy, and six (37.5%) with a combination of endocrine and targeted therapy. Median questionnaire collection rate for the total of 12 weeks was 84.6% (interquartile range 44.3-100). The reasons for missing data were worsening of disease, forgetting, and device malfunction., Conclusions: Compliance with electronic HRQOL data collection in this cohort was acceptable, considering the general ideal collection rate of 70-80%. We are conducting a prospective study to determine whether the use of CHES to input electronic real-time feedback of HRQOL ratings improves patients' overall HRQOL.

Cry46Ab is a Cry toxin derived from Bacillus thuringiensis TK-E6. Cry46Ab is not significantly homologous to other mosquitocidal Cry or Cyt toxins and is classified as an aerolysin-type pore-forming toxin based on structural similarity. In this study, the potency of Cry46Ab was assessed for its potential application to mosquito control. A synthetic Cry46Ab gene, cry46Ab-S1, was designed to produce recombinant Cry46Ab as a glutathione-S-transferase fusion in Escherichia coli. Recombinant Cry46Ab showed apparent toxicity to Culex pipiens larvae, with a 50% lethal dose of 1.02 μg/ml. In an artificial lipid bilayer, Cry46Ab activated by trypsin caused typical current transitions between open and closed states, suggesting it functions as a pore-forming toxin similar to other Cry and Cyt toxins. The single-channel conductance was 103.3 ± 4.1 pS in 150 mM KCl. Co-administration of recombinant Cry46Ab with other mosquitocidal Cry toxins, especially the combination of Cry4Aa and Cry46Ab, resulted in significant synergistic toxicity against C. pipiens larvae. Co-administration of multiple toxins exhibiting different modes of action is believed to prevent the onset of resistance in insects. Our data, taken in consideration with the differences in its structure, suggest that Cry46Ab could be useful in not only reducing resistance levels but also improving the insecticidal activity of Bt-based bio-insecticides., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Among the therapeutic avenues being explored for replacement of the functional islet β-cell mass lost in type 1 diabetes (T1D), reprogramming of adult cell types into new β-cells has been actively pursued. Notably, mouse islet α-cells will transdifferentiate into β-cells under conditions of near β-cell loss, a condition similar to T1D. Moreover, human islet α-cells also appear to poised for reprogramming into insulin-positive cells. Here we have generated transgenic mice conditionally expressing the islet β-cell-enriched Mafa and/or Pdx1 transcription factors to examine their potential to transdifferentiate embryonic pan-islet cell Ngn3-positive progenitors and the later glucagon-positive α-cell population into β-cells. Mafa was found to both potentiate the ability of Pdx1 to induce β-cell formation from Ngn3-positive endocrine precursors and enable Pdx1 to produce β-cells from α-cells. These results provide valuable insight into the fundamental mechanisms influencing islet cell plasticity in vivo., (© 2017 by the American Diabetes Association.)

Purpose: We investigated the prevalence of hypothyroidism (HT) in patients with breast cancer who received radiation therapy to the supraclavicular (SC) field to evaluate the effect of radiation on thyroid., Methods: Between April 2007 and May 2016, consecutive patients with invasive breast cancer who received SC radiation were recruited. Thyroid-stimulating hormone (TSH) and free thyroxine (fT4) were measured between April and August 2016. On the basis of the radiation-planning CT images, thyroid volume was calculated and dose-volume parameters were estimated. The endpoints were the prevalence of HT as determined by high levels of TSH and low levels of fT4 in serum, and the prevalence of subclinical HT, determined by high-serum TSH and normal fT4., Results: Among the 68 consecutive patients, 26 were excluded from evaluation (10 patients died, 6 had a history of previous thyroid disease and 10 were lost to follow-up). One (2.4%) and six (14.3%) of these patients had HT and subclinical HT, respectively, with a mean TSH level of 8.27 µU/mL. By univariate analysis, a predictive factor of HT and subclinical HT was a thyroid volume <8 cm 3 (OR 6.44, 95% CI 1.14 to 36.6; p=0.043). Multivariate analysis also showed an association between thyroid volume <8 cm 3 and HT or subclinical HT (OR 18.48, 95% CI 1.48 to 230.86; p=0.024)., Conclusions: The prevalence of HT in patients with breast cancer studied was relatively low. Although thyroid volume appeared to be a predictive marker of HT in this cohort, further prospective evaluation is needed., Competing Interests: Competing interests: None declared.

Pdx1, a β-cell-specific transcription factor, has been shown to play a crucial role in maintaining β-cell function through transactivation of β-cell-related genes. In addition, it has been reported that the expression levels of Pdx1 are compromised under diabetic conditions in human and rodent models. We therefore aimed to clarify the possible beneficial role of Pdx1 against β-cell failure and generated the transgenic mouse that expressed Pdx1 conditionally and specifically in β cells (βPdx1) and crossed these mice with Ins2 Akita diabetic mice. Whereas Pdx1 mRNA levels were reduced in Ins2 Akita mice compared with their non-diabetic littermates, the mRNA levels of Pdx1 were significantly recovered in the islets of βPdx1; Ins2 Akita mice. The βPdx1; Ins2 Akita mice exhibited significantly improved glucose tolerance, compared with control Ins2 Akita littermates, accompanied by increased insulin secretion after glucose loading. Furthermore, histological examination demonstrated that βPdx1; Ins2 Akita mice had improved localization of SLC2A2 (GLUT2), and quantitative RT-PCR showed the recovered expression of Mafa and Gck mRNAs in the islets of βPdx1; Ins2 Akita mice. These findings suggest that the sustained expression of Pdx1 improves β-cell failure in Ins2 Akita mice, at least partially through the preserving expression of β-cell-specific genes as well as improved localization of GLUT2., (Copyright © 2016 Elsevier Inc. All rights reserved.)

We studied the structure of fucoidans extracted from two brown seaweed species, Sargassum crassifolium and Padina australis, and their intestinal immunomodulating activity via Peyer's patch cells of C3H/HeJ mice. ESI-MS analysis indicated that the dominant structure of both fucoidans has a backbone of α-(1→4)-linked and α-(1→3)-linked l-fucose residues and sulfate groups are attached at the C-2 and C-4 positions; branches of fucoidan from S. crassifolium are galactose residues with (1→4)- linkage and branching points are at C-4 of fucose, while fucoidan from P. australis, branches are sulfated galactose-fucose disaccharides and sulfated galactose monosaccharides attached to the main chain through (1→3)- or (1→4)- linkages. According to small angle X-ray scattering (SAXS) measurements, the two fucoidans have a branched structure. We simulated them with molecular models based on our proposed primary structure. These fucoidan samples have the ability to stimulate intestinal immunological activity via Peyer's patch cells., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Escherichia coli non-heme-binding ferritin A (EcFtnA) is a spherical cagelike protein that is composed of 24 identical subunits. EcFtnA dissociates into 2-mers under acidic conditions and can reassemble into the native structure when the pH is increased. To understand how electrostatic interactions influence the assembly reaction, the dependence of the process on ionic strength and pH was investigated. The assembly reaction was initiated by stopped-flow mixing of the acid-dissociated EcFtnA solution and high-pH buffer solutions and monitored by time-resolved small-angle X-ray scattering. The rate of assembly increased with increasing ionic strength and decreased with increasing pH from 6 to 8. These dependences were thought to originate from repulsion between assembly units (2-mer in the case of EcFtnA) with the same net charge sign; therefore, to test this assumption, mutants with different net charges (net-charge mutants) were prepared. In buffers with a low ionic strength, the rate of assembly increased with a decreasing net charge. Thus, repulsion between the assembly unit net charges was demonstrated to be an important factor determining the rate of assembly. However, the difference in the assembly rate among net-charge mutants was not significant in buffers with an ionic strength of >0.1. Notably, under such high-ionic strength conditions, the assembly rate increased with an increasing ionic strength, suggesting that local electrostatic interactions are also responsible for the ionic strength dependence of the rate of assembly and are repulsive on average.

Alleviation of hyperglycaemia and hyperlipidemia improves pancreatic β-cell function in type 2 diabetes. However, the underlying molecular mechanisms are still not well clarified. In this study, we aimed to elucidate how the expression alterations of key β-cell factors are altered by the short-term selective alleviation of glucotoxicity or lipotoxicity. We treated db/db mice for one week with empagliflozin and/or bezafibrate to alleviate glucotoxicity and/or liptotoxicity, respectively. The gene expression levels of Pdx1 and Mafa, and their potential targets, insulin 1, Slc2a2, and Glp1r, were higher in the islets of empagliflozin-treated mice, and levels of insulin 2 were higher in mice treated with both reagents, than in untreated mice. Moreover, compared to the pretreatment levels, Mafa and insulin 1 expression increased in empagliflozin-treated mice, and Slc2a2 increased in combination-treated mice. In addition, empagliflozin treatment enhanced β-cell proliferation assessed by Ki-67 immunostaining. Our date clearly demonstrated that the one-week selective alleviation of glucotoxicity led to the better expression levels of the key β-cell factors critical for β-cell function over pretreatment levels, and that the alleviation of lipotoxicity along with glucotoxicity augmented the favorable effects under diabetic conditions., (Copyright © 2015 Elsevier Inc. All rights reserved.)

A 73-year-old man (164cm height 51 kg body weight) with a history of Parkinson's disease and dementia was scheduled for a cervical lymph node biopsy under general anesthesia. We induced anesthesia with thiamylal and fentanyl, and maintained with sevoflurane and remifentanil without any incident. The patient did not emerge from anesthesia after the surgery. He developed coma and did not respond to painful stimuli. However, his breathing was spontaneous with stable hemodynamics. Although naloxone was given, he was still comatose. His clinical neurological findings showed no organic abnormalities. Forty minutes after the surgery, he suddenly woke up and followed instructions. We learned that previously he had been diagnosed with dementia with Lewy bodies.

We encountered three cases of perioperative anaphylaxis identified by using skin-prick tests. [Case 1] A 43-year-old woman was scheduled to undergo elective laparoscopic subtotal gastrectomy under general anesthesia for gastric tumor. However, the procedure was cancelled because of anaphylaxis that was noted at the beginning of the surgery. We performed a skin-prick test and observed a positive reaction with ro- curonium. [Case 2] A 79-year-old man underwent laparoscopic colon resection under general anesthesia for colon cancer. Anaphylaxis was noted at the end of surgery. We performed a skin-prick test and observed a positive reaction with sugammadex. [Case 3] A 44-year-old woman underwent myomectomy under general anesthesia for a uterine fibroid. Anaphylaxis was noted approximately 10 minutes after the beginning of surgery. We performed a skin-prick test and noted a positive reaction with latex. It is difficult to identify the reason for anaphylaxis during surgery under general anesthesia because various agents may be responsible for the anaphylactic reaction. Anaphylaxis during surgery is a rare but life-threatening event and it is important to identify the causative agent for anaphylaxis.

While the exogenous expression of a combination of transcription factors have been shown to induce the conversion of non-β cells into insulin-producing cells, the reprogramming efficiency remains still low. In order to develop an in vitro screening system for an optimized reprogramming protocol, we generated the reporter cell line mPac-MIP-RFP in which the reprogramming efficiency can be quantified with red fluorescent protein expressed under the control of the insulin promoter. Analysis with mPac-MIP-RFP cells sequentially infected with adenoviruses expressing Pdx1, Neurog3, and Mafa revealed that expression of Pdx1 prior to Neurog3 or Mafa augments the reprogramming efficiency. Next, infection with a polycistronic adenoviral vector expressing Pdx1, Neurog3 and Mafa significantly increased the expression level of insulin compared with the simultaneous infection of three adenoviruses carrying each transcription factor, although excessive expression of Mafa together with the polycistronic vector dramatically inhibited the reprogramming into insulin-producing cells. Thus, in vitro screening with the mPac-MIP-RFP reporter cell line demonstrated that the timing and dosage of gene delivery with defined transcription factors influence the reprogramming efficiency. Further investigation should optimize the reprogramming conditions for the future cell therapy of diabetes., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Acidomonas methanolica (former name Acetobacter methanolicus) is a unique acetic acid bacterium capable to grow on methanol as a sole carbon source. Here we report the draft genome sequencing of A. methanolica type strain MB58, showing that it contains 3,270 protein-coding genes, including the genes involved in oxidation of methanol such as mxaFJGIRSACKL and hxlAB, and oxidation of ethanol such as adhAB and adhS. This article is protected by copyright. All rights reserved., (This article is protected by copyright. All rights reserved.)

The transcription factor MafA is a key regulator of insulin gene expression and maturation of islet β cells. Despite its importance, the regulatory mechanism of MafA gene expression is still unclear. To identify the transcriptional regulators of MafA, we examined various transcription factors, which are potentially involved in β cell differentiation. An adenovirus-mediated overexpression study clearly demonstrated that Onecut1 suppresses the promoter activity of MafA through the Foxa2-binding cis-element on the MafA enhancer region (named area A). However, ChIP analysis showed that Foxa2 but not Onecut1 could directly bind to area A. Furthermore, overexpression of Onecut1 inhibited the binding of Foxa2 onto area A upon ChIP analysis. Importantly, insertion of a mutation in the Foxa2-binding site of area A significantly decreased the promoter activity of MafA. These findings suggest that Onecut1 suppresses MafA gene expression through the Foxa2-binding site. In the mouse pancreas, MafA expression was first detected at the latest stage of β cell differentiation and was scarcely observed in Onecut1-positive cells during pancreas development. In addition, Onecut1 expression was significantly increased in the islets of diabetic db/db mice, whereas MafA expression was markedly decreased. The improved glucose levels of db/db mice with insulin injections significantly reduced Onecut1 expression and rescued the reduction of MafA expression. These in vivo experiments also suggest that Onecut1 is a negative regulator of MafA gene expression. This study implicates the novel role of Onecut1 in the control of normal β cell differentiation and its involvement in β cell dysfunction under diabetic conditions by suppressing MafA gene expression.

CoC(x) nanoparticles encapsulated in carbon shells were synthesized using a pulsed plasma in liquid ethanol. This is the first time that monolithic cubic phase cobalt carbide nanoparticles have been obtained. X-ray diffraction refinement of the nanoparticles showed that the lattice parameter of prepared cubic phase cobalt carbide is larger than that of CoC(x) (44-0962) and cubic phase Co (15-0806 and 01-1259). The x-ray absorption fine structure spectra near the Co K-edge of the synthesized sample indicated differences from commercial metallic cobalt and cobalt oxide samples. High resolution transmission electron microscopy revealed that a thin carbon coating covered the surface of the nanoparticles. These carbon layers might isolate core CoC(x) material from the outside environment, and allow functionalization by carboxyl groups for the further purpose of targeted drug delivery. The obtained CoC(x)@C particles, with a crystallite size of about 10 nm confirmed by the electron microscope, aggregate into 20-40 nm secondary particles in distilled water as shown by dynamic light scattering, and possess high saturation magnetization of about 120 emu g(-1). The sodium 3'-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis(4-methoxy-6-nitro)benzene sulfonic acid hydrate assay and defragmentation of deoxyribonucleic acid on MCF-7 cells after incubation with particles indicate relatively low cytotoxicity of CoC(x)@C nanoparticles, compared with micro-sized and nano-sized metallic cobalt particles and commonly used iron oxides. For the small sized CoC(x)@C particles, the release of cobalt ions was checked by a chelation method with ethylenediaminetetraacetic acid solution to be at a very low level compared with other reference materials.

Case 1: A 69-year-old man was diagnosed with rectal cancer and liver metastasis. After low anterior resection, mFOLFOX6 plus cetuximab therapy was started for resection of the liver metastasis. However, he had to forgo liver resection because he developed acute exacerbation of interstitial pneumonitis (IP) after 6 courses of chemotherapy. Despite beginning the second-line treatment with mFOLFOX6 plus bevacizumab, he died in June 2012. Case 2: A 71-year-old man had undergone sigmoidectomy for sigmoid colon cancer in 2005, and right lower lobe partial resection for metastatic lung cancer in 2006. Although radiofrequency ablation or transcatheter arterial chemoembolization had been performed for multiple liver metastases several times since 2007, his multiple liver metastases were uncontrollable. Therefore, FOLFOX4 therapy was started in 2010, and mFOLFOX6 plus cetuximab therapy was substituted for FOLFOX4 therapy in 2011. The patient died in March 2012 due to the rapid development of IP, and thus, it appears that IP was the cause of death in both patients. The general condition, including pulmonary function, of patients at risk of IP must be checked before starting cetuximab therapy for metastatic colorectal cancer.

Gene amplification is a major genetic alteration in human cancers. Amplicons, amplified genomic regions, are believed to contain "driver" genes responsible for tumorigenesis. However, the significance of co-amplified genes has not been extensively studied. We have established an integrated analysis system of amplicons using retrovirus-mediated gene transfer coupled with a human full-length cDNA set. Applying this system to 17q12-21 amplicon observed in breast cancer, we identified GRB7 as a context-dependent oncogene, which modulates the ERBB2 signaling pathway through enhanced phosphorylation of ERBB2 and Akt. Our work provides an insight into the biological significance of gene amplification in human cancers., (Copyright © 2012 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

The synthesis of wurtzite-type ZnS nanoparticles by an electric discharge submerged in molten sulfur is reported. Using a pulsed plasma between two zinc electrodes of diameter 5 mm in molten sulfur, we have synthesized high-temperature phase (wurtzite-type) ZnS nanocrystals with an average size of about 20 nm. The refined lattice parameters of the synthesized wurtzite-type ZnS nanoparticles were found to be larger than those of the reported ZnS (JCPDS card no 36-1450). Synthesis of ZnMgS (solid solution of ZnS and MgS) was achieved by using ZnMg alloys as both cathode and anode electrodes. UV-visible absorption spectroscopy analysis showed that the absorption peak of the as-prepared ZnS sample (319 nm) displays a blue-shift compared to the bulk ZnS (335 nm). Photoluminescence spectra of the samples revealed peaks at 340, 397, 423, 455 and 471 nm, which were related to excitonic emission and stoichiometric defects.

In hepatitis C virus (HCV) infection, the Th1-type immune response is involved in liver injury. A predominance of immunosuppressive regulatory T cells (Treg) is hypothesized in patients with persistently normal alanine aminotransferase (PNALT). Our aim was to clarify the role of Treg in the pathogenesis of PNALT. Fifteen chronically HCV-infected patients with PNALT, 21 with elevated ALT (CH) and 19 healthy subjects (HS) were enrolled. We determined naturally-occurring Treg (N-Treg) as CD4+CD25high+FOXP3+ T cells. The expression of FOXP3 and CTLA4 in CD4+CD25high+ cells was quantified by real-time reverse transcriptase-polymerase chain reaction. Bulk or CD25-depleted CD4+ T cells cultured with HCV-NS5 loaded dendritic cells were assayed for their proliferation and cytokine release. We examined CD127-CD25-FOXP3+ cells as distinct subsets other than CD25+ N-Treg. The frequencies of N-Treg in patients were significantly higher than those in HS. The FOXP3 and CTLA4 transcripts were higher in PNALT than those in CH. The depletion of CD25+ cells enhanced HCV-specific T cell responses, showing that co-existing CD25+ cells are suppressive. Such inhibitory capacity was more potent in PNALT. The frequency of CD4+CD127-CD25-FOXP3+ cells was higher in CH than those in PNALT. Treg are more abundant in HCV-infected patients, and their suppressor ability is more potent in patients with PNALT than in those with active hepatitis.

The PfPMT enzyme of Plasmodium falciparum, the agent of severe human malaria, is a member of a large family of known and predicted phosphoethanolamine methyltransferases (PMTs) recently identified in plants, worms, and protozoa. Functional studies in P. falciparum revealed that PfPMT plays a critical role in the synthesis of phosphatidylcholine via a plant-like pathway involving serine decarboxylation and phosphoethanolamine methylation. Despite their important biological functions, PMT structures have not yet been solved, and nothing is known about which amino acids in these enzymes are critical for catalysis and binding to S-adenosyl-methionine and phosphoethanolamine substrates. Here we have performed a mutational analysis of PfPMT focused on 24 residues within and outside the predicted catalytic motif. The ability of PfPMT to complement the choline auxotrophy of a yeast mutant defective in phospholipid methylation enabled us to characterize the activity of the PfPMT mutants. Mutations in residues Asp-61, Gly-83 and Asp-128 dramatically altered PfPMT activity and its complementation of the yeast mutant. Our analyses identify the importance of these residues in PfPMT activity and set the stage for advanced structural understanding of this class of enzymes.

A novel crystal protein that exhibited potent cytotoxicity against human leukemic T-cells was cloned from the Bacillus thuringiensis TK-E6 strain. The protein, designated as parasporin-2Ab (PS2Ab), was a polypeptide of 304 amino acid residues with a predicted molecular weight of 33,017. The deduced amino acid sequence of PS2Ab showed significant homology (84% identitiy) to parasporin-2Aa (PS2Aa) from the B. thuringiensis A1547 strain. Upon processing of PS2Ab with proteinase K, the active form of 29 kDa was produced. The activated PS2Ab showed potent cytotoxicity against MOLT-4 and Jurkat cells and the EC(50) values were estimated as 0.545 and 0.745 ng/mL, respectively. The cytotoxicity of PS2Ab was significantly higher than that of PS2Aa reported elsewhere. Although both cytotoxins were structurally related, it was thought that the minor differences found were responsible for the different cytotoxicities of PS2Ab and PS2Aa.

Plasmodium falciparum is the causative agent of severe human malaria, responsible for over 2 million deaths annually. Of the 5,300 polypeptides predicted to control the parasite life cycle in mosquitoes and humans, 60% are of unknown function. A major challenge of malaria postgenomic biology is to understand how the 5,300 predicted proteins coexist and interact to perform the essential tasks that define the complex life cycle of the parasite. One approach to assign function to these proteins is by identifying their physiological partners. Here we describe the use of tandem affinity purification (TAP) and mass spectrometry for identification of native protein interactions and purification of protein complexes in P. falciparum. Transgenic parasites were generated which express the translation elongation factor PfEF-1beta harboring a C-terminal PTP tag which consists of the protein C epitope, a tobacco etch virus protease cleavage site, and two protein A domains. Purification of PfEF-1beta-PTP from crude extracts followed by mass spectrometric analysis revealed, in addition to the tagged protein itself, the presence of the native PfEF-1beta, the G-protein PfEF-1alpha, and two new proteins that we named PfEF-1gamma and PfEF-1delta based on their homology to other eukaryotic gamma and delta translation elongation factor subunits. These data, which constitute the first application of TAP for purification of a protein complex under native conditions in P. falciparum, revealed that the translation elongation complex in this organism contains at least two subunits of PfEF-1beta. The success of this approach will set the stage for a systematic analysis of protein interactions in this important human pathogen.

Tctex1 is a light chain found in both cytoplasmic and flagellar dyneins and is involved in many fundamental cellular activities, including rhodopsin transport within photoreceptors, and may function in the non-Mendelian transmission of t haplotypes in mice. Here, we present the NMR solution structure for the Tctex1 dimer from Chlamydomonas axonemal inner dynein arm I1. Structural comparisons reveal a strong similarity with the LC8 dynein light chain dimer, including formation of a strand-switched beta sheet interface. Analysis of the Tctex1 structure enables the dynein intermediate chain binding site to be identified and suggests a mechanism by which cargo proteins might be attached to this microtubule motor complex. Comparison with the alternate dynein light chain rp3 reveals how the specificity of dynein-cargo interactions mediated by these dynein components is achieved. In addition, this structure provides insight into the consequences of the mutations found in the t haplotype forms of this protein.

The radial spokes are required for Ca(2+)-initiated intraflagellar signaling, resulting in modulation of inner and outer arm dynein activity. However, the mechanochemical properties of this signaling pathway remain unknown. Here, we describe a novel nucleoside diphosphate kinase (NDK) from the Chlamydomonas flagellum. This protein (termed p61 or RSP23) consists of an N-terminal catalytic NDK domain followed by a repetitive region that includes three IQ motifs and a highly acidic C-terminal segment. We find that p61 is missing in axonemes derived from the mutants pf14 (lacks radial spokes) and pf24 (lacks the spoke head and several stalk components) but not in those from pf17 (lacking only the spoke head). The p61 protein can be extracted from oda1 (lacks outer dynein arms) and pf17 axonemes with 0.5 M KI, and copurifies with radial spokes in sucrose density gradients. Furthermore, p61 contains two classes of calmodulin binding site: IQ1 interacts with calmodulin-Sepharose beads in a Ca(2+)-independent manner, whereas IQ2 and IQ3 show Ca(2+)-sensitive associations. Wild-type axonemes exhibit two distinct NDKase activities, at least one of which is stimulated by Ca(2+). This Ca(2+)-responsive enzyme, which accounts for approximately 45% of total axonemal NDKase, is missing from pf14 axonemes. We found that purified radial spokes also exhibit NDKase activity. Thus, we conclude that p61 is an integral component of the radial spoke stalk that binds calmodulin and exhibits Ca(2+)-controlled NDKase activity. These observations suggest that nucleotides other than ATP may play an important role in the signal transduction pathway that underlies the regulatory mechanism defined by the radial spokes.

The Roadblock/LC7 class of light chains associate with the intermediate chains at the base of the soluble dynein particle. In mammals, there are two Roadblock isoforms (Robl1 and Robl2), one of which (Robl2) is differentially expressed in a tissue-dependent manner and is especially prominent in testis. Here we define the alpha helical content of Robl and demonstrate using both the yeast two-hybrid system and in vitro biochemistry that Robl1 and Robl2 are capable of forming homo- and heterodimers. This is the first report of heterodimer formation by any cytoplasmic dynein component, and it further enlarges the number of potential cytoplasmic dynein isoforms available for binding specific cellular cargoes. In addition, we have generated an antibody that specifically recognizes Robl light chains and shows a 5-10 fold preference for Robl2 over Robl1. Using this antibody, we show that Robl is a ubiquitous cytoplasmic dynein component, being found in samples purified from brain, liver, kidney, and testis. Immunofluorescence analysis reveals that Robl is present in punctate organelles in rat neuroblastoma cells. In testis, Robl is found in Leydig cells, spermatocytes, and sperm flagella., (Copyright 2004 Wiley-Liss, Inc.)

Objective: To clarify the role of interleukin-4 (IL-4) in the expression of 15-lipoxygenase (15-LOX), whose metabolities are known to suppress the inflammatory reaction, in freshly prepared rheumatoid synovial cells., Methods: Adherent synovial cells were prepared by enzymatic digestion of synovia obtained from patients with rheumatoid arthritis (RA). Protein expression of 15-LOX was determined by Western blot analysis. The messenger RNAs of 15-LOX were determined by reverse transcription and the polymerase chain reaction (RT-PCR)., Results: Freshly prepared rheumatoid synovial cells did not express 15-LOX at either the mRNA or protein levels. IL-4 induced the protein expression of 15-LOX after 24 hours of culture. Although interleukin-1 alpha (IL-1 alpha) and tumor necrosis factor alpha (TNF alpha), major inflammatory cytokines in rheumatoid synovia, did not induce the expression of 15-LOX, IL-4 and these inflammatory cytokines synergistically enhanced the protein expression of 15-LOX. The synergistic effect was also observed at the level of mRNA., Conclusions: We demonstrate that IL-4 cooperated with the inflammatory cytokines IL-1 alpha and TNF alpha to enhance the expression of 15-LOX in rheumatoid synovial cells. Since 15-LOX metabolites have potent anti-inflammatory actions, our data suggest that IL-4 might downregulate rheumatoid inflammation via the induction of 15-LOX and its metabolites.

The autocatalytic processing of the streptococcal cysteine protease zymogen (proSCP) to active streptococcal cysteine protease (SCP) was investigated in vitro using purified protein from Streptococcus pyogenes strain B220. It was found that the autocatalytic maturation of the zymogen proceeds through the sequential appearance of at least six intermediates, five of which were characterized through a combination of N-terminal sequencing and MS. Intermediates were identified as resulting from cleavages after Lys26, Asn41, Lys101, Ala112, and Lys118. Time-course studies of the proSCP processing gave a sigmoidal activity profile and indicated that proSCP catalyses its own transformation, mainly via an intermolecular processing mechanism. A similar sequential appearance of intermediates was observed when inactive Cys192Ser proSCP was treated with native, enzymatically active SCP, thus demonstrating that the maturation can exclusively proceed by a bimolecular mechanism. It was shown that proSCP, but not mature SCP, immobilized on a Sepharose resin is capable of liberating itself from the column, indicating that the zymogen is also capable of intramolecular processing. In order to test whether the amino acid sequences at the processing sites could be used for developing new, specific substrates, 3-amino benzoic acid octapeptide derivatives based on all five characterized amino acid sequences from the autoprocessing cleavage sites were synthesized and tested for activity. The 3-amino benzoic acid derivatives have kcat/KM values ranging from 1200 to 7700.M-1.s-1, making them very good endopeptidase substrates for SCP.

The autocatalytic processing of procathepsin L was investigated in vitro using purified recombinant proenzyme expressed in Pichia pastoris. Pure intermolecular processing was studied by incubating the mutant procathepsin L (C25S), which cannot autoactivate with a small amount of mature active cathepsin L. The results clearly establish that, contrary to recent reports, intermolecular processing of procathepsin L is possible. The main cleavage sites are located at or near the N terminus of the mature enzyme, in an accessible portion of the proregion, which contains sequences corresponding to the known substrate specificity of cathepsin L. Contrary to procathepsins B, K, and S, autocatalytic processing of procathepsin L can generate the natural mature form of the enzyme. A continuous assay using the substrate benzyloxycarbonyl-Phe-Arg 4-methylcoumarinyl-7-amide hydrochloride has also been used to obtain information on the nature of the steps involved in the autocatalytic processing of wild-type procathepsin L. Processing is initiated by decreasing the pH from 8.0 to 5.3. The influence of proenzyme concentration on the rate of processing indicates the existence of both unimolecular and bimolecular steps in the mechanism of processing. The nature of the unimolecular event that triggers processing remains elusive. Circular dichroism and fluorescence measurements indicate the absence of large scale conformational change in the structure of procathepsin L on reduction of pH. However, the bimolecular reaction can be attributed to intermolecular processing of the zymogen.