Absence of Nceh1 augments 25-hydroxycholesterol-induced ER stress and apoptosis in macrophages

An excess of cholesterol and/or oxysterols induces apoptosis in macrophages, contributing to the development of advanced atherosclerotic lesions. In foam cells, these sterols are stored in esterified forms, which are hydrolyzed by two enzymes: neutral cholesterol ester hydrolase 1 (Nceh1) and hormone-sensitive lipase (Lipe). A deficiency in either enzyme leads to accelerated growth of atherosclerotic lesions in mice. However, it is poorly understood how the esterification and hydrolysis of sterols are linked to apoptosis. Remarkably, Nceh1-deficient thioglycollate-elicited peritoneal macrophages (TGEMs), but not Lipe-deficient TGEMs, were more susceptible to apoptosis induced by oxysterols, particularly 25-hydroxycholesterol (25-HC), and incubation with 25-HC caused massive accumulation of 25-HC ester in the endoplasmic reticulum (ER) due to its defective hydrolysis, thereby activating ER stress signaling such as induction of CCAAT/enhancer-binding protein-homologous protein (CHOP). These changes were nearly reversed by inhibition of ACAT1. In conclusion, deficiency of Nceh1 augments 25-HC-induced ER stress and subsequent apoptosis in TGEMs. In addition to reducing the cholesteryl ester content of foam cells, Nceh1 may protect against the pro-apoptotic effect of oxysterols and modulate the development of atherosclerosis.     

A generalizable NLP framework for fast development of pattern-based biomedical relation extraction systems

Background

Text mining is increasingly used in the biomedical domain because of its ability to automatically gather information from large amount of scientific articles. One important task in biomedical text mining is relation extraction, which aims to identify designated relations among biological entities reported in literature. A relation extraction system achieving high performance is expensive to develop because of the substantial time and effort required for its design and implementation. Here, we report a novel framework to facilitate the development of a pattern-based biomedical relation extraction system. It has several unique design features: (1) leveraging syntactic variations possible in a language and automatically generating extraction patterns in a systematic manner, (2) applying sentence simplification to improve the coverage of extraction patterns, and (3) identifying referential relations between a syntactic argument of a predicate and the actual target expected in the relation extraction task.

Results

A relation extraction system derived using the proposed framework achieved overall F-scores of 72.66% for the Simple events and 55.57% for the Binding events on the BioNLP-ST 2011 GE test set, comparing favorably with the top performing systems that participated in the BioNLP-ST 2011 GE task. We obtained similar results on the BioNLP-ST 2013 GE test set (80.07% and 60.58%, respectively). We conducted additional experiments on the training and development sets to provide a more detailed analysis of the system and its individual modules. This analysis indicates that without increasing the number of patterns, simplification and referential relation linking play a key role in the effective extraction of biomedical relations.

Conclusions

In this paper, we present a novel framework for fast development of relation extraction systems. The framework requires only a list of triggers as input, and does not need information from an annotated corpus. Thus, we reduce the involvement of domain experts, who would otherwise have to provide manual annotations and help with the design of hand crafted patterns. We demonstrate how our framework is used to develop a system which achieves state-of-the-art performance on a public benchmark corpus.     

Psychiatrists' Attitudes toward Metabolic Adverse Events in Patients with Schizophrenia

Background

There is growing concern about the metabolic abnormalities in patients with schizophrenia.

Aims

The aim of this study was to assess the attitudes of psychiatrists toward metabolic adverse events in patients with schizophrenia.

Method

A brief questionnaire was constructed to cover the following broad areas: the psychiatrists'' recognition of the metabolic risk of antipsychotic therapy, pattern of monitoring patients for physical risks, practice pattern for physical risks, and knowledge of metabolic disturbance. In March 2012, the questionnaire was mailed to 8,482 psychiatrists who were working at hospitals belonging to the Japan Psychiatric Hospitals Association.

Results

The overall response rate was 2,583/8,482 (30.5%). Of the respondents, 85.2% (2,200/2,581) reported that they were concerned about prescribing antipsychotics that have a risk of elevating blood sugar; 47.6% (1,201/2,524) stated that their frequency of monitoring patients under antipsychotic treatment was based on their own experiences; and only 20.6% (5,22/2,534) of respondents answered that the frequency with which they monitored their patients was sufficient to reduce the metabolic risks.

Conclusions

Psychiatrists practicing in Japan were generally aware and concerned about the metabolic risks for patients being treated with antipsychotics. Although psychiatrists should monitor their patients for metabolic abnormalities to balance these risks, a limited number of psychiatrists answered that the frequency with which they monitored patients to reduce the metabolic risks was sufficient. Promotion of the best practices of pharmacotherapy and monitoring is needed for psychiatrists treating patients with schizophrenia.     

Preclinical Validation of Talaporfin Sodium-Mediated Photodynamic Therapy for Esophageal Squamous Cell Carcinoma

Photodynamic therapy (PDT) kills cancer cells via a photochemical reaction mediated by an oncotropic photosensitizer. Herein, we performed an experimental preclinical study to validate the anti-tumour effect of talaporfin sodium-mediated PDT (t-PDT) for esophageal squamous cell carcinoma (ESCC) cells. We used human ESCC cells derived from various differentiation grades or resistant to 5-fluorouracil (5-FU). The cytotoxic effect of t-PDT was determined by evaluating cell viability, apoptosis and generation of reactive oxygen species (ROS) and DNA double-strand breaks. Furthermore, the anti-tumour effect of t-PDT was assessed using an anchorage-independent cell-growth assay and xenograft transplantation models. t-PDT induced potent cytotoxicity in ESCC cells independent of their differentiation grade or 5-FU resistance. Moreover, t-PDT induced robust apoptosis, as indicated by cell shrinkage, perinuclear vacuolization, nuclear fragmentation and induction of annexin V-positive cells. This apoptotic response was accompanied by concurrent activation of ROS, and induction of DNA double-strand breakage. Importantly, t-PDT suppressed efficiently anchorage-independent cell growth as well as ESCC-xenografted tumor formation. In aggregate, t-PDT showed anti-tumor potential for ESCC cells with various histological grades or chemoresistance, providing a novel translational rationale of t-PDT for the treatment of ESCC.     

Ephexin4 and EphA2 mediate cell migration through a RhoG-dependent mechanism

EphA2, a member of the Eph receptor family, is frequently overexpressed in a variety of human cancers, including breast cancers, and promotes cancer cell motility and invasion independently of its ligand ephrin stimulation. In this study, we identify Ephexin4 as a guanine nucleotide exchange factor (GEF) for RhoG that interacts with EphA2 in breast cancer cells, and knockdown and rescue experiments show that Ephexin4 acts downstream of EphA2 to promote ligand-independent breast cancer cell migration and invasion toward epidermal growth factor through activation of RhoG. The activation of RhoG recruits its effector ELMO2 and a Rac GEF Dock4 to form a complex with EphA2 at the tips of cortactin-rich protrusions in migrating breast cancer cells. In addition, the Dock4-mediated Rac activation is required for breast cancer cell migration. Our findings reveal a novel link between EphA2 and Rac activation that contributes to the cell motility and invasiveness of breast cancer cells.     

Isolation and characterization of a novel 2-<Emphasis Type=Italic>sec</Emphasis>-butylphenol-degrading bacterium <Emphasis Type=Italic>Pseudomonas</Emphasis> sp. strain MS-1

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites—3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid—by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho ≫ meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).     

Blocking LC3 lipidation and ATG12 conjugation reactions by ATG7 mutant protein containing C572S

Autophagy, a system for the bulk degradation of intracellular components, is essential for homeostasis and the healthy physiology and development of cells and tissues. Its deregulation is associated with human disease. Thus, methods to modulate autophagic activity are critical for analysis of its role in mammalian cells and tissues. Here we report a method to inhibit autophagy using a mutant variant of the protein ATG7, a ubiquitin E1-like enzyme essential for autophagosome formation. During autophagy, ATG7 activates the conjugation of LC3 (ATG8) with phosphatidylethanolamine (PE) and ATG12 with ATG5. Human ATG7 interactions with LC3 or ATG12 require a thioester bond involving the ATG7 cysteine residue at position 572. We generated TetOff cells expressing mutant ATG7 protein carrying a serine substitution of this critical cysteine residue (ATG7C572S). Because ATG7C572S forms stable intermediate complexes with LC3 or ATG12, its expression resulted in a strong blockage of the ATG-conjugation system and suppression of autophagosome formation. Consequently, ATG7C572S mutant protein can be used as an inhibitor of autophagy.     

Clinical Utility of an Enzyme-Linked Immunosorbent Assay for Detecting Anti-Melanoma Differentiation-Associated Gene 5 Autoantibodies

ObjectiveAutoantibodies to melanoma differentiation-associated gene 5 (MDA5) are specifically expressed in patients with dermatomyositis (DM) and are associated with a subset of DM patients with rapidly progressive interstitial lung disease (RP-ILD). Here, we examined the clinical utility of a newly developed enzyme-linked immunosorbent assay (ELISA) system for detecting these antibodies.MethodsHere we developed an improved ELISA for detecting anti-MDA5 antibodies. We then performed a multicenter clinical study involving 8 medical centers and enrolled 242 adult patients with polymyositis (PM)/DM, 190 with non-PM/DM connective tissue disease (CTD), 154 with idiopathic interstitial pneumonia (IIP), and 123 healthy controls. Anti-MDA5 antibodies in the patients’ serum samples were quantified using our newly developed ELISA, and the results were compared to those obtained using the gold-standard immunoprecipitation (IP) assay. In addition, correlations between the ELISA-quantified anti-MDA5 antibodies and clinical characteristics were evaluated.ResultsIn patients with PM/DM, the anti-MDA5 antibody measurements obtained from the ELISA and IP assay were highly concordant; the ELISA exhibited an analytical sensitivity of 98.2%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 99.5% (compared to the IP assay). Anti-MDA5 antibodies were detected in 22.7% of the DM patients, but not in any of the patients with PM, non-PM/DM CTD, or IIP. Clinically amyopathic DM, RP-ILD, arthritis, and fever were more prevalent in DM patients who were anti-MDA5 antibody-positive than in those who were antibody-negative (P ≤ 0.0002 for all comparisons). In addition, anti-MDA5 antibody-positive patients with RP-ILD exhibited higher antibody levels than those without RP-ILD (P = 0.006).ConclusionOur newly developed ELISA can detect anti-MDA5 antibodies as efficiently as the gold standard IP assay and has the potential to facilitate the routine clinical measurement of anti-MDA5 antibodies in patients who suspected to have DM.     

Desulforubrerythrin from <Emphasis Type="Italic">Campylobacter jejuni</Emphasis>, a novel multidomain protein

A novel multidomain metalloprotein from Campylobacter jejuni was overexpressed in Escherichia coli, purified, and extensively characterized. This protein is isolated as a homotetramer of 24-kDa monomers. According to the amino acid sequence, each monomer was predicted to contain three structural domains: an N-terminal desulforedoxin-like domain, followed by a four-helix bundle domain harboring a non-sulfur μ-oxo diiron center, and a rubredoxin-like domain at the C-terminus. The three predicted iron sites were shown to be present and were studied by a combination of UV–vis, EPR, and resonance Raman spectroscopies, which allowed the determination of the electronic and redox properties of each site. The protein contains two FeCys₄ centers with reduction potentials of +240 mV (desulforedoxin-like center) and +185 mV (rubredoxin-like center). These centers are in the high-spin configuration in the as-isolated ferric form. The protein further accommodates a μ-oxo-bridged diiron site with reduction potentials of +270 and +235 mV for the two sequential redox transitions. The protein is rapidly reoxidized by hydrogen peroxide and has a significant NADH-linked hydrogen peroxide reductase activity of 1.8 μmol H₂O₂ min⁻¹ mg⁻¹. Owing to its building blocks and its homology to the rubrerythrin family, the protein is named desulforubrerythrin. It represents a novel example of the large diversity of the organization of domains exhibited by this enzyme family.     

Aminoglycoside antibiotics reduce glucose reabsorption in kidney through down-regulation of SGLT1

Nephrotoxicity is known to be a major clinical side effect of aminoglycoside antibiotics. Aminoglycosides cause damage to proximal tubular cells in kidney, however the mechanism of toxicity is still unclear. In order to elucidate the mechanism of nephrotoxicity, we studied the effect of aminoglycoside antibiotics on glucose transport systems in vitro and in vivo. As a result, we found that the aminoglycosides significantly reduced Na(+)/glucose cotransporter (SGLT1)-dependent glucose transport and also down-regulated mRNA and protein levels of the SGLT1 in pig proximal tubular LLC-PK(1) cells. To obtain evidence about SGLT1 down-regulation in vivo, we studied the mRNA expression of SGLT1 using gentamicin C-treated murine kidney and found that gentamicin C down-regulated SGLT1 in vivo as well as in vitro. Furthermore, the gentamicin C-treated mice showed significant rise in urinary glucose excretion. These results indicate that one of the mechanisms of aminoglycoside nephrotoxicity is the down-regulation of SGLT1, which causes reduction in glucose reabsorption in kidney.