Quantitative Identification of Mutant Alleles Derived from Lung Cancer in Plasma Cell-Free DNA via Anomaly Detection Using Deep Sequencing Data

The detection of rare mutants using next generation sequencing has considerable potential for diagnostic applications. Detecting circulating tumor DNA is the foremost application of this approach. The major obstacle to its use is the high read error rate of next-generation sequencers. Rather than increasing the accuracy of final sequences, we detected rare mutations using a semiconductor sequencer and a set of anomaly detection criteria based on a statistical model of the read error rate at each error position. Statistical models were deduced from sequence data from normal samples. We detected epidermal growth factor receptor (EGFR) mutations in the plasma DNA of lung cancer patients. Single-pass deep sequencing (>100,000 reads) was able to detect one activating mutant allele in 10,000 normal alleles. We confirmed the method using 22 prospective and 155 retrospective samples, mostly consisting of DNA purified from plasma. A temporal analysis suggested potential applications for disease management and for therapeutic decision making to select epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKI).     

Glucagon-Like Peptide-1 Secretory Function as an Independent Determinant of Blood Pressure: Analysis in the Tanno-Sobetsu Study

Aims

Roles of glucagon-like peptide-1 (GLP-1) in extra-pancreatic tissues remain unclear. The aim of this study was to examine determinants of GLP-1 secretory function and possible contribution of GLP-1 to blood pressure (BP) regulation.

Methods and Results

We recruited 128 subjects who received annual examinations and 75g-oral glucose tolerance tests (OGTT) in the Tanno-Sobetsu cohort. Subjects on regular medications for cardiovascular and/or metabolic diseases were excluded, and data for the remaining 103 subjects were used for the univariate and multivariate analyses. Age, plasma glucose (PG), hemoglobin A1c (HbA1c), plasma insulin, and serum lipids were not selected as independent determinants of fasting GLP-1 level by multiple linear regression analysis. However, age and female sex were selected as independent positive determinants of the area under the curve of GLP-1 level during OGTT (AUC_GLP-1), an index of GLP-1 secretory function. Multiple linear regression analysis indicated that AUC_GLP-1 was an independent negative predictor of systolic BP (SBP), while AUC_GLP-1 was not correlated with fasting PG or HbA1c level. In subgroup analyses using the median of AUC_GLP-1 to divide the study subjects into high and low GLP-1 response groups, AUC_GLP-1 was significantly correlated with both SBP and diastolic BP (r = 0.40 and 0.28, respectively) in the low GLP-1 response group but not in the high GLP-1 response group.

Conclusions

The results of the present study suggest that GLP-1 secretory function is involved in prevention of BP elevation and that the GLP-1 response to oral glucose rather increases with aging perhaps as an adaptive phenomenon.     

Rates of Serious Intracellular Infections in Autoimmune Disease Patients Receiving Initial Glucocorticoid Therapy

Background/Aims

The Japanese National Hospital Organization evidence-based medicine (EBM) Study group for Adverse effects of Corticosteroid therapy (J-NHOSAC) is a Japanese hospital-based cohort study investigating the safety of the initial use of glucocorticoids (GCs) in patients with newly diagnosed autoimmune diseases. Using the J-NHOSAC registry, the purpose of this observational study is to analyse the rates, characteristics and associated risk factors of intracellular infections in patients with newly diagnosed autoimmune diseases who were initially treated with GCs.

Methodology/Principal Findings

A total 604 patients with newly diagnosed autoimmune diseases treated with GCs were enrolled in this registry between April 2007 and March 2009. Cox proportional-hazards regression was used to determine independent risk factors for serious intracellular infections with covariates including sex, age, co-morbidity, laboratory data, use of immunosuppressants and dose of GCs. Survival was analysed according to the Kaplan-Meier method and was assessed by the log-rank test. There were 127 serious infections, including 43 intracellular infections, during 1105.8 patient-years of follow-up. The 43 serious intracellular infections resulted in 8 deaths. After adjustment for covariates, diabetes (Odds ratio [OR]: 2.5, 95% confidence interval [95% CI] 1.1–5.9), lymphocytopenia (≦1000/μl, OR: 2.5, 95% CI 1.2–5.2) and use of high-dose (≧30 mg/day) GCs (OR: 2.4, 95% CI 1.1–5.3) increased the risk of intracellular infections. Survival curves showed lower intracellular infection-free survival rate in patients with diabetes, lymphocytopaenia and high-dose GCs treatments.

Conclusions/Significance

Patients with newly diagnosed autoimmune diseases were at high risk of developing intracellular infection during initial treatment with GCs. Our findings provide background data on the risk of intracellular infections of patients with autoimmune diseases. Clinicians showed remain vigilant for intracellular infections in patients with autoimmune diseases who are treated with GCs.     

Circulating Levels of Fatty Acid-Binding Protein Family and Metabolic Phenotype in the General Population

Objective

Fatty acid-binding proteins (FABPs) are a family of 14-15-kDa proteins, and some FABPs have been to be used as biomarkers of tissue injury by leak from cells. However, recent studies have shown that FABPs can be secreted from cells into circulation. Here we examined determinants and roles of circulating FABPs in a general population.

Methods

From the database of the Tanno-Sobetsu Study, a study with a population-based cohort design, data in 2011 for 296 subjects on no medication were retrieved, and FABP1∼5 in their serum samples were assayed.

Results

Level of FABP4, but not the other isoforms, showed a gender difference, being higher in females than in males. Levels of all FABPs were negatively correlated with estimated glomerular filtration rate (eGFR), but a distinct pattern of correlation with other clinical parameters was observed for each FABP isoform; significant correlates were alanine aminotransferase (ALT), blood pressure (BP), and brain natriuretic peptide (BNP) for FABP1, none besides eGFR for FABP2, age, BP, and BNP for FABP3, age, waist circumference (WC), BP, BNP, lipid variables, high-sensitivity C-reactive protein (hsCRP), and HOMA-R for FABP4, and age, WC, BP, ALT, BNP, and HOMA-R for FABP5. FABP4 is the most strongly related to metabolic markers among FABPs. In a multivariate regression analysis, FABP4 level was an independent predictor of HOMA-R after adjustment of age, gender, WC, BP, HDL cholesterol, and hsCRP.

Conclusions

Each FABP isoform level showed a distinct pattern of correlation with clinical parameters, although levels of all FABPs were negatively determined by renal function. Circulating FABP4 appears to be a useful biomarker for detecting pre-clinical stage of metabolic syndrome, especially insulin resistance, in the general population.     

Extracts of Adipose Derived Stem Cells Slows Progression in the R6/2 Model of Huntington's Disease

Stem cell therapy is a promising treatment for incurable disorders including Huntington''s disease (HD). Adipose-derived stem cell (ASC) is an easily available source of stem cells. Since ASCs can be differentiated into nervous stem cells, it has clinically feasible potential for neurodegenerative disease. In addition, ASCs secrete various anti-apoptotic growth factors, which improve the symptoms of disease from transplanted ASCs. Thus, cell-free extracts of ASCs (ASCs-E) could be a potential candidate for treatment of HD. Here, we investigated effects of ASCs-E on R6/2 HD mouse model and neuronal cells. In R6/2 HD model, injection of ASCs-E improved the performance in Rotarod test. ASCs-E also ameliorated striatal atrophy and mutant huntingtin aggregation in the striatum. In Western blot increased expressions of p-Akt, p-CREB and PGC1α were noted by injection of ASCs-E, when comparing to the R6/2 HD model. Neuro2A neuroblastoma cells treated with ASCs-E showed increased expression of p-CREB and PGC1α. In conclusion, ASCs-E delayed disease progression in animal model of HD by restoring of CREB-PGC1α pathway and could be a potential resource for treatment of HD.     

Cooperative Functions of ZnT1, Metallothionein and ZnT4 in the Cytoplasm Are Required for Full Activation of TNAP in the Early Secretory Pathway

The activation process of secretory or membrane-bound zinc enzymes is thought to be a highly coordinated process involving zinc transport, trafficking, transfer and coordination. We have previously shown that secretory and membrane-bound zinc enzymes are activated in the early secretory pathway (ESP) via zinc-loading by the zinc transporter 5 (ZnT5)-ZnT6 hetero-complex and ZnT7 homo-complex (zinc transport complexes). However, how other proteins conducting zinc metabolism affect the activation of these enzymes remains unknown. Here, we investigated this issue by disruption and re-expression of genes known to be involved in cytoplasmic zinc metabolism, using a zinc enzyme, tissue non-specific alkaline phosphatase (TNAP), as a reporter. We found that TNAP activity was significantly reduced in cells deficient in ZnT1, Metallothionein (MT) and ZnT4 genes (ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells), in spite of increased cytosolic zinc levels. The reduced TNAP activity in ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells was not restored when cytosolic zinc levels were normalized to levels comparable with those of wild-type cells, but was reversely restored by extreme zinc supplementation via zinc-loading by the zinc transport complexes. Moreover, the reduced TNAP activity was adequately restored by re-expression of mammalian counterparts of ZnT1, MT and ZnT4, but not by zinc transport-incompetent mutants of ZnT1 and ZnT4. In ZnT1 ^−/− MT ^−/− ZnT4 ^−/− cells, the secretory pathway normally operates. These findings suggest that cooperative zinc handling of ZnT1, MT and ZnT4 in the cytoplasm is required for full activation of TNAP in the ESP, and present clear evidence that the activation process of zinc enzymes is elaborately controlled.     

CLE2 regulates light-dependent carbohydrate metabolism in Arabidopsis shoots

Plant Molecular Biology - This study focused on the role of CLE1-CLE7 peptides as environmental mediators and indicated that root-induced CLE2 functions systemically in light-dependent carbohydrate...     

Tertiary structure of bacterial selenocysteine tRNA

Selenocysteine (Sec) is translationally incorporated into proteins in response to the UGA codon. The tRNA specific to Sec (tRNA^Sec) is first ligated with serine by seryl-tRNA synthetase (SerRS). In the present study, we determined the 3.1 Å crystal structure of the tRNA^Sec from the bacterium Aquifex aeolicus, in complex with the heterologous SerRS from the archaeon Methanopyrus kandleri. The bacterial tRNA^Sec assumes the L-shaped structure, from which the long extra arm protrudes. Although the D-arm conformation and the extra-arm orientation are similar to those of eukaryal/archaeal tRNA^Secs, A. aeolicus tRNA^Sec has unique base triples, G14:C21:U8 and C15:G20a:G48, which occupy the positions corresponding to the U8:A14 and R15:Y48 tertiary base pairs of canonical tRNAs. Methanopyrus kandleri SerRS exhibited serine ligation activity toward A. aeolicus tRNA^Sec in vitro. The SerRS N-terminal domain interacts with the extra-arm stem and the outer corner of tRNA^Sec. Similar interactions exist in the reported tRNA^Ser and SerRS complex structure from the bacterium Thermus thermophilus. Although the catalytic C-terminal domain of M. kandleri SerRS lacks interactions with A. aeolicus tRNA^Sec in the present complex structure, the conformational flexibility of SerRS is likely to allow the CCA terminal region of tRNA^Sec to enter the SerRS catalytic site.     

Structure and Function of Nucleoside Hydrolases from Physcomitrella patens and Maize Catalyzing the Hydrolysis of Purine,Pyrimidine, and Cytokinin Ribosides

We present a comprehensive characterization of the nucleoside N-ribohydrolase (NRH) family in two model plants, Physcomitrella patens (PpNRH) and maize (Zea mays; ZmNRH), using in vitro and in planta approaches. We identified two NRH subclasses in the plant kingdom; one preferentially targets the purine ribosides inosine and xanthosine, while the other is more active toward uridine and xanthosine. Both subclasses can hydrolyze plant hormones such as cytokinin ribosides. We also solved the crystal structures of two purine NRHs, PpNRH1 and ZmNRH3. Structural analyses, site-directed mutagenesis experiments, and phylogenetic studies were conducted to identify the residues responsible for the observed differences in substrate specificity between the NRH isoforms. The presence of a tyrosine at position 249 (PpNRH1 numbering) confers high hydrolase activity for purine ribosides, while an aspartate residue in this position confers high activity for uridine. Bud formation is delayed by knocking out single NRH genes in P. patens, and under conditions of nitrogen shortage, PpNRH1-deficient plants cannot salvage adenosine-bound nitrogen. All PpNRH knockout plants display elevated levels of certain purine and pyrimidine ribosides and cytokinins that reflect the substrate preferences of the knocked out enzymes. NRH enzymes thus have functions in cytokinin conversion and activation as well as in purine and pyrimidine metabolism.Nucleoside hydrolases or nucleoside N-ribohydrolases (NRHs; EC 3.2.2.-) are glycosidases that catalyze the cleavage of the N-glycosidic bond in nucleosides to enable the recycling of the nucleobases and Rib (Fig. 1A). The process by which nucleosides and nucleobases are recycled is also known as salvaging and is a way of conserving energy, which would otherwise be needed for the de novo synthesis of purine- and pyrimidine-containing compounds. During the salvage, bases and nucleosides can be converted into nucleoside monophosphates by the action of phosphoribosyltransferases and nucleoside kinases, respectively, and further phosphorylated into nucleoside diphosphates and triphosphates (Moffatt et al., 2002; Zrenner et al., 2006; Fig. 1B). Uridine kinase and uracil phosphoribosyl transferase are key enzymes in the pyrimidine-salvaging pathway in plants (Mainguet et al., 2009; Chen and Thelen, 2011). Adenine phosphoribosyltransferase and adenosine kinase (ADK) are important in purine salvaging (Moffatt and Somerville, 1988; Moffatt et al., 2002), and their mutants cause reductions in fertility or sterility, changes in transmethylation, and the formation of abnormal cell walls. In addition, both enzymes were also reported to play roles in cytokinin metabolism (Moffatt et al., 1991, 2000; von Schwartzenberg et al., 1998; Schoor et al., 2011). Cytokinins (N⁶-substituted adenine derivatives) are plant hormones that regulate cell division and numerous developmental events (Mok and Mok, 2001; Sakakibara, 2006). Cytokinin ribosides are considered to be transport forms and have little or no activity.Open in a separate windowFigure 1.A, Scheme of the reactions catalyzed by plant NRHs when using purine (inosine), pyrimidine (uridine), and cytokinin (iPR) ribosides as the substrates. B, Simplified schematic overview of cytokinin, purine, and pyrimidine metabolism in plants. The diagram is adapted from the work of Stasolla et al. (2003) and Zrenner et al. (2006) with modifications. The metabolic components shown are as follows: 1, cytokinin nucleotide phosphoribohydrolase; 2, adenine phosphoribosyltransferase; 3, adenosine kinase; 4, 5′-nucleotidase; 5, adenosine phosphorylase; 6, purine/pyrimidine nucleoside ribohydrolase; 7, cytokinin oxidase/dehydrogenase; 8, AMP deaminase; 9, hypoxanthine phosphoribosyltransferase; 10, inosine kinase; 11, inosine-guanosine phosphorylase; 12, IMP dehydrogenase; 13, xanthine dehydrogenase; 14, 5′-nucleotidase; 15, GMP synthase; 16, hypoxanthine-guanine phosphoribosyltransferase; 17, guanosine deaminase; 18, guanine deaminase; 19, guanosine kinase; 20, uracil phosphoribosyltransferase; 21, uridine cytidine kinase; 22, pyrimidine 5′-nucleotidase; 23, cytidine deaminase; 24, adenosine/adenine deaminase. CK, Cytokinin; CKR, cytokinin riboside; CKRMP, cytokinin riboside monophosphate.NRHs are metalloproteins first identified and characterized in parasitic protozoa such as Trypanosoma, Crithidia, and Leishmania species that rely on the import and salvage of nucleotide derivatives. They have since been characterized in other organisms such as bacteria, yeast, and insects (Versées and Steyaert, 2003) but never in mammals (Parkin et al., 1991). They have been divided into four classes based on their substrate specificity: nonspecific NRHs, which hydrolyze inosine and uridine (IU-NRHs; Parkin et al., 1991; Shi et al., 1999); purine-specific inosine/adenosine/guanosine NRHs (Parkin, 1996); the 6-oxopurine-specific guanosine/inosine NRHs (Estupiñán and Schramm, 1994); and the pyrimidine nucleoside-specific cytidine/uridine NRHs (CU-NRHs; Giabbai and Degano, 2004). All NRHs exhibit a stringent specificity for the Rib moiety and differ in their preferences regarding the nature of the nucleobase. Crystal structures are available for empty NRH or in complex with inhibitors from Crithidia fasciculata (CfNRH; Degano et al., 1998), Leishmania major (LmNRH; Shi et al., 1999), and Trypanosoma vivax (TvNRH; Versées et al., 2001, 2002). The structures of two CU-NRHs from Escherichia coli, namely YeiK (Iovane et al., 2008) and YbeK (rihA; Muzzolini et al., 2006; Garau et al., 2010), are also available. NRHs are believed to catalyze N-glycosidic bond cleavage by a direct displacement mechanism. An Asp from a conserved motif acts as a general base and abstracts a proton from a catalytic water molecule, which then attacks the C1′ atom of the Rib moiety of the nucleoside. Kinetic isotope-effect studies on CfNRH (Horenstein et al., 1991) showed that the substrate’s hydrolysis proceeds via an oxocarbenium ion-like transition state and is preceded by protonation at the N7 atom of the purine ring, which lowers the electron density on the purine ring and destabilizes the N-glycosidic bond. A conserved active-site His is a likely candidate for this role in IU-NRHs and CU-NRHs. In the transition state, the C1′-N9 glycosidic bond is almost 2 Å long, with the C1′ atom being sp² hybridized while the C3′ atom adopts an exo-conformation, and the whole ribosyl moiety carries a substantial positive charge (Horenstein et al., 1991).Several NRH enzymes have been identified in plants, including a uridine-specific NRH from mung bean (Phaseolus radiatus; Achar and Vaidyanathan, 1967), an inosine-specific NRH (EC 3.2.2.2) and a guanosine-inosine-specific NRH, both from yellow lupine (Lupinus luteus; Guranowski, 1982; Szuwart et al., 2006), and an adenosine-specific NRH (EC 3.2.2.7) from coffee (Coffea arabica), barley (Hordeum vulgare), and wheat (Triticum aestivum; Guranowski and Schneider, 1977; Chen and Kristopeit, 1981; Campos et al., 2005). However, their amino acid sequences have not been reported so far. A detailed study of the NRH gene family from Arabidopsis (Arabidopsis thaliana) has recently been reported (Jung et al., 2009, 2011). The AtNRH1 enzyme exhibits highest hydrolase activity toward uridine and xanthosine. It can also hydrolyze the cytokinin riboside N⁶-(2-isopentenyl)adenosine (iPR), which suggests that it may also play a role in cytokinin homeostasis. However, Riegler et al. (2011) analyzed the phenotypes of homozygous nrh1 and nrh2 single mutants along with the homozygous double mutants and concluded that AtNRHs are probably unimportant in cytokinin metabolism.Here, we identify and characterize plant IU-NRHs from two different model organisms, Physcomitrella patens and maize (Zea mays), combining structural, enzymatic, and in planta functional approaches. The moss P. patens was chosen to represent the bryophytes, which can be regarded as being evolutionarily basal terrestrial plants, and is suitable for use in developmental and metabolic studies (Cove et al., 2006; von Schwartzenberg, 2009), while maize is an important model system for cereal crops. We report the crystal structures of NRH enzymes from the two plant species, PpNRH1 and ZmNRH3. Based on these structures, we performed site-directed mutagenesis experiments and kinetic analyses of point mutants of PpNRH1 in order to identify key residues involved in nucleobase interactions and catalysis. To analyze the physiological role of the PpNRHs, single knockout mutants were generated. NRH deficiency caused significant changes in the levels of purine, pyrimidine, and cytokinin metabolites relative to those seen in the wild type, illustrating the importance of these enzymes in nucleoside and cytokinin metabolism.