High-Throughput Screening of Acyl-CoA Thioesterase I Mutants Using a Fluid Array Platform

Screening target microorganisms from a mutated recombinant library plays a crucial role in advancing synthetic biology and metabolic engineering. However, conventional screening tools have several limitations regarding throughput, cost, and labor. Here, we used the fluid array platform to conduct high-throughput screening (HTS) that identified Escherichia coli ???TesA thioesterase mutants producing elevated yields of free fatty acids (FFAs) from a large (106) mutant library. A growth-based screening method using a TetA-RFP fusion sensing mechanism and a reporter-based screening method using high-level FFA producing mutants were employed to identify these mutants via HTS. The platform was able to cover >95% of the mutation library, and it screened target cells from many arrays of the fluid array platform so that a post-analysis could be conducted by gas chromatography. The ???TesA mutation of each isolated mutant showing improved FFA production in E. coli was characterized, and its enhanced FFA production capability was confirmed

Mitochondrial ion channels as therapeutic targets

AbstractThe study of mitochondrial ion channels changed our perception of these double-wrapped organelles from being just the power house of a cell to the guardian of a cell’s fate. Mitochondria communicate with the cell through these special channels. Most of the time, the message is encoded by ion flow across the mitochondrial outer and inner membranes. Potassium, sodium, calcium, protons, nucleotides, and proteins traverse the mitochondrial membranes in an exquisitely regulated manner to control a myriad of processes, from respiration and mitochondrial morphology to cell proliferation and cell death. This review is an update on both well established and putative mitochondrial channels regarding their composition, function, regulation, and therapeutic potential

A genetic screen for modifiers of Drosophila caspase Dcp-1 reveals caspase involvement in autophagy and novel caspase-related genes

BACKGROUND: Caspases are cysteine proteases with essential functions in the apoptotic pathway; their proteolytic activity toward various substrates is associated with the morphological changes of cells. Recent reports have described non-apoptotic functions of caspases, including autophagy. In this report, we searched for novel modifiers of the phenotype of Dcp-1 gain-of-function (GF) animals by screening promoter element- inserted Drosophila melanogaster lines (EP lines). RESULTS: We screened ~15,000 EP lines and identified 72 Dcp-1-interacting genes that were classified into 10 groups based on their functions and pathways: 4 apoptosis signaling genes, 10 autophagy genes, 5 insulin/IGF and TOR signaling pathway genes, 6 MAP kinase and JNK signaling pathway genes, 4 ecdysone signaling genes, 6 ubiquitination genes, 11 various developmental signaling genes, 12 transcription factors, 3 translation factors, and 11 other unclassified genes including 5 functionally undefined genes. Among them, insulin/IGF and TOR signaling pathway, MAP kinase and JNK signaling pathway, and ecdysone signaling are known to be involved in autophagy. Together with the identification of autophagy genes, the results of our screen suggest that autophagy counteracts Dcp-1-induced apoptosis. Consistent with this idea, we show that expression of eGFP-Atg5 rescued the eye phenotype caused by Dcp-1 GF. Paradoxically, we found that over-expression of full-length Dcp-1 induced autophagy, as Atg8b-GFP, an indicator of autophagy, was increased in the eye imaginal discs and in the S2 cell line. Taken together, these data suggest that autophagy suppresses Dcp-1-mediated apoptotic cell death, whereas Dcp-1 positively regulates autophagy, possibly through feedback regulation. CONCLUSIONS: We identified a number of Dcp-1 modifiers that genetically interact with Dcp-1-induced cell death. Our results showing that Dcp-1 and autophagy-related genes influence each other will aid future investigations of the complicated relationships between apoptosis and autophagy

GAIT ASYMMETRY IN CHILDREN WITH DOWN SYNDROME

It has been reported that approximately 80% of children with Down syndrome (OS) exhibit gait problems (Matteo, 2002), characterized by flat-footed contact with no heel-strike, excessive abduction of the leg in the swing phase, and abnormal knee and hip f1exions throughout the gait cycle (Parker et aI., 1986). Due to muscle hypotonia, muscle weakness, !igamentous laXity, and other orthopedic abnormalities, different gait characteristics exist. The purpose of this study was to investigate the gait (walking and running) asymmetry in children with OS

Mitochondrial ion channels/transporters as sensors and regulators of cellular redox signaling.

SIGNIFICANCE: Mitochondrial ion channels/transporters and the electron transport chain (ETC) serve as key sensors and regulators for cellular redox signaling, the production of reactive oxygen species (ROS) and nitrogen species (RNS) in mitochondria, and balancing cell survival and death. Although the functional and pharmacological characteristics of mitochondrial ion transport mechanisms have been extensively studied for several decades, the majority of the molecular identities that are responsible for these channels/transporters have remained a mystery until very recently. RECENT ADVANCES: Recent breakthrough studies uncovered the molecular identities of the diverse array of major mitochondrial ion channels/transporters, including the mitochondrial Ca2+ uniporter pore, mitochondrial permeability transition pore, and mitochondrial ATP-sensitive K+ channel. This new information enables us to form detailed molecular and functional characterizations of mitochondrial ion channels/transporters and their roles in mitochondrial redox signaling. CRITICAL ISSUES: Redox-mediated post-translational modifications of mitochondrial ion channels/transporters and ETC serve as key mechanisms for the spatiotemporal control of mitochondrial ROS/RNS generation. FUTURE DIRECTIONS: Identification of detailed molecular mechanisms for redox-mediated regulation of mitochondrial ion channels will enable us to find novel therapeutic targets for many diseases that are associated with cellular redox signaling and mitochondrial ion channels/transporters

Reinforcing effects of methamphetamine in an animal model of Attention-Deficit/Hyperactivity Disorder-the Spontaneously Hypertensive Rat

Substrains of the Spontaneously Hypertensive rat (SHR), a putative animal model of Attention-Deficit/Hyperactivity Disorder (ADHD), have demonstrated increased sensitivity to many drugs of abuse, including psychostimulants. Therefore, it was suggested that studies in SHR may help elucidate ADHD and comorbidity with substance use disorder (SUD). However, the drug intake profile of the SHR in the most relevant animal model of drug addiction, the self-administration (SA) test, and its response on the conditioned place preference (CPP) paradigm are not yet determined. In the present study, we employed SA and CPP tests to investigate the reinforcing effects of the psychostimulant methamphetamine in an SHR substrain obtained from Charles River, Japan (SHR/NCrlCrlj). Concurrent tests were also performed in Wistar rats, the strain representing "normal" heterogeneous population. To address if the presence of ADHD behaviors further increases sensitivity to the rewarding effect of methamphetamine during adolescence, a critical period for the onset of drug abuse, CPP tests were especially conducted in adolescent Wistar and SHR/NCrlCrlj. We found that the SHR/NCrlCrlj also acquired methamphetamine SA and CPP, indicating reinforcing effects of methamphetamine in this ADHD animal model. However, we did not observe increased responsiveness of the SHR/NCrlCrlj to methamphetamine in both SA and CPP assays. This indicates that the reinforcing effects of methamphetamine may be similar in strains and that the SHR/NCrlCrlj may not adequately model ADHD and increased sensitivity to methamphetamine

Mitochondrial ryanodine receptors and other mitochondrial Ca2+ permeable channels

AbstractCa2+ channels that underlie mitochondrial Ca2+ transport first reported decades ago have now just recently been precisely characterized electrophysiologically. Numerous data indicate that mitochondrial Ca2+ uptake via these channels regulates multiple intracellular processes by shaping cytosolic and mitochondrial Ca2+ transients, as well as altering the cellular metabolic and redox state. On the other hand, mitochondrial Ca2+ overload also initiates a cascade of events that leads to cell death. Thus, characterization of mitochondrial Ca2+ channels is central to a comprehensive understanding of cell signaling. Here, we discuss recent progresses in the biophysical and electrophysiological characterization of several distinct mitochondrial Ca2+ channels

Identification of protein functions using a machine-learning approach based on sequence-derived properties

<p>Abstract</p> <p>Background</p> <p>Predicting the function of an unknown protein is an essential goal in bioinformatics. Sequence similarity-based approaches are widely used for function prediction; however, they are often inadequate in the absence of similar sequences or when the sequence similarity among known protein sequences is statistically weak. This study aimed to develop an accurate prediction method for identifying protein function, irrespective of sequence and structural similarities.</p> <p>Results</p> <p>A highly accurate prediction method capable of identifying protein function, based solely on protein sequence properties, is described. This method analyses and identifies specific features of the protein sequence that are highly correlated with certain protein functions and determines the combination of protein sequence features that best characterises protein function. Thirty-three features that represent subtle differences in local regions and full regions of the protein sequences were introduced. On the basis of 484 features extracted solely from the protein sequence, models were built to predict the functions of 11 different proteins from a broad range of cellular components, molecular functions, and biological processes. The accuracy of protein function prediction using random forests with feature selection ranged from 94.23% to 100%. The local sequence information was found to have a broad range of applicability in predicting protein function.</p> <p>Conclusion</p> <p>We present an accurate prediction method using a machine-learning approach based solely on protein sequence properties. The primary contribution of this paper is to propose new <it>PNPRD </it>features representing global and/or local differences in sequences, based on positively and/or negatively charged residues, to assist in predicting protein function. In addition, we identified a compact and useful feature subset for predicting the function of various proteins. Our results indicate that sequence-based classifiers can provide good results among a broad range of proteins, that the proposed features are useful in predicting several functions, and that the combination of our and traditional features may support the creation of a discriminative feature set for specific protein functions.</p

Type 1 ryanodine receptor in cardiac mitochondria: Transducer of excitation–metabolism coupling

AbstractMitochondria in a variety of cell types respond to physiological Ca2+ oscillations in the cytosol dynamically with Ca2+ uptakes. In heart cells, mitochondrial Ca2+ uptakes occur by a ruthenium red-sensitive Ca2+ uniporter (CaUP), a rapid mode of Ca2+ uptake (RaM) and a ryanodine receptor (RyR) localized in the inner mitochondrial membrane (IMM). Three subtypes of RyRs have been described and cloned, however, the subtype identity of the mitochondrial ryanodine receptor (mRyR) is unknown. Using subtype specific antibodies, we characterized the mRyR in the IMM from rat heart as RyR1. These results are substantiated by the absence of RyR protein in heart mitochondria from RyR1 knockout mice. The bell-shape Ca2+-dependent [3H]ryanodine binding curve and its modulation by caffeine and adenylylmethylenediphosphonate (AMPPCP) give further evidence that mRyR functions pharmacologically like RyR1. Ryanodine prevents mitochondrial Ca2+ uptake induced by raising extramitochondrial Ca2+ to 10 μM. Similarly, ryanodine inhibits oxidative phosphorylation stimulated by 10 μM extramitochondrial Ca2+. In summary, our results show that the mRyR in cardiac muscle has similar biochemical and pharmacological properties to the RyR1 in the sarcoplasmic reticulum (SR) of skeletal muscle. These results could also suggest an efficient mechanism by which mitochondria sequesters Ca2+ via mRyR during excitation–contraction coupling to stimulate oxidative phosphorylation for ATP production to meet metabolic demands. Thus, the mRyR functions as a transducer for excitation–metabolism coupling

Sleep Duration, Sleep Quality, and the Development of Nonalcoholic Fatty Liver Disease:A Cohort Study

INTRODUCTION: The longitudinal relationship between sleep duration, sleep quality, and the risk of nonalcoholic fatty liver disease (NAFLD) is unknown. We aimed to examine the association between sleep duration, sleep quality, and NAFLD development.METHODS: Using the Pittsburgh Sleep Quality Index, sleep duration and quality were evaluated for 143,306 NAFLD-free Korean adults with a mean age of 36.6 years, who were followed for an average of 4.0 years. Hepatic steatosis (HS) was assessed using ultrasonography and liver fibrosis by the fibrosis-4 index (FIB-4) or the NAFLD fibrosis score. Flexible parametric proportional hazard models were used to determine the hazard ratios (HRs) and 95% confidence intervals.RESULTS: There were 27,817 subjects with incident HS, of whom 1,471 had incident HS plus intermediate/high FIB-4. Multivariable-adjusted HRs (95% confidence intervals) for incident HS comparing sleep durations of ≤5, 6, 8, and ≥ 9 hours with 7 hours were 1.19 (1.14-1.23), 1.07 (1.04-1.10), 0.98 (0.94-1.02), and 0.95 (0.87-1.03), respectively. The corresponding HRs for incident HS plus intermediate/high FIB-4 were 1.30 (1.11-1.54), 1.14 (1.01-1.29), 1.11 (0.93-1.33), and 1.08 (0.71-1.63). The association between sleep duration and HS plus intermediate/high FIB-4 was inverse in individuals with good sleep quality but tended to be U-shaped in those with poor sleep quality. The results were similar if FIB-4 was replaced by the NAFLD fibrosis score.DISCUSSION: In young adults, short sleep duration was independently associated with an increased risk of incident NAFLD with or without intermediate/high fibrosis score, suggesting a role for inadequate sleep quantity in NAFLD risk and severity.</p