ALBERT

Description: For cancer and many other complex diseases, a large number of gene signatures have been generated. In this study, we use cancer as an example and note that other diseases can be analyzed in a similar manner. For signatures generated in multiple independent studies on the same cancer type and outcome, and for signatures on different cancer types, it is of interest to evaluate their degree of overlap. Many of the existing studies simply count the number (or percentage) of overlapped genes shared by two signatures. Such an approach has serious limitations. In this study, as a demonstrating example, we consider cancer prognosis data under the Cox model. Lasso, which is representative of a large number of regularization methods, is adopted for generating gene signatures. We examine two families of measures for quantifying the degree of overlap. The first family is based on the Cox-Lasso estimates at the optimal tunings, and the second family is based on estimates across the whole solution paths. Within each family, multiple measures, which describe the overlap from different perspectives, are introduced. The analysis of TCGA (The Cancer Genome Atlas) data on five cancer types shows that the degree of overlap varies across measures, cancer types and types of (epi)genetic measurements. More investigations are needed to better describe and understand the overlaps among gene signatures.

Description: Ferritins and other cage proteins have been utilized as models to understand the fundamentals of protein folding and self-assembly. The bacterioferritin (BFR) from Escherichia coli, a maxi-ferritin made up of 24 subunits, was chosen as the basis for a mutagenesis study to investigate the role of electrostatic intermolecular interactions mediated through charged amino acids. Through structural and computational analyses, three charged amino acids R30, D56 and E60 which involved in an electrostatic interaction network were mutated to the opposite charge. Four mutants, R30D, D56R, E60H and D56R-E60H, were expressed, purified and characterized. All of the mutants fold into α-helical structures. Consistent with the computational prediction, they all show a lowered thermostability; double mutant D56R-E60H was found to be 16°C less stable than the wild type. Except for the mutant E60H, all the other mutations completely shut down the formation of protein cages to favour the dimer state in solution. The mutants, however, retain their ability to form cage-like nanostructures in the dried, surface immobilized conditions of transmission electron microscopy. Our findings confirm that even a single charge-inversion mutation at the 2-fold interface of BFR can affect the quaternary structure of its dimers and their ability to self-assemble into cage structures.

Description: Most of bacteria can swim by rotating flagella bidirectionally. The C ring, located at the bottom of the flagellum and in the cytoplasmic space, consists of FliG, FliM and FliN, and has an important function in flagellar protein secretion, torque generation and rotational switch of the motor. FliG is the most important part of the C ring that interacts directly with a stator subunit. Here, we introduced a three-amino acids in-frame deletion mutation (PSA) into FliG from Vibrio alginolyticus , whose corresponding mutation in Salmonella confers a switch-locked phenotype, and examined its phenotype. We found that this FliG mutant could not produce flagellar filaments in a fliG null strain but the FliG(PSA) protein could localize at the cell pole as does the wild-type protein. Unexpectedly, when this mutant was expressed in a wild-type strain, cells formed flagella efficiently but the motor could not rotate. We propose that this different phenotype in Vibrio and Salmonella might be due to distinct interactions between FliG mutant and FliM in the C ring between the bacterial species.

Description: Sulphation is known to be critically involved in the metabolism of acetaminophen in vivo . This study aimed to systematically identify the major human cytosolic sulfotransferase (SULT) enzyme(s) responsible for the sulphation of acetaminophen. A systematic analysis showed that three of the twelve human SULTs, SULT1A1, SULT1A3 and SULT1C4, displayed the strongest sulphating activity towards acetaminophen. The pH dependence of the sulphation of acetaminophen by each of these three SULTs was examined. Kinetic parameters of these three SULTs in catalysing acetaminophen sulphation were determined. Moreover, sulphation of acetaminophen was shown to occur in HepG2 human hepatoma cells and Caco-2 human intestinal epithelial cells under the metabolic setting. Of the four human organ samples tested, liver and intestine cytosols displayed considerably higher acetaminophen-sulphating activity than those of lung and kidney. Collectively, these results provided useful information concerning the biochemical basis underlying the metabolism of acetaminophen in vivo previously reported.

Description: In this study, the physicochemical and enzymatic properties of recombinant human ubiquitin (Ub)-specific protease (USP) 47, a novel member of the C19 family of de-ubiquitinating enzymes (DUB), were characterized for the first time. Recombinant human USP47 was expressed in a baculovirus expression system and purified to homogeneity. The purified protein was shown to be a monomeric protein with a molecular mass of ~146 kDa on sodium dodecyl sulphate—polyacrylamide gel electrophoresis. USP47 released Ub from Ub-aminoacyl-4-metheylcoumaryl-7-amide and Ub-tagged granzyme B. The substitution of the potential nucleophile Cys109 with Ser severely abrogated the Ub-releasing activity of USP47, indicating that USP47 is indeed a cysteine DUB. An assay using Ub dimer substrates showed that the enzyme cleaved a variety of isopeptide bonds between 2 Ub molecules, including the Lys48- and Lys63-linked isopeptide bonds. USP47 also released a Ub moiety from Lys48- and Lys63-linked polyUb chains. Of the inhibitors tested, N -ethylmaleimide, Zn ion and Ub aldehyde revealed a dose-dependent inhibition of USP47. In this study, clear differences in the enzymatic properties between USP47 and USP7 (the most closely related proteins among DUBs) were also found. Therefore, our results suggest that USP47 may play distinct roles in Ub-mediated cellular processes via DUB activity.

Description: P24 antigen is the main structural protein of HIV-1, its detection provide a means to aid the early diagnosis of HIV-1 infection. The aim of this study was to improve the selectivity and sensitivity of the HIV P24 diagnostic assay by developing a cohort of 9E8 affinity-matured antibodies through in vitro phage affinity maturation which was performed by complementarity determining region (CDR)-hot spot mutagenesis strategy. Antibody 9E8-491 had an affinity constant of 5.64 x 10 –11 M, which was 5.7-fold higher than that of the parent antibody (9E8). Furthermore, the affinity, sensitivity and specificity of 9E8-491 were higher than those of 9E8, which indicate that 9E8-491 is a good candidate detection antibody for HIV P24 assay. Structure analysis of matured variants revealed that most hydrogen bonds resided in HCDR3. Among the antibody–antigen predicted binding residues, Tyr 100A/100B was the original conserved residue that was commonly present in HCDR3 of 9E8 and variants. Arg 100 /Asp 100C was the major variant substitution that most likely influenced the binding differences among variants and 9E8 monoclonal antibody. Both efficient library panning and predicted structural data were in agreement that the binding residues were mostly located in HCDR3 and enabled identification of key residues that influence antibody affinity.

Description: The past two decades of microRNA (miRNA) research has solidified the role of these small non-coding RNAs as key regulators of many biological processes and promising biomarkers for disease. The concurrent development in high-throughput profiling technology has further advanced our understanding of the impact of their dysregulation on a global scale. Currently, next-generation sequencing is the platform of choice for the discovery and quantification of miRNAs. Despite this, there is no clear consensus on how the data should be preprocessed before conducting downstream analyses. Often overlooked, data preprocessing is an essential step in data analysis: the presence of unreliable features and noise can affect the conclusions drawn from downstream analyses. Using a spike-in dilution study, we evaluated the effects of several general-purpose aligners (BWA, Bowtie, Bowtie 2 and Novoalign), and normalization methods (counts-per-million, total count scaling, upper quartile scaling, Trimmed Mean of M, DESeq, linear regression, cyclic loess and quantile) with respect to the final miRNA count data distribution, variance, bias and accuracy of differential expression analysis. We make practical recommendations on the optimal preprocessing methods for the extraction and interpretation of miRNA count data from small RNA-sequencing experiments.

Description: Parameter estimation is a challenging computational problem in the reverse engineering of biological systems. Because advances in biotechnology have facilitated wide availability of time-series gene expression data, systematic parameter estimation of gene circuit models from such time-series mRNA data has become an important method for quantitatively dissecting the regulation of gene expression. By focusing on the modeling of gene circuits, we examine here the performance of three types of state-of-the-art parameter estimation methods: population-based methods, online methods and model-decomposition-based methods. Our results show that certain population-based methods are able to generate high-quality parameter solutions. The performance of these methods, however, is heavily dependent on the size of the parameter search space, and their computational requirements substantially increase as the size of the search space increases. In comparison, online methods and model decomposition-based methods are computationally faster alternatives and are less dependent on the size of the search space. Among other things, our results show that a hybrid approach that augments computationally fast methods with local search as a subsequent refinement procedure can substantially increase the quality of their parameter estimates to the level on par with the best solution obtained from the population-based methods while maintaining high computational speed. These suggest that such hybrid methods can be a promising alternative to the more commonly used population-based methods for parameter estimation of gene circuit models when limited prior knowledge about the underlying regulatory mechanisms makes the size of the parameter search space vastly large.

Description: Human housekeeping genes are often confused with essential human genes, and several studies regard both types of genes as having the same level of evolutionary conservation. However, this is not necessarily the case. To clarify this, we compared the differences between human housekeeping genes and essential human genes with respect to four aspects: the evolutionary rate (dN/dS), protein sequence identity, single-nucleotide polymorphism (SNP) density and level of linkage disequilibrium (LD). The results showed that housekeeping genes had lower evolutionary rates, higher sequence identities, lower SNP densities and higher levels of LD compared with essential genes. Together, these findings indicate that housekeeping and essential genes are two distinct types of genes, and that housekeeping genes have a higher level of evolutionary conservation. Therefore, we suggest that researchers should pay careful attention to the distinctions between housekeeping genes and essential genes. Moreover, it is still controversial whether we should substitute human orthologs of mouse essential genes for human essential genes. Therefore, we compared the evolutionary features between human orthologs of mouse essential genes and human housekeeping genes and we got inconsistent results in long-term and short-term evolutionary characteristics implying the irrationality of simply replacing human essential genes with human orthologs of mouse essential genes.

Description: Many skeletal diseases have common pathological phenotype of defective osteogenesis of bone marrow stromal cells (BMSCs), in which histone modifications play an important role. However, few studies have examined the dynamics of distinct histone modifications during osteogenesis. In this study, we examined the dynamics of H3K9/K14 and H4K12 acetylation; H3K4 mono-, di- and tri-methylation; H3K9 di-methylation and H3K27 tri-methylation in osteogenic genes, runt-related transcription factor 2 (Runx2), osterix (Osx), alkaline phosphatase, bone sialoprotein and osteocalcin, during C3H10T1/2 osteogenesis. H3 and H4 acetylation and H3K4 di-methylation were elevated, and H3K9 di-methylation and H3K27 tri-methylation were reduced in osteogenic genes during C3H10T1/2 osteogenesis. C3H10T1/2 osteogenesis could be modulated by altering the patterns of H3 and H4 acetylation and H3K27 tri-methylation. In a glucocorticoid-induced osteoporosis mouse model, we observed the attenuation of osteogenic potential of osteoporotic BMSCs in parallel with H3 and H4 hypo-acetylation and H3K27 hyper-tri-methylation in Runx2 and Osx genes. When H3 and H4 acetylation was elevated, and H3K27 tri-methylation was reduced, the attenuated osteogenic potential of osteoporotic BMSCs was rescued effectively. These observations provide a deeper insight into the mechanisms of osteogenic differentiation and the pathophysiology of osteoporosis and can be used to design new drugs and develop new therapeutic methods to treat skeletal diseases.

Description: Dihydrouridine (D) is formed by tRNA dihydrouridine synthases (Dus). In mesophiles, multiple Dus enzymes bring about D modifications at several positions in tRNA. The extreme-thermophilic eubacterium Thermus thermophilus , in contrast, has only one dus gene in its genome and only two D modifications (D20 and D20a) in tRNA have been identified. Until now, an in vitro assay system for eubacterial Dus has not been reported. In this study, therefore, we constructed an in vitro assay system using purified Dus. Recombinant T. thermophilus Dus lacking bound tRNA was successfully purified. The in vitro assay revealed that no other factors in living cells were required for D formation. A dus gene disruptant ( dus ) strain of T. thermophilus verified that the two D20 and D20a modifications in tRNA were derived from one Dus protein. The dus strain did not show growth retardation at any temperature. The assay system showed that Dus modified tRNA Phe transcript at 60°C, demonstrating that other modifications in tRNA are not essential for Dus activity. However, a comparison of the formation of D in native tRNA Phe purified from the dus strain and tRNA Phe transcript revealed that other tRNA modifications are required for D formation at high temperatures.

Description: Human lactate dehydrogenase (LDH) has attracted attention as a potential target for cancer therapy and contraception. In this study, we reconstituted human lactic acid fermentation in Saccharomyces cerevisiae , with the goal of constructing a yeast cell-based LDH assay system. pdc null mutant yeast (mutated in the endogenous pyruvate decarboxylase genes) are unable to perform alcoholic fermentation; when grown in the presence of an electron transport chain inhibitor, pdc null strains exhibit a growth defect. We found that introduction of the human gene encoding LDHA complemented the pdc growth defect; this complementation depended on LDHA catalytic activity. Similarly, introduction of the human LDHC complemented the pdc growth defect, even though LDHC did not generate lactate at the levels seen with LDHA. In contrast, the human LDHB did not complement the yeast pdc null mutant, although LDHB did generate lactate in yeast cells. Expression of LDHB as a red fluorescent protein (RFP) fusion yielded blebs in yeast, whereas LDHA-RFP and LDHC-RFP fusion proteins exhibited cytosolic distribution. Thus, LDHB exhibits several unique features when expressed in yeast cells. Because yeast cells are amenable to genetic analysis and cell-based high-throughput screening, our pdc /LDH strains are expected to be of use for versatile analyses of human LDH.

Description: RelB is activated by the non-canonical NF-B pathway, which is crucial for immunity by establishing lymphoid organogenesis and B-cell and dendritic cell (DC) maturation. To elucidate the mechanism of the RelB-mediated immune cell maturation, a precise understanding of the relationship between cell maturation and RelB expression and activation at the single-cell level is required. Therefore, we generated knock-in mice expressing a fusion protein between RelB and fluorescent protein (RelB-Venus) from the Relb locus. The Relb Venus / Venus mice developed without any abnormalities observed in the Relb –/– mice, allowing us to monitor RelB-Venus expression and nuclear localization as RelB expression and activation. Relb Venus / Venus DC analyses revealed that DCs consist of RelB – , RelB low and RelB high populations. The RelB high population, which included mature DCs with projections, displayed RelB nuclear localization, whereas RelB in the RelB low population was in the cytoplasm. Although both the RelB low and RelB – populations barely showed projections, MHC II and co-stimulatory molecule expression were higher in the RelB low than in the RelB – splenic conventional DCs. Taken together, our results identify the RelB low population as a possible novel intermediate maturation stage of cDCs and the Relb Venus / Venus mice as a useful tool to analyse the dynamic regulation of the non-canonical NF-B pathway.

Description: Hyperthermophilic bacteria Thermotoga maritima and Thermotoga hypogea produce ethanol as a metabolic end product, which is resulted from acetaldehyde reduction catalysed by an alcohol dehydrogenase (ADH). However, the enzyme that is involved in the production of acetaldehyde from pyruvate is not well characterized. An oxygen sensitive and coenzyme A-dependent pyruvate decarboxylase (PDC) activity was found to be present in cell free extracts of T. maritima and T. hypogea . Both enzymes were purified and found to have pyruvate ferredoxin oxidoreductase (POR) activity, indicating their bifunctionality. Both PDC and POR activities from each of the purified enzymes were characterized in regards to their optimal assay conditions including pH dependency, oxygen sensitivity, thermal stability, temperature dependency and kinetic parameters. The close relatedness of the PORs that was shown by sequence analysis could be an indication of the presence of such bifunctionality in other hyperthermophilic bacteria. This is the first report of a bifunctional PDC/POR enzyme in hyperthermophilic bacteria. The PDC and the previously reported ADHs are most likely the key enzymes catalysing the production of ethanol from pyruvate in bacterial hyperthermophiles.

Description: Whole-genome search of genes is an essential approach to dissecting complex traits, but a marginal one-single-nucleotide polymorphism (SNP)/one-phenotype regression analysis widely used in current genome-wide association studies fails to estimate the net and cumulative effects of SNPs and reveal the developmental pattern of interplay between genes and traits. Here we describe a computational framework, which we refer to as two-side high-dimensional genome-wide association studies (2HiGWAS), to associate an ultrahigh dimension of SNPs with a high dimension of developmental trajectories measured across time and space. The model is implemented with a dual dimension-reduction procedure for both predictors and responses to select a sparse but full set of significant loci from an extremely large pool of SNPs and estimate their net time-varying effects on trait development. The model can not only help geneticists to precisely identify an entire set of genes underlying complex traits but also allow them to elucidate a global picture of how genes control developmental and dynamic processes of trait formation. We investigated the statistical properties of the model via extensive simulation studies. With the increasing availability of GWAS in various organisms, 2HiGWAS will have important implications for genetic studies of developmental compelx traits.

Description: Three principal approaches have been proposed for inferring the set of transcripts expressed in RNA samples using RNA-seq. The simplest approach uses curated annotations, which assumes the transcripts in a sample are a subset of the transcripts listed in a curated database. A more ambitious method involves aligning reads to a reference genome and using the alignments to infer the transcript structures, possibly with the aid of a curated transcript database. The most challenging approach is to assemble reads into putative transcripts de novo without the aid of reference data. We have systematically assessed the properties of these three approaches through a simulation study. We have found that the sensitivity of computational transcript set estimation is severely limited. Computational approaches (both genome-guided and de novo assembly) produce a large number of artefacts, which are assigned large expression estimates and absorb a substantial proportion of the signal when performing expression analysis. The approach using curated annotations shows good expression correlation even when the annotations are incomplete. Furthermore, any incorrect transcripts present in a curated set do not absorb much signal, so it is preferable to have a curation set with high sensitivity than high precision. Software to simulate transcript sets, expression values and sequence reads under a wider range of parameter values and to compare sensitivity, precision and signal-to-noise ratios of different methods is freely available online ( https://github.com/boboppie/RSSS ) and can be expanded by interested parties to include methods other than the exemplars presented in this article.

Description: Significant efforts have been made recently to improve data throughput and data quality in screening technologies related to drug design. The modern pharmaceutical industry relies heavily on high-throughput screening (HTS) and high-content screening (HCS) technologies, which include small molecule, complementary DNA (cDNA) and RNA interference (RNAi) types of screening. Data generated by these screening technologies are subject to several environmental and procedural systematic biases, which introduce errors into the hit identification process. We first review systematic biases typical of HTS and HCS screens. We highlight that study design issues and the way in which data are generated are crucial for providing unbiased screening results. Considering various data sets, including the publicly available ChemBank data, we assess the rates of systematic bias in experimental HTS by using plate-specific and assay-specific error detection tests. We describe main data normalization and correction techniques and introduce a general data preprocessing protocol. This protocol can be recommended for academic and industrial researchers involved in the analysis of current or next-generation HTS data.

Description: De novo motif discovery is a difficult computational task. Historically, dedicated algorithms always reported a high percentage of false positives. Their performance did not improve considerably even after they adapted to handle large amounts of chromatin immunoprecipitation sequencing (ChIP-Seq) data. Several studies have advocated aggregating complementary algorithms, combining their predictions to increase the accuracy of the results. This led to the development of ensemble methods. To form a better view on modern ensembles, we review all compound tools designed for ChIP-Seq. After a brief introduction to basic algorithms and early ensembles, we describe the most recent tools. We highlight their limitations and strengths by presenting their architecture, the input options and their output. To provide guidance for next-generation sequencing practitioners, we observe the differences and similarities between them. Last but not least, we identify and recommend several features to be implemented by any novel ensemble algorithm.

Description: The scaffolding protein Salvador (Sav) plays a key role in the Hippo (Hpo) signalling pathway, which controls tissue growth by inhibiting cell proliferation and promoting apoptosis. Dysregulation of the Hippo pathway contributes to cancer development. Since the identification of the first Sav gene in 2002, very little is known regarding the molecular basis of Sav-SARAH mediating interactions due to its insolubility. In this study, refolding of the first Sav (known as WW45)-SARAH provided insight into the biochemical and biophysical properties, indicating that WW45-SARAH exhibits properties of a disordered protein, when the domain was refolded at a neutral pH. Interestingly, WW45-SARAH shows folded and rigid conformations relative to the decrease in pH. Further, diffracting crystals were obtained from protein refolded under acidic pH, suggesting that the refolded WW45 protein at low pH has a homogeneous and stable conformation. A comparative analysis of molecular properties found that the acidic-stable fold of WW45-SARAH enhances a heterotypic interaction with Mst2-SARAH. In addition, using an Mst2 mutation that disrupts homotypic dimerization, we showed that the monomeric Mst2-SARAH domain could form a stable complex of 1:1 stoichiometric ratio with WW45 refolded under acidic pH.

Description: Hypercholesterolemia is one of the factors contributing to cardiovascular problems. Erythrocytes are known to contribute its cholesterol to atherosclerotic plaque. Our earlier study showed that erythrocytes overexpress chondroitin sulphate/dermatan sulphate (CS/DS), a linear co-polymer, during diabetes which resulted in increased cytoadherence to extracellular matrix (ECM) components. This study was carried out to determine whether diet-induced hypercholesterolemia had any effect on erythrocyte CS/DS and impacted cytoadherence to ECM components. Unlike in diabetes, diet-induced hypercholesterolemia did not show quantitative changes in erythrocyte CS/DS but showed difference in proportion of un-sulphated and 4- O -sulphated disaccharides. Erythrocytes from hypercholesterolemic rats showed increased adhesion to ECM components which was abrogated to various extents when subjected to chondroitinase ABC digestion. However, isolated CS/DS chains showed a different pattern of binding to ECM components indicating that orientation of CS/DS chains could be playing a role in binding.

Description: The antigen-binding domain of camelid dimeric heavy chain antibodies, known as VHH or Nanobody, has much potential in pharmaceutical and industrial applications. To establish the isolation process of antigen-specific VHH, a VHH phage library was constructed with a diversity of 8.4 x 10 7 from cDNA of peripheral blood mononuclear cells of an alpaca ( Lama pacos ) immunized with a fragment of IZUMO1 (IZUMO1 PFF ) as a model antigen. By conventional biopanning, 13 antigen-specific VHHs were isolated. The amino acid sequences of these VHHs, designated as N-group VHHs, were very similar to each other (〉93% identity). To find more diverse antibodies, we performed high-throughput sequencing (HTS) of VHH genes. By comparing the frequencies of each sequence between before and after biopanning, we found the sequences whose frequencies were increased by biopanning. The top 100 sequences of them were supplied for phylogenic tree analysis. In total 75% of them belonged to N-group VHHs, but the other were phylogenically apart from N-group VHHs (Non N-group). Two of three VHHs selected from non N-group VHHs showed sufficient antigen binding ability. These results suggested that biopanning followed by HTS provided a useful method for finding minor and diverse antigen-specific clones that could not be identified by conventional biopanning.

Description: The autophosphorylation of specific tyrosine residues occurs in the cytoplasmic region of the insulin receptor (IR) upon insulin binding, and this in turn initiates signal transduction. The R3 subfamily (Ptprb, Ptprh, Ptprj and Ptpro) of receptor-like protein tyrosine phosphatases (RPTPs) is characterized by an extracellular region with 6–17 fibronectin type III-like repeats and a cytoplasmic region with a single phosphatase domain. We herein identified the IR as a substrate for R3 RPTPs by using the substrate-trapping mutants of R3 RPTPs. The co-expression of R3 RPTPs with the IR in HEK293T cells suppressed insulin-induced tyrosine phosphorylation of the IR. In vitro assays using synthetic phosphopeptides revealed that R3 RPTPs preferentially dephosphorylated a particular phosphorylation site of the IR: Y960 in the juxtamembrane region and Y1146 in the activation loop. Among four R3 members, only Ptprj was co-expressed with the IR in major insulin target tissues, such as the skeletal muscle, liver and adipose tissue. Importantly, the activation of IR and Akt by insulin was enhanced, and glucose and insulin tolerance was improved in Ptprj -deficient mice. These results demonstrated Ptprj as a physiological enzyme that attenuates insulin signalling in vivo , and indicate that an inhibitor of Ptprj may be an insulin-sensitizing agent.

Description: The diazotrophic cyanobacterium Anabaena sp. strain PCC 7120 (A.7120) differentiates into specialized heterocyst cells that fix nitrogen under nitrogen starvation conditions. Although reducing equivalents are essential for nitrogen fixation, little is known about redox systems in heterocyst cells. In this study, we investigated thioredoxin (Trx) networks in Anabaena using TrxM, and identified 16 and 38 candidate target proteins in heterocysts and vegetative cells, respectively, by Trx affinity chromatography (Motohashi et al. (Comprehensive survey of proteins targeted by chloroplast thioredoxin. Proc Natl Acad Sci USA , 2001; 98 , 11224–11229)). Among these, the Fe–S cluster scaffold protein NifU that facilitates functional expression of nitrogenase in heterocysts was found to be a potential TrxM target. Subsequently, we observed that the scaffold activity of N-terminal catalytic domain of NifU is enhanced in the presence of Trx-system, suggesting that TrxM is involved in the Fe–S cluster biogenesis.

Description: The combination of DNA bisulfite treatment with high-throughput sequencing technologies has enabled investigation of genome-wide DNA methylation beyond CpG sites and CpG islands. These technologies have opened new avenues to understand the interplay between epigenetic events, chromatin plasticity and gene regulation. However, the processing, managing and mining of this huge volume of data require specialized computational tools and statistical methods that are yet to be standardized. Here, we describe a complete bisulfite sequencing analysis workflow, including recently developed programs, highlighting each of the crucial analysis steps required, i.e. sequencing quality control, reads alignment, methylation scoring, methylation heterogeneity assessment, genomic features annotation, data visualization and determination of differentially methylated cytosines. Moreover, we discuss the limitations of these technologies and considerations to perform suitable analyses.

Description: Understanding the genetic basis of human traits/diseases and the underlying mechanisms of how these traits/diseases are affected by genetic variations is critical for public health. Current genome-wide functional genomics data uncovered a large number of functional elements in the noncoding regions of human genome, providing new opportunities to study regulatory variants (RVs). RVs play important roles in transcription factor bindings, chromatin states and epigenetic modifications. Here, we systematically review an array of methods currently used to map RVs as well as the computational approaches in annotating and interpreting their regulatory effects, with emphasis on regulatory single-nucleotide polymorphism. We also briefly introduce experimental methods to validate these functional RVs.

Description: The detection of parent-of-origin effects aims to identify whether the functionality of alleles, and in turn associated phenotypic traits, depends on the parental origin of the alleles. Different parent-of-origin effects have been identified through a variety of mechanisms and a number of statistical methodologies for their detection have been proposed, in particular for genome-wide association studies (GWAS). GWAS have had limited success in explaining the heritability of many complex disorders and traits, but successful identification of parent-of-origin effects using trio (mother, father and offspring) GWAS may help shed light on this missing heritability. However, it is important to choose the most appropriate parent-of-origin test or methodology, given knowledge of the phenotype, amount of available data and the type of parent-of-origin effect(s) being considered. This review brings together the parent-of-origin detection methodologies available, comparing them in terms of power and type I error for a number of different simulated data scenarios, and finally offering guidance as to the most appropriate choice for the different scenarios.

Description: With the increasing recognition of its role in trait and disease development, it is crucial to account for genetic imprinting to illustrate the genetic architecture of complex traits. Genetic mapping can be innovated to test and estimate effects of genetic imprinting in a segregating population derived from experimental crosses. Here, we describe and assess a design for imprinting detection in natural plant populations. This design is to sample maternal plants at random from a natural population and collect open-pollinated (OP) seeds randomly from each maternal plant and germinate them into seedlings. A two-stage hierarchical platform is constructed to jointly analyze maternal and OP progeny markers. Through tracing the segregation and transmission of alleles from the parental to progeny generation, this platform allows parent-of-origin-dependent gene expression to be discerned, providing an avenue to estimate the effect of imprinting genes on a quantitative trait. The design is derived to estimate imprinting effects expressed at the haplotype level. Its usefulness and utilization were validated through computer simulation. This OP-based design provides a tool to detect the genomic distribution and pattern of imprinting genes as an important component of heritable variation that is neglected in traditional genetic studies of complex traits.

Description: Breast cancer was traditionally perceived as a single disease; however, recent advances in gene expression and genomic profiling have revealed that breast cancer is in fact a collection of diseases exhibiting distinct anatomical features, responses to treatment and survival outcomes. Consequently, a number of schemes have been proposed for subtyping of breast cancer to bring out the biological and clinically relevant characteristics of the subtypes. Although some of these schemes capture underlying molecular differences, others predict variations in response to treatment and survival patterns. However, despite this diversity in the approaches, it is clear that molecular mechanisms drive clinical outcomes, and therefore an effective scheme should integrate molecular as well as clinical parameters to enable deeper understanding of cancer mechanisms and allow better decision making in the clinic. Here, using a large cohort of ~550 breast tumours from The Cancer Genome Atlas, we systematically evaluate a number of expression-based schemes including at least eight molecular pathways implicated in breast cancer and three prognostic signatures, across a variety of classification scenarios covering molecular characteristics, biomarker status, tumour stages and survival patterns. We observe that a careful combination of these schemes yields better classification results compared with using them individually, thus confirming that molecular mechanisms and clinical outcomes are related and that an effective scheme should therefore integrate both these parameters to enable a deeper understanding of the cancer.

Description: microRNAs (miRNAs) are important gene regulators. They control a wide range of biological processes and are involved in several types of cancers. Thus, exploring miRNA functions is important for diagnostics and therapeutics. To date, there are few feasible experimental techniques for discovering miRNA regulatory mechanisms. Alternatively, predictions of miRNA–mRNA regulatory relationships by computational methods have increasingly achieved promising results. Computational approaches are proving their ability as effective tools in reducing the number of biological experiments that must be conducted and to assist with the design of the experiments. In this review, we categorize and review different computational approaches to identify miRNA activities and functions, including the co-regulation of miRNAs and transcription factors. Our main focuses are on the recent approaches that use multiple data types for exploring miRNA functions. We discuss the remaining challenges in the evaluation and selection of models based on the results from a case study. Finally, we analyse the remaining challenges of each computational approach and suggest some future research directions.

Description: Technological advances in next-generation sequencing have uncovered a wide spectrum of aberrations in cancer genomes. The extreme diversity in cancer mutations necessitates computational approaches to differentiate between the ‘drivers’ with vital function in cancer progression and those nonfunctional ‘passengers’. Although individual driver mutations are routinely identified, mutational profiles of different tumors are highly heterogeneous. There is growing consensus that pathways rather than single genes are the primary target of mutations. Here we review extant bioinformatics approaches to identifying oncogenic drivers at different mutational levels, highlighting the strategies for discovering driver pathways and networks from cancer mutation data. These approaches will help reduce the mutation complexity, thus providing a simplified picture of cancer.

Description: Copy number variants (CNVs) play important roles in a number of human diseases and in pharmacogenetics. Powerful methods exist for CNV detection in whole genome sequencing (WGS) data, but such data are costly to obtain. Many disease causal CNVs span or are found in genome coding regions (exons), which makes CNV detection using whole exome sequencing (WES) data attractive. If reliably validated against WGS-based CNVs, exome-derived CNVs have potential applications in a clinical setting. Several algorithms have been developed to exploit exome data for CNV detection and comparisons made to find the most suitable methods for particular data samples. The results are not consistent across studies. Here, we review some of the exome CNV detection methods based on depth of coverage profiles and examine their performance to identify problems contributing to discrepancies in published results. We also present a streamlined strategy that uses a single metric, the likelihood ratio, to compare exome methods, and we demonstrated its utility using the VarScan 2 and eXome Hidden Markov Model (XHMM) programs using paired normal and tumour exome data from chronic lymphocytic leukaemia patients. We use array-based somatic CNV (SCNV) calls as a reference standard to compute prevalence-independent statistics, such as sensitivity, specificity and likelihood ratio, for validation of the exome-derived SCNVs. We also account for factors known to influence the performance of exome read depth methods, such as CNV size and frequency, while comparing our findings with published results.

Description: In this study, we examined the role of aminopeptidases with reference to endoplasmic reticulum aminopeptidase 1 (ERAP1) in nitric oxide (NO) synthesis employing murine macrophage cell line RAW264.7 cells activated by lipopolysaccharide (LPS) and interferon (IFN)- and LPS-activated peritoneal macrophages derived from ERAP1 knockout mouse. When NO synthesis was measured in the presence of peptides having N-terminal Arg, comparative NO synthesis was seen with that measured in the presence of Arg. In the presence of an aminopeptidase inhibitor amastatin, NO synthesis in activated RAW264.7 cells was significantly decreased. These results suggest that aminopeptidases are involved in the NO synthesis in activated RAW264.7 cells. Subsequently, significant reduction of NO synthesis was observed in ERAP1 knockdown cells compared with wild-type cells. This reduction was rescued by exogenously added ERAP1. Furthermore, when peritoneal macrophages prepared from ERAP1 knockout mouse were employed, reduction of NO synthesis in knockout mouse macrophages was also attributable to ERAP1. In the presence of amastatin, further reduction was observed in knockout mouse-derived macrophages. Taken together, these results suggest that several aminopeptidases play important roles in the maximum synthesis of NO in activated macrophages in a substrate peptide-dependent manner and ERAP1 is one of the aminopeptidases involved in the NO synthesis.

Description: Glycogen phosphorylase (GP) is biologically active as a dimer of identical subunits. Each subunit has two distinct maltooligosaccharide binding sites: a storage site and a catalytic site. Our characterization of the properties of these sites suggested that GP activity consists of two activities: (i) binding to the glycogen molecule and (ii) phosphorolysis of the non-reducing-end glucose residues. Activity (i) is mainly due to the activities of the two storage sites, which depended on the ionic strength of the medium and were directly inhibited by cyclodextrins (CDs). Activity (i) is of benefit to GP because a high concentration of non-reducing-end glucose residues is localized on the surface of the glycogen molecule. Activity (ii), the total activity of the two catalytic sites, exhibited relatively little ionic strength dependence. Because the combined activity of (i) and (ii) is deduced using glycogen as an assay substrate, the sole activity of (ii) must be measured using small maltooligosyl-substrates. By using a very low concentration of pyridylaminated maltohexaose, we demonstrated that the GP catalytic sites are active even in the presence of CDs, and that the actions of the catalytic site and the storage site are independent of each other.

Description: O -GlcNAcylation is a ubiquitous, dynamic and reversible post-translational protein modification in metazoans, and it is catalysed and removed by O -GlcNAc transferase (OGT) and O -GlcNAcase, respectively. Prokaryotes lack endogenous OGT activity. It has been reported that coexpression of mammalian OGT with its target substrates in Escherichia coli produce O -GlcNAcylated recombinant proteins, but the plasmids used were not compatible, and the expression of both OGT and its target protein were induced by the same inducer. Here, we describe a compatible dual plasmid system for coexpression of OGT and its target substrate for O -GlcNAcylated protein production in E. coli . The approach was validated using the CKII and p53 protein as control. This compatible dual plasmid system contains an arabinose-inducible OGT expression vector with a pUC origin and an isopropyl β - d -thiogalactopyranoside-inducible OGT target substrate expression vector bearing a p15A origin. The dual plasmid system produces recombinant proteins with varying O -GlcNAcylation levels by altering the inducer concentration. More importantly, the O -GlcNAcylation efficiency was much higher than the previously reported system. Altogether, we established an adjustable compatible dual plasmid system that can effectively yield O -GlcNAcylated proteins in E. coli .

Description: Active equi-paritioning of the F plasmid is achieved by its sopABC gene. SopA binds to the sopAB promoter region and SopB binds to sopC . SopA also polymerizes in the presence of ATP and Mg(II), which is stimulated by SopB. Non-specific DNA is known to inhibit SopA polymerization and disassemble SopA polymer. This study followed kinetics of polymerization and de-polymerization of SopA by turbidity measurement and found new effects by DNA and SopB. Plasmid DNA, at low concentrations, shortened the lag (nucleation) phase of SopA polymerization and also caused an initial ‘burst’ of turbidity. Results with two non-specific 20-bp DNAs indicated sequence/length dependence of these effects. sopAB operator DNA only showed inhibition of SopA polymerization. Results of turbidity decrease of pre-formed SopA polymer in the presence of ethylenediaminetetraacetic acid showed that SopB also accelerates disassembly of the SopA polymer. The steady-state level of turbidity in the presence of SopB and plasmid DNA indicated synergy between SopB and DNA in the disassembly. SopB protein showed no effect on SopA polymerization, when SopB was specifically bound to DNA. This result and others with truncation mutants of SopB suggested that a proper configuration of the domains of SopB is important for SopA-SopB interactions.

Description: Influenza A virus (IAV) has been raising public health and safety concerns worldwide. Cyanovirin-N (CVN) is a prominent anti-IAV candidate, but both cytotoxicity and immunogenicity have hindered the development of this protein as a viable therapy. In this article, linker-CVN (LCVN) with a flexible and hydrophilic polypeptide at the N-terminus was efficiently produced from the cytoplasm of Escherichia coli at a 〉15-l scale. PEGylation at the N-terminal α-amine of LCVN was also reformed as 20 kDa PEGylated linkered Cyanovirin-N (PEG 20k –LCVN). The 50% effective concentrations of PEG 20k –LCVN were 0.43 ± 0.11 µM for influenza A/HK/8/68 (H3N2) and 0.04 ± 0.02 µM for A/Swan/Hokkaido/51/96 (H5N3), dramatically lower than that of the positive control, Ribavirin (2.88 ± 0.66 x 10 3 µM and 1.79 ± 0.62 x 10 3 µM, respectively). A total of 12.5 µM PEG 20k –LCVN effectively inactivate the propagation of H3N2 in chicken embryos. About 2.0 mg/kg/day PEG 20k –LCVN increased double the survival rate (66.67%, P = 0.0378) of H3N2 infected mice, prolonged the median survival period, downregulated the mRNA level of viral nuclear protein and decreased (attenuated) the pathology lesion in mice lung. A novel PEGylated CVN derivative, PEG 20k –LCVN, exhibited potent and strain-dependent anti-IAV activity in nanomolar concentrations in vitro, as well as in micromolar concentration in vivo .

Description: The semi-filamentous multicellular cyanobacterium Limnothrix / Pseudanabaena sp. strain ABRG5-3 undergoes autolysis, which involves the accumulation of polyphosphate compounds and disintegration of thylakoid membranes in cells, as a unique feature that occurs due to growth conditions. In this study, the overexpression and easy recovery of alkane (a saturated hydrocarbon, C 17 H 36 ) as a biofuel were examined in recombinants of the cyanobacteria ABRG5-3 and Synechocystis sp. strain PCC6803. The results obtained indicated that the accumulated mass of alkane accounted for ~50 or 60% of the dry weight of ABRG5-3 or PCC6803 recombinant cells, respectively. Furthermore, cultivating cells in liquid medium BG11 in which the nitrogen resource had been depleted promoted the production of alkane and cell lysis, resulting in the easy recovery of target products from the supernatant.

Description: Leucine-rich repeat kinase 2 (LRRK2) has been identified as a causative gene for Parkinson’s disease (PD). LRRK2 contains a kinase and a GTPase domain, both of which provide critical intracellular signal-transduction functions. We showed previously that Rab5b, a small GTPase protein that regulates the motility and fusion of early endosomes, interacts with LRRK2 and co-regulates synaptic vesicle endocytosis. Using recombinant proteins, we show here that LRRK2 phosphorylates Rab5b at its Thr6 residue in in vitro kinase assays with mass spectrophotometry analysis. Phosphorylation of Rab5b by LRRK2 on the threonine residue was confirmed by western analysis using cells stably expressing LRRK2 G2019S. The phosphomimetic T6D mutant exhibited stronger GTPase activity than that of the wild-type Rab5b. In addition, phosphorylation of Rab5b by LRRK2 also exhibited GTPase activity stronger than that of the unphosphorylated Rab5b protein. Two assays testing Rab5’s activity, neurite outgrowth analysis and epidermal growth factor receptor degradation assays, showed that Rab5b T6D exhibited phenotypes that were expected to be observed in the inactive Rab5b, including longer neurite length and less degradation of EGFR. These results suggest that LRRK2 kinase activity functions as a Rab5b GTPase activating protein and thus, negatively regulates Rab5b signalling.

Description: The cellular Src (c-Src) tyrosine kinase is upregulated and believed to play a pivotal role in various human cancers. However, the molecular mechanism underlying c-Src-mediated tumour progression remains elusive. Recent studies have revealed that several microRNAs (miRNAs) function as tumour suppressors by regulating the malignant expression of signalling molecules. Aberrant expression of miRNAs is frequently observed in human cancers and should be exploited to seek related molecular targets. In this review, we focus on miRNAs found to be involved in Src signalling in various cancers. We summarize recent findings on Src-related miRNAs, their target genes, mechanisms behind their interplay and their implications for cancer therapeutics.

Description: Tail-anchored (TA) proteins, a class of membrane proteins having an N-terminal cytoplasmic region anchored to the membrane by a single C-terminal transmembrane domain, are posttranslationally inserted into the endoplasmic reticulum (ER) membrane. In yeasts, the posttranslational membrane insertion is mediated by the Guided Entry of TA Proteins (GET) complex. Get3, a cytosolic ATPase, targets newly synthesized TA proteins to the ER membrane, where Get2 and Get3 constitute the Get3 receptor driving the membrane insertion. While mammalian cells employ TRC40 and WRB, mammalian homologs of Get3 and Get1, respectively, they lack the gene homologous to Get2. We recently identified calcium-modulating cyclophilin ligand (CAML) as a TRC40 receptor, indicating that CAML was equivalent to Get2 in the context of the membrane insertion. On the other hand, CAML has been well characterized as a signaling molecule that regulates various biological processes, raising the question of how the two distinct actions of CAML, the membrane insertion and the signal transduction, are assembled. In this review, we summarize recent progress of the molecular mechanism of the membrane insertion of TA proteins and discuss the possibility that CAML could sense the various signals at the ER membrane, thereby controlling TA protein biogenesis.

Description: In bacterial organisms, the oriC -independent primosome plays an essential role in replication restart after dissociation of the replication DNA-protein complex following DNA damage. PriC is a key protein component in the oriC -independent replication restart primosome. Our previous study suggested that PriC was divided into an N-terminal domain and a C-terminal domain, with the latter domain being the major contributor to single-stranded DNA (ssDNA) binding capacity. In this study, we prepared several PriC mutants in which basic and aromatic amino acid residues were mutated to alanine. Five of these residues, Arg107, Lys111, Phe118, Arg121 and Lys165 in the C-terminal domain, were shown to be involved in ssDNA binding. Moreover, we evaluated the binding of the PriC mutants to the ssDNA-binding protein (SSB) complex. Five residues, Phe118, Arg121, Arg129, Tyr152 and Arg155 in the C-terminal domain of PriC, were shown to be involved in SSB binding in the presence of ssDNA. On the basis of these results, we propose a structural model of the C-terminal domain of PriC and discuss how the interactions of PriC with SSB and ssDNA may contribute to the regulation of PriC-dependent replication restart.

Description: L -Lysine α-oxidase (LysOX) from Trichoderma viride is a homodimeric 112 kDa flavoenzyme that catalyzes the oxidative deamination of L -lysine to form α-keto--aminocaproate. LysOX severely inhibited growth of cancer cells but showed relatively low cytotoxicity for normal cells. We have determined the cDNA nucleotide sequence encoding LysOX from T. viride. The full-length cDNA consists of 2,119 bp and encodes a possible signal peptide (Met1-Arg77) and the mature protein (Ala78-Ile617). The LysOX gene have been cloned and heterologously expressed in Streptomyces lividans TK24 with the enzyme activity up to 9.8 U/ml. The enzymatic properties of the purified recombinant LysOX, such as substrate specificity and thermal stability, are same as those of native LysOX. The crystal structure of LysOX at 1.9 Å resolution revealed that the overall structure is similar to that of snake venom L -amino acid oxidase (LAAO), and the residues involved in the interaction with the amino or carboxy group of the substrate are structurally conserved. However, the entrance and the inner surface structures of the funnel to the active site, as well as the residues involved in the substrate side-chain recognition, are distinct from LAAOs. These structural differences well explain the unique substrate specificity of LysOX.

Description: For a multistep pre-targeting method using antibodies, a streptavidin mutant with low immunogenicity, termed low immunogenic streptavidin mutant No. 314 (LISA-314), was produced previously as a drug delivery tool. However, endogenous biotins (BTNs) with high affinity ( K d 〈 10 –10 M) for the binding pocket of LISA-314 prevents access of exogenous BTN-labelled anticancer drugs. In this study, we improve the binding pocket of LISA-314 to abolish its affinity for endogenous BTN species, therefore ensuring that the newly designed LISA-314 binds only artificial BTN analogue. The replacement of three amino acid residues was performed in two steps to develop a mutant termed V212, which selectively binds to 6-(5-((3a S ,4 S ,6a R )-2-iminohexahydro-1 H -thieno[3,4- d ]imidazol-4-yl)pentanamido)hexanoic acid (iminobiotin long tail, IMNtail). Surface plasmon resonance results showed that V212 has a K d value of 5.9 x 10 –7 M towards IMNtail, but no binding affinity for endogenous BTN species. This V212/IMNtail system will be useful as a novel delivery tool for anticancer therapy.

Description: A number of gene mutations are detected in cells derived from human cancer tissues, but roles of these mutations in cancer cell development are largely unknown. We examined G364R mutation of MCM4 detected in human skin cancer cells. Formation of MCM4/6/7 complex is not affected by the mutation. Consistent with this notion, the binding to MCM6 is comparable between the mutant MCM4 and wild-type MCM4. Nuclear localization of this mutant MCM4 expressed in HeLa cells supports this conclusion. Purified MCM4/6/7 complex containing the G364R MCM4 exhibited similar levels of single-stranded DNA binding and ATPase activities to the complex containing wild-type MCM4. However, the mutant complex showed only 30–50% of DNA helicase activity of the wild-type complex. When G364R MCM4 was expressed in HeLa cells, it was fractionated into nuclease-sensitive chromatin fraction, similar to wild-type MCM4. These results suggest that this mutation does not affect assembly of MCM2-7 complex on replication origins but it interferes some step at function of MCM2-7 helicase. Thus, this mutation may contribute to cancer cell development by disturbing DNA replication.

Description: The detailed, atomistic-level understanding of molecular signaling along the tumor-suppressive Hippo signaling pathway that controls tissue homeostasis by balancing cell proliferation and death through apoptosis is a promising avenue for the discovery of novel anticancer drug targets. The activation of kinases such as Mammalian STE20-Like Protein Kinases 1 and 2 (MST1 and MST2)—modulated through both homo- and heterodimerization (e.g. interactions with Ras association domain family, RASSF, enzymes)—is a key upstream event in this pathway and remains poorly understood. On the other hand, RASSFs (such as RASSF1A or RASSF5) act as important apoptosis activators and tumor suppressors, although their exact regulatory roles are also unclear. We present recent molecular studies of signaling along the Ras-RASSF-MST pathway, which controls growth and apoptosis in eukaryotic cells, including a variety of modern molecular modeling and simulation techniques. Using recently available structural information, we discuss the complex regulatory scenario according to which RASSFs perform dual signaling functions, either preventing or promoting MST2 activation, and thus control cell apoptosis. Here, we focus on recent studies highlighting the special role being played by the specific interactions between the helical Salvador/RASSF/Hippo (SARAH) domains of MST2 and RASSF1a or RASSF5 enzymes. These studies are crucial for integrating atomistic-level mechanistic information about the structures and conformational dynamics of interacting proteins, with information available on their system-level functions in cellular signaling.

Description: Big-data-based edge biomarker is a new concept to characterize disease features based on biomedical big data in a dynamical and network manner, which also provides alternative strategies to indicate disease status in single samples. This article gives a comprehensive review on big-data-based edge biomarkers for complex diseases in an individual patient, which are defined as biomarkers based on network information and high-dimensional data. Specifically, we firstly introduce the sources and structures of biomedical big data accessible in public for edge biomarker and disease study. We show that biomedical big data are typically ‘small-sample size in high-dimension space', i.e. small samples but with high dimensions on features (e.g. omics data) for each individual, in contrast to traditional big data in many other fields characterized as ‘large-sample size in low-dimension space', i.e. big samples but with low dimensions on features. Then, we demonstrate the concept, model and algorithm for edge biomarkers and further big-data-based edge biomarkers. Dissimilar to conventional biomarkers, edge biomarkers, e.g. module biomarkers in module network rewiring-analysis, are able to predict the disease state by learning differential associations between molecules rather than differential expressions of molecules during disease progression or treatment in individual patients. In particular, in contrast to using the information of the common molecules or edges (i.e.molecule-pairs) across a population in traditional biomarkers including network and edge biomarkers, big-data-based edge biomarkers are specific for each individual and thus can accurately evaluate the disease state by considering the individual heterogeneity. Therefore, the measurement of big data in a high-dimensional space is required not only in the learning process but also in the diagnosing or predicting process of the tested individual. Finally, we provide a case study on analyzing the temporal expression data from a malaria vaccine trial by big-data-based edge biomarkers from module network rewiring-analysis. The illustrative results show that the identified module biomarkers can accurately distinguish vaccines with or without protection and outperformed previous reported gene signatures in terms of effectiveness and efficiency.

Description: Molecular interrogation of a biological sample through DNA sequencing, RNA and microRNA profiling, proteomics and other assays, has the potential to provide a systems level approach to predicting treatment response and disease progression, and to developing precision therapies. Large publicly funded projects have generated extensive and freely available multi-assay data resources; however, bioinformatic and statistical methods for the analysis of such experiments are still nascent. We review multi-assay genomic data resources in the areas of clinical oncology, pharmacogenomics and other perturbation experiments, population genomics and regulatory genomics and other areas, and tools for data acquisition. Finally, we review bioinformatic tools that are explicitly geared toward integrative genomic data visualization and analysis. This review provides starting points for accessing publicly available data and tools to support development of needed integrative methods.

Description: One of the major challenges in biology concerns the integration of data across length and time scales into a consistent framework: how do macroscopic properties and functionalities arise from the molecular regulatory networks—and how can they change as a result of mutations? Morphogenesis provides an excellent model system to study how simple molecular networks robustly control complex processes on the macroscopic scale despite molecular noise, and how important functional variants can emerge from small genetic changes. Recent advancements in three-dimensional imaging technologies, computer algorithms and computer power now allow us to develop and analyse increasingly realistic models of biological control. Here, we present our pipeline for image-based modelling that includes the segmentation of images, the determination of displacement fields and the solution of systems of partial differential equations on the growing, embryonic domains. The development of suitable mathematical models, the data-based inference of parameter sets and the evaluation of competing models are still challenging, and current approaches are discussed.

Description: Owing greatly to the advancement of next-generation sequencing (NGS), the amount of NGS data is increasing rapidly. Although there are many NGS applications, one of the most commonly used techniques ‘RNA sequencing (RNA-seq)’ is rapidly replacing microarray-based techniques in laboratories around the world. As more and more of such techniques are standardized, allowing technicians to perform these experiments with minimal hands-on time and reduced experimental/operator-dependent biases, the bottleneck of such techniques is clearly visible; that is, data analysis. Further complicating the matter, increasing evidence suggests most of the genome is transcribed into RNA; however, the majority of these RNAs are not translated into proteins. These RNAs that do not become proteins are called ‘noncoding RNAs (ncRNAs)’. Although some time has passed since the discovery of ncRNAs, their annotations remain poor, making analysis of RNA-seq data challenging. Here, we examine the current limitations of RNA-seq analysis using case studies focused on the detection of novel transcripts and examination of their characteristics. Finally, we validate the presence of novel transcripts using biological experiments, showing novel transcripts can be accurately identified when a series of filters is applied. In conclusion, novel transcripts that are identified from RNA-seq must be examined carefully before proceeding to biological experiments.

Description: Functional genomics has enormous potential to facilitate our understanding of normal and disease-specific physiology. In the past decade, intensive research efforts have been focused on modeling functional relationship networks, which summarize the probability of gene co-functionality relationships. Such modeling can be based on either expression data only or heterogeneous data integration. Numerous methods have been deployed to infer the functional relationship networks, while most of them target the global (non-context-specific) functional relationship networks. However, it is expected that functional relationships consistently reprogram under different tissues or biological processes. Thus, advanced methods have been developed targeting tissue-specific or developmental stage-specific networks. This article brings together the state-of-the-art functional relationship network modeling methods, emphasizes the need for heterogeneous genomic data integration and context-specific network modeling and outlines future directions for functional relationship networks.

Description: Identification of drug–target interactions is an important process in drug discovery. Although high-throughput screening and other biological assays are becoming available, experimental methods for drug–target interaction identification remain to be extremely costly, time-consuming and challenging even nowadays. Therefore, various computational models have been developed to predict potential drug–target associations on a large scale. In this review, databases and web servers involved in drug–target identification and drug discovery are summarized. In addition, we mainly introduced some state-of-the-art computational models for drug–target interactions prediction, including network-based method, machine learning-based method and so on. Specially, for the machine learning-based method, much attention was paid to supervised and semi-supervised models, which have essential difference in the adoption of negative samples. Although significant improvements for drug–target interaction prediction have been obtained by many effective computational models, both network-based and machine learning-based methods have their disadvantages, respectively. Furthermore, we discuss the future directions of the network-based drug discovery and network approach for personalized drug discovery based on personalized medicine, genome sequencing, tumor clone-based network and cancer hallmark-based network. Finally, we discussed the new evaluation validation framework and the formulation of drug–target interactions prediction problem by more realistic regression formulation based on quantitative bioactivity data.

Description: Atherosclerosis is one of the principle pathologies of cardiovascular disease with blood cholesterol a significant risk factor. The World Health Organization estimates that approximately 2.5 million deaths occur annually because of the risk from elevated cholesterol, with 39% of adults worldwide at future risk. Atherosclerosis emerges from the combination of many dynamical factors, including haemodynamics, endothelial damage, innate immunity and sterol biochemistry. Despite its significance to public health, the dynamics that drive atherosclerosis remain poorly understood. As a disease that depends on multiple factors operating on different length scales, the natural framework to apply to atherosclerosis is mathematical and computational modelling. A computational model provides an integrated description of the disease and serves as an in silico experimental system from which we can learn about the disease and develop therapeutic hypotheses. Although the work completed in this area to date has been limited, there are clear signs that interest is growing and that a nascent field is establishing itself. This article discusses the current state of modelling in this area, bringing together many recent results for the first time. We review the work that has been done, discuss its scope and highlight the gaps in our understanding that could yield future opportunities.

Description: We present Bioinformatics Autodiscovery of Training Materials (BATMat), an open-source, Google-based, targeted, automatic search tool for training materials related to bioinformatics. BATMat helps gain access with one click to filtered and portable information containing links to existing materials (when present). It also offers functionality to sort results according to source site or title. Availability: http://imbatmat.com Contact: piar301@gmail.com

Description: Cancer is often driven by the accumulation of genetic alterations, including single nucleotide variants, small insertions or deletions, gene fusions, copy-number variations, and large chromosomal rearrangements. Recent advances in next-generation sequencing technologies have helped investigators generate massive amounts of cancer genomic data and catalog somatic mutations in both common and rare cancer types. So far, the somatic mutation landscapes and signatures of 〉10 major cancer types have been reported; however, pinpointing driver mutations and cancer genes from millions of available cancer somatic mutations remains a monumental challenge. To tackle this important task, many methods and computational tools have been developed during the past several years and, thus, a review of its advances is urgently needed. Here, we first summarize the main features of these methods and tools for whole-exome, whole-genome and whole-transcriptome sequencing data. Then, we discuss major challenges like tumor intra-heterogeneity, tumor sample saturation and functionality of synonymous mutations in cancer, all of which may result in false-positive discoveries. Finally, we highlight new directions in studying regulatory roles of noncoding somatic mutations and quantitatively measuring circulating tumor DNA in cancer. This review may help investigators find an appropriate tool for detecting potential driver or actionable mutations in rapidly emerging precision cancer medicine.

Description: Recent literature has highlighted the advantages of haplotype association methods for detecting rare variants associated with common diseases. As several new haplotype association methods have been proposed in the past few years, a comparison of new and standard methods is important and timely for guidance to the practitioners. We consider nine methods—Haplo.score, Haplo.glm, Hapassoc, Bayesian hierarchical Generalized Linear Model (BhGLM), Logistic Bayesian LASSO (LBL), regularized GLM (rGLM), Haplotype Kernel Association Test, wei-SIMc-matching and Weighted Haplotype and Imputation-based Tests. These can be divided into two types—individual haplotype-specific tests and global tests depending on whether there is just one overall test for a haplotype region (global) or there is an individual test for each haplotype in the region. Haplo.score is the only method that tests for both; Haplo.glm, Hapassoc, BhGLM and LBL are individual haplotype-specific, while the rest are global tests. For comparison, we also apply a popular collapsing method—Sequence Kernel Association Test (SKAT) and its two variants—SKAT-O (Optimal) and SKAT-C (Combined). We carry out an extensive comparison on our simulated data sets as well as on the Genetic Analysis Workshop (GAW) 18 simulated data. Further, we apply the methods to GAW18 real hypertension data and Dallas Heart Study sequence data. We find that LBL, Haplo.score (global test) and rGLM perform well over the scenarios considered here. Also, haplotype methods are more powerful (albeit more computationally intensive) than SKAT and its variants in scenarios where multiple causal variants act interactively to produce haplotype effects.

Description: The purpose of this article is to inform readers about technical challenges that we encountered when assembling exome sequencing data from the ‘Simplifying Complex Exomes' (SIMPLEXO) consortium—whose mandate is the discovery of novel genes predisposing to breast and ovarian cancers. Our motivation is to share these obstacles—and our solutions to them—as a means of communicating important technical details that should be discussed early in projects involving massively parallel sequencing.

Description: Predictive, preventive, personalized and participatory (P4) medicine is an emerging medical model that is based on the customization of all medical aspects (i.e. practices, drugs, decisions) of the individual patient. P4 medicine presupposes the elucidation of the so-called omic world, under the assumption that this knowledge may explain differences of patients with respect to disease prevention, diagnosis and therapies. Here, we elucidate the role of some selected omics sciences for different aspects of disease management, such as early diagnosis of diseases, prevention of diseases, selection of personalized appropriate and optimal therapies based on molecular profiling of patients. After introducing basic concepts of P4 medicine and omics sciences, we review some computational tools and approaches for analysing selected omics data, with a special focus on microarray and mass spectrometry data, which may be used to support P4 medicine. Some applications of biomarker discovery and pharmacogenomics and some experiences on the study of drug reactions are also described.

Description: A wide variety of large-scale data have been produced in bioinformatics. In response, the need for efficient handling of biomedical big data has been partly met by parallel computing. However, the time demand of many bioinformatics programs still remains high for large-scale practical uses because of factors that hinder acceleration by parallelization. Recently, new generations of storage devices have emerged, such as NAND flash-based solid-state drives (SSDs), and with the renewed interest in near-data processing, they are increasingly becoming acceleration methods that can accompany parallel processing. In certain cases, a simple drop-in replacement of hard disk drives by SSDs results in dramatic speedup. Despite the various advantages and continuous cost reduction of SSDs, there has been little review of SSD-based profiling and performance exploration of important but time-consuming bioinformatics programs. For an informative review, we perform in-depth profiling and analysis of 23 key bioinformatics programs using multiple types of devices. Based on the insight we obtain from this research, we further discuss issues related to design and optimize bioinformatics algorithms and pipelines to fully exploit SSDs. The programs we profile cover traditional and emerging areas of importance, such as alignment, assembly, mapping, expression analysis, variant calling and metagenomics. We explain how acceleration by parallelization can be combined with SSDs for improved performance and also how using SSDs can expedite important bioinformatics pipelines, such as variant calling by the Genome Analysis Toolkit and transcriptome analysis using RNA sequencing. We hope that this review can provide useful directions and tips to accompany future bioinformatics algorithm design procedures that properly consider new generations of powerful storage devices.

Description: Importin α performs the indispensable role of ferrying proteins from the cytoplasm into the nucleus with a transport carrier, importin β1. Mammalian cells from mouse or human contain either six or seven importin α subtypes, respectively, each with a tightly regulated expression. Therefore, the combination of subtype expression in a cell defines distinct signaling pathways to achieve progressive changes in gene expression essential for cellular events, such as differentiation. Recent studies reveal that, in addition to nucleocytoplasmic transport, importin αs also serve non-transport functions. In this review, we first discuss the physiological significance of importin α as a nuclear transport regulator, and then focus on the functional diversities of importin αs based on their specific subcellular and cellular localizations, such as the nucleus and plasma membrane. These findings enrich our knowledge of how importin αs actively contribute to various cellular events.

Description: The structure of the complex of maize sulfite reductase (SiR) and ferredoxin (Fd) has been determined by X-ray crystallography. Co-crystals of the two proteins prepared under different conditions were subjected to the diffraction analysis and three possible structures of the complex were solved. Although topological relationship of SiR and Fd varied in each of the structures, two characteristics common to all structures were found in the pattern of protein-protein interactions and positional arrangements of redox centres; (i) a few negative residues of Fd contact with a narrow area of SiR with positive electrostatic surface potential and (ii) [2Fe-2S] cluster of Fd and [4Fe-4S] cluster of SiR are in a close proximity with the shortest distance around 12 Å. Mutational analysis of a total of seven basic residues of SiR distributed widely at the interface of the complex showed their importance for supporting an efficient Fd-dependent activity and a strong physical binding to Fd. These combined results suggest that the productive electron transfer complex of SiR and Fd could be formed through multiple processes of the electrostatic intermolecular interaction and this implication is discussed in terms of the multi-functionality of Fd in various redox metabolisms.

Description: Analysis of replicating mammalian mitochondrial DNA (mtDNA) suggested that initiation of the replication occurs not only at the specific position, Ori-H but also across a broad zone in mtDNA. We investigated relationship of mitochondrial transcription initiation which takes place upstream of Ori-H and mtDNA replication initiation through analysing the effect of knockdown of mitochondrial transcription factor B2, TFB2M and mitochondrial RNA polymerase, POLRMT, components of the transcription initiation complexes in cultured human cells. Under the conditions where suppression of the transcription initiation complexes was achieved by simultaneous depletion of TFB2M and POLRMT, decrease of replication intermediates of mtDNA RITOLS replication mode accompanied reduction in mtDNA copy number. On the other hand, replication intermediates of coupled leading and lagging strand DNA replication, another proposed replication mode, appeared to be less affected. The findings support the view that the former mode involves transcription from the light strand promoter (LSP), and suggest that initiation of the latter mode is independent from the transcription and has distinct regulation. Further, knockdown of TFB2M alone caused significant decrease of 7S DNA, which implies that transcription initiation complexes formed at the LSP engage 7S DNA synthesis more frequently than the initiation of productive replication and transcription.

Description: Cycas revoluta leaf lectin (CRLL) of mannose-recognizing jacalin-related lectin (mJRL) has two tandem repeated carbohydrate recognition domains, and shows the characteristic sugar-binding specificity toward high mannose-glycans, compared with other mJRLs. We expressed the N-terminal domain and C-terminal domain (CRLL-N and CRLL-C) separately, to determine the fine sugar-binding specificity of each domain, using frontal affinity chromatography, glycan array and equilibrium dialysis. The specificity of CRLL toward high mannose was basically derived from CRLL-N, whereas CRLL-C had affinity for α1-6 extended mono-antennary complex-type glycans. Notably, the affinity of CRLL-N was most potent to one of three Man 8 glycans and Man 9 glycan, whereas the affinity of CRLL-C decreased with the increase in the number of extended α1-2 linked mannose residue. The recognition of the Man 8 glycans by CRLL-N has not been found for other mannose recognizing lectins. Glycan array reflected these specificities of the two domains. Furthermore, it was revealed by equilibrium dialysis method that the each domain had two sugar-binding sites, similar with Banlec, banana mannose-binding Jacalin-related lectin.

Description: State-of-the-art next-generation sequencing, transcriptomics, proteomics and other high-throughput ‘omics' technologies enable the efficient generation of large experimental data sets. These data may yield unprecedented knowledge about molecular pathways in cells and their role in disease. Dimension reduction approaches have been widely used in exploratory analysis of single omics data sets. This review will focus on dimension reduction approaches for simultaneous exploratory analyses of multiple data sets. These methods extract the linear relationships that best explain the correlated structure across data sets, the variability both within and between variables (or observations) and may highlight data issues such as batch effects or outliers. We explore dimension reduction techniques as one of the emerging approaches for data integration, and how these can be applied to increase our understanding of biological systems in normal physiological function and disease.

Description: There is much interest in using high-throughput DNA sequencing methodology to monitor microorganisms, complex plant and animal communities. However, there are experimental and analytical issues to consider before applying a sequencing technology, which was originally developed for genome projects, to ecological projects. Many of these issues have been highlighted by recent microbial studies. Understanding how high-throughput sequencing is best implemented is important for the interpretation of recent results and the success of future applications. Addressing complex biological questions with metagenomics requires the interaction of researchers who bring different skill sets to problem solving. Educators can help by nurturing a collaborative interdisciplinary approach to genome science, which is essential for effective problem solving. Educators are in a position to help students, teachers, the public and policy makers interpret the new knowledge that metagenomics brings. To do this, they need to understand, not only the excitement of the science but also the pitfalls and shortcomings of methodology and research designs. We review these issues and some of the research directions that are helping to move the field forward.

Description: We believe that undergraduate biology students must acquire a foundational background in computing including how to formulate a computational problem; develop an algorithmic solution; implement their solution in software and then test, document and use their code to explore biological phenomena. Moreover, by learning these skills in the first year, students acquire a powerful tool set that they can use and build on throughout their studies. To address this need, we have developed a first-year undergraduate course that teaches students the foundations of computational thinking and programming in the context of problems in biology. This article describes the structure and content of the course and summarizes assessment data on both affective and learning outcomes.

Description: Bioinformatics is an integral part of modern life sciences. It has revolutionized and redefined how research is carried out and has had an enormous impact on biotechnology, medicine, agriculture and related areas. Yet, it is only rarely integrated into high school teaching and learning programs, playing almost no role in preparing the next generation of information-oriented citizens. Here, we describe the design principles of bioinformatics learning environments, including our own, that are aimed at introducing bioinformatics into senior high school curricula through engaging learners in scientifically authentic inquiry activities. We discuss the bioinformatics-related benefits and challenges that high school teachers and students face in the course of the implementation process, in light of previous studies and our own experience. Based on these lessons, we present a new approach for characterizing the questions embedded in bioinformatics teaching and learning units, based on three criteria: the type of domain-specific knowledge required to answer each question (declarative knowledge, procedural knowledge, strategic knowledge, situational knowledge), the scientific approach from which each question stems (biological, bioinformatics, a combination of the two) and the associated cognitive process dimension (remember, understand, apply, analyze, evaluate, create). We demonstrate the feasibility of this approach using a learning environment, which we developed for the high school level, and suggest some of its implications. This review sheds light on unique and critical characteristics related to broader integration of bioinformatics in secondary education, which are also relevant to the undergraduate level, and especially on curriculum design, development of suitable learning environments and teaching and learning processes.

Description: Next-generation sequencing (NGS) is increasingly being adopted as the backbone of biomedical research. With the commercialization of various affordable desktop sequencers, NGS will be reached by increasing numbers of cellular and molecular biologists, necessitating community consensus on bioinformatics protocols to tackle the exponential increase in quantity of sequence data. The current resources for NGS informatics are extremely fragmented. Finding a centralized synthesis is difficult. A multitude of tools exist for NGS data analysis; however, none of these satisfies all possible uses and needs. This gap in functionality could be filled by integrating different methods in customized pipelines, an approach helped by the open-source nature of many NGS programmes. Drawing from community spirit and with the use of the Wikipedia framework, we have initiated a collaborative NGS resource: The NGS WikiBook. We have collected a sufficient amount of text to incentivize a broader community to contribute to it. Users can search, browse, edit and create new content, so as to facilitate self-learning and feedback to the community. The overall structure and style for this dynamic material is designed for the bench biologists and non-bioinformaticians. The flexibility of online material allows the readers to ignore details in a first read, yet have immediate access to the information they need. Each chapter comes with practical exercises so readers may familiarize themselves with each step. The NGS WikiBook aims to create a collective laboratory book and protocol that explains the key concepts and describes best practices in this fast-evolving field.

Description: A requirement for advancing antibody-based medicine is the development of proteins that can bind with high affinity to a specific epitope related to a critical protein activity site. As a part of generating such proteins, we have succeeded in creating a binding protein without changing epitope by complementarity-determining region 3 (CDR3) grafting (Inoue et al. , Affinity transfer to a human protein by CDR3 grafting of camelid VHH. Protein Sci. 20, 1971–1981). However, the affinity of the target-binding protein was low. In this manuscript, the affinity maturation of a target-binding protein was examined using CDR3-grafted camelid single domain antibody (VHH) as a model protein. Several amino acids in the CDR1 and CDR2 regions of VHH were mutated to tyrosines and/or serines and screened for affinity-matured proteins by using in silico analysis. The mutation of two amino acids in the CDR2 region to arginine and/or aspartic acid increased the affinity by decreasing the dissociation rate. The affinity of designed mutant increased by ~20-fold over that of the original protein. In the present study, candidate mutants were narrowed down using in silico screening and computational modelling, thus avoiding much in vitro analytical effort. Therefore, the method used in this study is expected to be one of the useful for promoting affinity maturation of antibodies.

Description: A method was previously established for evaluating Asn deamidation by matrix-assisted laser desorption/ionization time of flight-mass spectrometry using endoproteinase Asp-N. In this study, we demonstrated that this method could be applied to the identification of the deamidation site of the humanized fragment antigen-binding (Fab). First, a system for expressing humanized Fab from methylotrophic yeast Pichia pastoris was constructed, resulting in the preparation of ~30 mg of the purified humanized Fab from 1 l culture. Analysis of the L-chain derived from recombinant humanized Fab that was heated at pH 7 and 100°C for 1 h showed the deamidation at Asn138 in the constant region. Then, we prepared L-N138D Fab and L-N138A Fab and examined their properties. The circular dichroism (CD) spectrum of the L-N138D Fab was partially different from that of the wild-type Fab. The measurement of the thermostability showed that L-N138D caused a significant decrease in the thermostability of Fab. On the other hand, the CD spectrum and thermostability of L-N138A Fab showed the same behaviour as the wild-type Fab. Thus, it was suggested that the introduction of a negative charge at position 138 in the L-chain by the deamidation significantly affected the stability of humanized Fab.

Description: The tripartite motif (TRIM) or RBCC proteins are characterized by the TRIM composed of a RING finger, B-box and coiled-coil domains. TRIM proteins often play roles in the post-translational protein modification, including ubiquitylation and other ubiquitin-like modifications. Evidence has accumulated in regard to the contribution of TRIM proteins to diverse cellular processes, including such as cell cycle progression, apoptosis, immunity and transcriptional regulation. In particular, some of the TRIM proteins have been characterized to exert oncogenic or tumour suppressor-like functions depending on the context. A recent report by Inoue and his colleagues has revealed that Terf/TRIM17 stimulates the degradation of a kinetochore protein ZWINT and regulates the proliferation of breast cancer cells. Terf has also been paid attention as a factor promoting neuronal apoptosis, by degrading a Bcl2-like anti-apoptotic protein Mcl-1. Like aircraft trim tabs, TRIM proteins trim the balance of homoeostasis by modulating various biological pathways through protein–protein interactions.

Description: Cyclin-dependent kinase (CDK) that plays a central role in preventing re-replication of DNA phosphorylates several replication proteins to inactivate them. MCM4 in MCM2-7 and RPA2 in RPA are phosphorylated with CDK in vivo . There are inversed correlations between the phosphorylation of these proteins and their chromatin binding. Here, we examined in vitro phosphorylation of human replication proteins of MCM2-7, RPA, TRESLIN, CDC45 and RECQL4 with CDK2/cyclinE, CDK2/cyclinA, CDK1/cyclinB, CHK1, CHK2 and CDC7/DBF4 kinases. MCM4, RPA2, TRESLIN and RECQL4 were phosphorylated with CDKs. Effect of the phosphorylation by CDK2/cyclinA on DNA-binding abilities of MCM2-7 and RPA was examined by gel-shift analysis. The phosphorylation of RPA did not affect its DNA-binding ability but that of MCM4 inhibited the ability of MCM2-7. Change of six amino acids of serine and threonine to alanines in the amino-terminal region of MCM4 rendered the mutant MCM2-7 insensitive to the inhibition with CDK. These biochemical data suggest that phosphorylation of MCM4 at these sites by CDK plays a direct role in dislodging MCM2-7 from chromatin and/or preventing re-loading of the complex to chromatin.

Description: To determine the effects of alcohols on the low-frequency local motions that control slow changes in structural dynamics of native-like compact states of proteins, we have studied the effects of alcohols on structural fluctuation of M80-containing -loop by measuring the rate of thermally driven CO dissociation from a natively folded carbonmonoxycytochrome c under varying concentrations of alcohols (methanol, ethanol, 1-propanol, 2-propanol, 3°-butanol, 2,2,2-trifluoroethanol). As alcohol is increased, the rate coefficient of CO dissociation ( k diss ) first decreases in subdenaturing region and then increases on going from subdenaturing to denaturing milieu. This decrease in k diss is more for 2,2,2-trifluroethanol and 1-propanol and least for methanol, indicating that the first phase of motional constraint is due to the hydrophobicity of alcohols and intramolecular protein cross-linking effect of alcohols, which results in conformational entropy loss of protein. The thermal denaturation midpoint for ferrocytochrome c decreases with increase in alcohol, indicating that alcohol decrease the global stability of protein. The stabilization free energy ( G ) in alcohols’ solution was calculated from the slope of the Wyman–Tanford plot and water activity. The m -values obtained from the slope of G versus alcohols plot were found to be more negative for longer and linear chain alcohols, indicating destabilization of proteins by alcohols through disturbance of hydrophobic interactions and hydrogen bonding.

Description: Ubiquitination is a post-translational modification involved in the regulation of a broad variety of cellular functions, such as protein degradation and signal transduction, including nuclear factor-B (NF-B) signalling. NF-B is crucial for inflammatory and immune responses, and aberrant NF-B signalling is implicated in multiple disorders. We found that linear ubiquitin chain assembly complex (LUBAC), composed of HOIL-1L, HOIP and SHARPIN, generates a novel type of Met1 (M1)-linked linear polyubiquitin chain and specifically regulates the canonical NF-B pathway. Moreover, specific deubiquitinases, such as CYLD, A20 (TNFAIP3) and OTULIN/gumby, inhibit LUBAC-induced NF-B activation by different molecular mechanisms, and several M1-linked ubiquitin-specific binding domains have been structurally defined. LUBAC and these linear ubiquitination-regulating factors contribute to immune and inflammatory processes and apoptosis. Functional impairments of these factors are correlated with multiple disorders, including autoinflammation, immunodeficiencies, dermatitis, B-cell lymphomas and Parkinson’s disease. This review summarizes the molecular basis and the pathophysiological implications of the linear ubiquitination-mediated NF-B activation pathway regulation by LUBAC.

Description: We screened circadian-regulated genes in rat cartilage by using a DNA microarray analysis. In rib growth-plate cartilage, numerous genes showed statistically significant circadian mRNA expression under both 12:12 h light–dark and constant darkness conditions. Type II collagen and aggrecan genes—along with several genes essential for post-translational modifications of collagen and aggrecan, including prolyl 4-hydroxylase 1, lysyl oxidase, lysyl oxidase-like 2 and 3'-phosphoadenosine 5'-phosphosulphate synthase 2—showed the same circadian phase. In addition, the mRNA level of SOX9, a master transcription factor for the synthesis of type II collagen and aggrecan, has a similar phase of circadian rhythms. The circadian expression of the matrix-related genes may be critical in the development and the growth of various cartilages, because similar circadian expression of the matrix-related genes was observed in hip joint cartilage. However, the circadian phase of the major matrix-related genes in the rib permanent cartilage was almost the converse of that in the rib growth-plate cartilage under light–dark conditions. We also found that half of the oscillating genes had conserved clock-regulatory elements, indicating contribution of the elements to the clock outputs. These findings suggest that the synthesis of the cartilage matrix macromolecules is controlled by cell-autonomous clocks depending upon the in vivo location of cartilage.

Description: Human chromosome 7 open reading frame 24 (C7orf24)/-glutamyl cyclotransferase has been suggested to be a potential diagnostic marker for several cancers, including carcinomas in the bladder urothelium, breast and endometrial epithelium. We here investigated the epigenetic regulation of the human C7orf24 promoter in normal diploid ARPE-19 and IMR-90 cells and in the MCF-7 and HeLa cancer cell lines to understand the transcriptional basis for the malignant-associated high expression of C7orf24. Chromatin immunoprecipitation analysis revealed that histone modifications associated with active chromatin were enriched in the proximal region but not in the distal region of the C7orf24 promoter in HeLa and MCF-7 cells. In contrast, elevated levels of histone modifications leading to transcriptional repression and accumulation of heterochromatin proteins in the C7orf24 promoter were observed in the ARPE-19 and IMR-90 cells, compared to the levels in HeLa and MCF-7 cancer cells. In parallel, the CpG island of the C7orf24 promoter was methylated to a greater extent in the normal cells than in the cancer cells. These results suggest that the transcriptional silencing of the C7orf24 gene in the non-malignant cells is elicited through heterochromatin formation in its promoter region; aberrant expression of C7orf24 associated with malignant alterations results from changes in chromatin dynamics.

Description: Abscisic acid (ABA) is a stress-inducible plant hormone comprising an inevitable component of the human diet. Recently, stress-induced accumulation of autocrine ABA was shown in humans, as well as ABA-mediated modulation of a number of disease-associated systems. Now, the application of a chemical proteomics approach to gain further insight into ABA mechanisms of action in mammalian cells is reported. An ABA mimetic photoaffinity probe was applied to intact mammalian insulinoma and embryonic cells, leading to the identification of heat shock protein 70 (HSP70) family members, (including GRP78 and HSP70-2) as putative human ABA-binding proteins. In vitro characterization of the ABA–HSP70 interactions yielded K d s in the 20–60 µM range, which decreased several fold in the presence of co-chaperone. However, ABA was found to have only variable- and co-chaperone-independent effects on the ATPase activity of these proteins. The potential implications of these ABA–HSP70 interactions are discussed with respect to the intracellular protein folding and extracellular receptor-like activities of these stress-inducible proteins. While mechanistic and functional relevance remain enigmatic, we conclude that ABA can bind to human HSP70 family members with physiologically relevant affinities and in a co-chaperone-dependent manner.

Description: With the advent of YouTube channels in bioinformatics, open platforms for problem solving in bioinformatics, active web forums in computing analyses and online resources for learning to code or use a bioinformatics tool, the more traditional continuing education bioinformatics training programs have had to adapt. Bioinformatics training programs that solely rely on traditional didactic methods are being superseded by these newer resources. Yet such face-to-face instruction is still invaluable in the learning continuum. Bioinformatics.ca, which hosts the Canadian Bioinformatics Workshops, has blended more traditional learning styles with current online and social learning styles. Here we share our growing experiences over the past 12 years and look toward what the future holds for bioinformatics training programs.

Description: Teaching students with very diverse backgrounds can be extremely challenging. This article uses the Bioinformatics and Systems Biology MSc in Amsterdam as a case study to describe how the knowledge gap for students with heterogeneous backgrounds can be bridged. We show that a mix in backgrounds can be turned into an advantage by creating a stimulating learning environment for the students. In the MSc Programme, conversion classes help to bridge differences between students, by mending initial knowledge and skill gaps. Mixing students from different backgrounds in a group to solve a complex task creates an opportunity for the students to reflect on their own abilities. We explain how a truly interdisciplinary approach to teaching helps students of all backgrounds to achieve the MSc end terms. Moreover, transferable skills obtained by the students in such a mixed study environment are invaluable for their later careers.

Description: The number of bioinformatics tools and resources that support molecular and cell biology approaches is continuously expanding. Moreover, systems and network biology analyses are accompanied more and more by integrated bioinformatics methods. Traditional information-centered university teaching methods often fail, as (1) it is impossible to cover all existing approaches in the frame of a single course, and (2) a large segment of the current bioinformation can become obsolete in a few years. Signaling network offers an excellent example for teaching bioinformatics resources and tools, as it is both focused and complex at the same time. Here, we present an outline of a university bioinformatics course with four sample practices to demonstrate how signaling network studies can integrate biochemistry, genetics, cell biology and network sciences. We show that several bioinformatics resources and tools, as well as important concepts and current trends, can also be integrated to signaling network studies. The research-type hands-on experiences we show enable the students to improve key competences such as teamworking, creative and critical thinking and problem solving. Our classroom course curriculum can be re-formulated as an e-learning material or applied as a part of a specific training course. The multi-disciplinary approach and the mosaic setup of the course have the additional benefit to support the advanced teaching of talented students.

Description: Today, Bioinformatics has become a scientific discipline with great relevance for the Molecular Biosciences and for the Omics sciences in general. Although developed countries have progressed with large strides in Bioinformatics education and research, in other regions, such as Central America, the advances have occurred in a gradual way and with little support from the Academia, either at the undergraduate or graduate level. To address this problem, the University of Costa Rica’s Medical School, a regional leader in Bioinformatics in Central America, has been conducting a series of Bioinformatics workshops, seminars and courses, leading to the creation of the region’s first Bioinformatics Master’s Degree. The recent creation of the Central American Bioinformatics Network (BioCANET), associated to the deployment of a supporting computational infrastructure (HPC Cluster) devoted to provide computing support for Molecular Biology in the region, is providing a foundational stone for the development of Bioinformatics in the area. Central American bioinformaticians have participated in the creation of as well as co-founded the Iberoamerican Bioinformatics Society (SOIBIO). In this article, we review the most recent activities in education and research in Bioinformatics from several regional institutions. These activities have resulted in further advances for Molecular Medicine, Agriculture and Biodiversity research in Costa Rica and the rest of the Central American countries. Finally, we provide summary information on the first Central America Bioinformatics International Congress, as well as the creation of the first Bioinformatics company (Indromics Bioinformatics), spin-off the Academy in Central America and the Caribbean.

Description: Pattern recognition is concerned with the development of systems that learn to solve a given problem using a set of example instances, each represented by a number of features. These problems include clustering, the grouping of similar instances; classification, the task of assigning a discrete label to a given instance; and dimensionality reduction, combining or selecting features to arrive at a more useful representation. The use of statistical pattern recognition algorithms in bioinformatics is pervasive. Classification and clustering are often applied to high-throughput measurement data arising from microarray, mass spectrometry and next-generation sequencing experiments for selecting markers, predicting phenotype and grouping objects or genes. Less explicitly, classification is at the core of a wide range of tools such as predictors of genes, protein function, functional or genetic interactions, etc., and used extensively in systems biology. A course on pattern recognition (or machine learning) should therefore be at the core of any bioinformatics education program. In this review, we discuss the main elements of a pattern recognition course, based on material developed for courses taught at the BSc, MSc and PhD levels to an audience of bioinformaticians, computer scientists and life scientists. We pay attention to common problems and pitfalls encountered in applications and in interpretation of the results obtained.

Description: The mountains of data thrusting from the new landscape of modern high-throughput biology are irrevocably changing biomedical research and creating a near-insatiable demand for training in data management and manipulation and data mining and analysis. Among life scientists, from clinicians to environmental researchers, a common theme is the need not just to use, and gain familiarity with, bioinformatics tools and resources but also to understand their underlying fundamental theoretical and practical concepts. Providing bioinformatics training to empower life scientists to handle and analyse their data efficiently, and progress their research, is a challenge across the globe. Delivering good training goes beyond traditional lectures and resource-centric demos, using interactivity, problem-solving exercises and cooperative learning to substantially enhance training quality and learning outcomes. In this context, this article discusses various pragmatic criteria for identifying training needs and learning objectives, for selecting suitable trainees and trainers, for developing and maintaining training skills and evaluating training quality. Adherence to these criteria may help not only to guide course organizers and trainers on the path towards bioinformatics training excellence but, importantly, also to improve the training experience for life scientists.

Description: High-throughput technologies are widely used in the field of functional genomics and used in an increasing number of applications. For many ‘wet lab’ scientists, the analysis of the large amount of data generated by such technologies is a major bottleneck that can only be overcome through very specialized training in advanced data analysis methodologies and the use of dedicated bioinformatics software tools. In this article, we wish to discuss the challenges related to delivering training in the analysis of high-throughput sequencing data and how we addressed these challenges in the hands-on training courses that we have developed at the European Bioinformatics Institute.

Description: The widespread adoption of high-throughput next-generation sequencing (NGS) technology among the Australian life science research community is highlighting an urgent need to up-skill biologists in tools required for handling and analysing their NGS data. There is currently a shortage of cutting-edge bioinformatics training courses in Australia as a consequence of a scarcity of skilled trainers with time and funding to develop and deliver training courses. To address this, a consortium of Australian research organizations, including Bioplatforms Australia, the Commonwealth Scientific and Industrial Research Organisation and the Australian Bioinformatics Network, have been collaborating with EMBL-EBI training team. A group of Australian bioinformaticians attended the train-the-trainer workshop to improve training skills in developing and delivering bioinformatics workshop curriculum. A 2-day NGS workshop was jointly developed to provide hands-on knowledge and understanding of typical NGS data analysis workflows. The road show–style workshop was successfully delivered at five geographically distant venues in Australia using the newly established Australian NeCTAR Research Cloud. We highlight the challenges we had to overcome at different stages from design to delivery, including the establishment of an Australian bioinformatics training network and the computing infrastructure and resource development. A virtual machine image, workshop materials and scripts for configuring a machine with workshop contents have all been made available under a Creative Commons Attribution 3.0 Unported License. This means participants continue to have convenient access to an environment they had become familiar and bioinformatics trainers are able to access and reuse these resources.

Description: The patterns of variation within a molecular sequence data set result from the interplay between population genetic, molecular evolutionary and macroevolutionary processes—the standard purview of evolutionary biologists. Elucidating these patterns, particularly for large data sets, requires an understanding of the structure, assumptions and limitations of the algorithms used by bioinformatics software—the domain of mathematicians and computer scientists. As a result, bioinformatics often suffers a ‘two-culture’ problem because of the lack of broad overlapping expertise between these two groups. Collaboration among specialists in different fields has greatly mitigated this problem among active bioinformaticians. However, science education researchers report that much of bioinformatics education does little to bridge the cultural divide, the curriculum too focused on solving narrow problems (e.g. interpreting pre-built phylogenetic trees) rather than on exploring broader ones (e.g. exploring alternative phylogenetic strategies for different kinds of data sets). Herein, we present an introduction to the mathematics of tree enumeration, tree construction, split decomposition and sequence alignment. We also introduce off-line downloadable software tools developed by the BioQUEST Curriculum Consortium to help students learn how to interpret and critically evaluate the results of standard bioinformatics analyses.

Description: Sequencing by ligation (SBL) is a straightforward enzymatic method for interrogating DNA sequence, in which the ligation efficiency and specificity of each probe play an essential role. Here, the number of labelled dyes in the probe, probe length and probe constituent were investigated to optimize the ligation efficiency and specificity. First, the performance of double- and single-labelled fluorescent probes in SBL was evaluated. The experimental results showed that double-labelled fluorescent probes could yield a remarkable increase in the fluorescence intensities and avoid higher background compared with single-labelled fluorescent probes. Second, probes between 7- and 9-mers in length were designed to uniform T m difference. We hoped the uniformed probes with smaller T m difference could improve the ligation efficiency. However, 8-mer probes with larger T m difference showed stronger fluorescence intensities. Third, we evaluated whether probes containing deoxyinosines either in the 5' or the 3' end had influence on the ligation efficiency. Consequently, probes containing deoxyinosines at the 5' termini might decrease the ligation efficiency, and the accumulation of 3' terminal deoxyinosines in the sequencing primers was likely to reduce the fluorescence intensity and the ligation efficiency, which was inconsistent with the traditional viewpoint. The optimized probes will improve the ligation efficiency and accuracy in SBL.

Description: In this study, bleomycin-Fe 3+ steadily oxidized tetramethylbenzidine (TMB) in the presence of peroxides. However, the ability of bleomycin-Fe 3+ to function as a peroxidase was extremely low compared with that of other peroxidases. A characteristic property of bleomycin-Fe 3+ different from that observed for other peroxidases is its ability to oxidize TMB at the similar rate at both a pH 5 and 8 in the presence of lipid hydroperoxide (LOOH). In the present experiments, hydroxyl radicals (HO•) were generated only when bleomycin-Fe 3+ was incubated with H 2 O 2 at a pH of 5. No generation of HO• was observed during the incubation of bleomycin-Fe 3+ with LOOH. Meanwhile, bleomycin-Fe 3+ induced the formation of LOOH from linoleic acid and alcohol dehydrogenase was inactivated by bleomycin-Fe 3+ with peroxides. Thiobarbituric acid reactive substances were formed from DNA by bleomycin-Fe 3+ with H 2 O 2 , and strand breaks were caused by bleomycin-Fe 3+ with LOOH. The oxidative substrates for bleomycin-Fe 3+ blocked the damage to biological components induced by bleomycin-Fe 3+ . These results suggest that compound I-like species contribute to the process of damage to biological components induced by bleomycin-Fe 3+ .

Description: The Ral guanosine triphosphatases (GTPases), RalA and RalB, are members of the Ras superfamily of small GTPases. Research on Ral GTPases and their functions over the past 25 years has revealed the essential involvement of these GTPases in unique and diverse cellular processes including exocyst-mediated exocytosis and related cellular activities. Moreover, it is increasingly appreciated that the aberrant activation of Ral GTPases is one of the major causes of human tumourigenesis induced by oncogenic Ras. Recent evidence suggests that Ral signalling pathways may be potential therapeutic targets for the treatment of human cancers. This review summarizes recent advance in the investigation of Ral GTPases.

Description: Heparin-binding epidermal growth factor-like growth factor (HB-EGF), a ligand of EGF receptor, is involved in the growth and malignant progression of cancers. Cross-reacting material 197, CRM197, a non-toxic mutant of diphtheria toxin (DT), specifically binds to the EGF-like domain of HB-EGF and inhibits its mitogenic activity, thus CRM197 is currently under evaluation in clinical trials for cancer therapy. To develop more potent DT mutants than CRM197, we screened various mutant proteins of R domain of DT, the binding site for HB-EGF. A variety of R-domain mutant proteins fused with maltose-binding protein were produced and their inhibitory activity was evaluated in vitro . We found four R domain mutants that showed much higher inhibitory activity against HB-EGF than wild-type (WT) R domain. These R domain mutants suppressed HB-EGF-dependent cell proliferation more effectively than WT R domain. Surface plasmon resonance revealed their higher affinity to HB-EGF than WT R domain. CRM197(R460H) carrying the newly identified mutation showed increased cell proliferation inhibitory activity and affinity to HB-EGF. These results suggest that CRM197(R460H) or other recombinant proteins carrying newly identified mutation(s) in the R domain are potential therapeutics targeting HB-EGF.

Description: Osteoclasts are giant multinucleated cells that differentiate from hematopoietic cells in the bone marrow and carry out important physiological functions in the regulation of skeletal homeostasis as well as hematopoiesis. Osteoclast biology shares many features and components with cells of the immune system, including cytokine-receptor interactions (RANKL-RANK), intracellular signalling molecules (TRAF6) and transcription factors (NFATc1). Although the roles of these molecules in osteoclast differentiation are well known, fundamental questions remain unsolved, including the exact location of the RANKL-RANK interaction and the in vivo temporal and spatial information on the transformation of hematopoietic cells into bone-resorbing osteoclasts. This review focuses on the importance of cell-cell contact and metabolic adaptation for differentiation, relatively overlooked aspects of osteoclast biology and biochemistry.

Description: Although monoclonal antibodies have been used not only as analytical tools but also as biologic therapeutics, they cannot target intracellular proteins due to their large molecular size and low membrane permeability, which limit their applications. During previous attempts to delivery antibodies intracellularly, the low efficiency of escape from endosomes to the cytosol reduced the bioavailability of antibodies or antibody-conjugated effectors. Recently, we found that the fusogenic peptides (FPs) B18 and B55 from bindin, a sea urchin gamete recognition protein, facilitated the endosomal escape of FP-fused enhanced green fluorescent protein (eGFP) and/or of co-administered cargos such as dextrans [Niikura et al. A fusogenic peptide from a sea urchin fertilization protein promotes intracellular delivery of biomacromolecules by facilitating endosomal escape. J. Control. Release 2015;212:85-93]. In this study, we constructed FP-fused anti-epidermal growth factor receptor (EGFR) single-chain Fv (αEGFR[scFv]) proteins and evaluated their endosomal escape efficiency by utilizing a nuclear localization signal). When the FP-fused αEGFR[scFv] proteins were incubated with A431 cells, the estimated endosomal escape efficiency of αEGFR[scFv]-B18 was significantly higher than that of αEGFR[scFv] alone, suggesting that the B18 peptide facilitates endosomal escape of the conjugated scFv in cis . Moreover, αEGFR[scFv]-B55 promoted the intracellular uptake of co-administered eGFP and dextrans in trans . These results imply that B18- and B55-fused antibodies may be useful for the cell-specific intracellular delivery of biomacromolecules.

Description: Methylobacterium extorquens AM1 is an aerobic facultative methylotroph known to secrete pyrroloquinoline quinone (PQQ), a cofactor of a number of bacterial dehydrogenases, into the culture medium. To elucidate the molecular mechanism of PQQ biosynthesis, we are focusing on PqqE which is believed to be the enzyme catalysing the first reaction of the pathway. PqqE belongs to the radical S -adenosyl- l -methionine (SAM) superfamily, in which most, if not all, enzymes are very sensitive to dissolved oxygen and rapidly inactivated under aerobic conditions. We here report that PqqE from M. extorquens AM1 is markedly oxygen-tolerant; it was efficiently expressed in Escherichia coli cells grown aerobically and affinity-purified to near homogeneity. The purified and reconstituted PqqE contained multiple (likely three) iron-sulphur clusters and showed the reductive SAM cleavage activity that was ascribed to the consensus [4Fe-4S] 2+ cluster bound at the N-terminus region. Mössbauer spectrometric analyses of the as-purified and reconstituted enzymes revealed the presence of [4Fe-4S] 2+ and [2Fe-2S] 2+ clusters as the major forms with the former being predominant in the reconstituted enzyme. PqqE from M.extorquens AM1 may serve as a convenient tool for studying the molecular mechanism of PQQ biosynthesis, avoiding the necessity of establishing strictly anaerobic conditions.

Description: Pho Pop5 and Pho Rpp30 in the hyperthermophilic archaeon Pyrococcus horikoshii , homologues of human ribonuclease P (RNase P) proteins hPop5 and Rpp30, respectively, fold into a heterotetramer [ Pho Rpp30–( Pho Pop5) 2 – Pho Rpp30], which plays a crucial role in the activation of RNase P RNA ( Pho pRNA). Here, we examined the functional implication of Pho Pop5 and Pho Rpp30 in the tetramer. Surface plasmon resonance (SPR) analysis revealed that the tetramer strongly interacts with an oligonucleotide including the nucleotide sequence of a stem-loop SL3 in Pho pRNA. In contrast, Pho Pop5 had markedly reduced affinity to SL3, whereas Pho Rpp30 had little affinity to SL3. SPR studies of Pho Pop5 mutants further revealed that the C-terminal helix (α4) in Pho Pop5 functions as a molecular recognition element for SL3. Moreover, gel filtration indicated that Pho Rpp30 exists as a monomer, whereas Pho Pop5 is an oligomer in solution, suggesting that Pho Rpp30 assists Pho Pop5 in attaining a functionally active conformation by shielding hydrophobic surfaces of Pho Pop5. These results, together with available data, allow us to generate a structural and mechanistic model for the Pho pRNA activation by Pho Pop5 and Pho Rpp30, in which the two C-terminal helices (α4) of Pho Pop5 in the tetramer whose formation is assisted by Pho Rpp30 act as binding elements and bridge SL3 and SL16 in Pho pRNA.

Description: Wnt ligands play a central role in the development and homeostasis of various organs through β-catenin-dependent and -independent signalling. The crucial roles of Wnt/β-catenin signals in bone mass have been established by a large number of studies since the discovery of a causal link between mutations in the low-density lipoprotein receptor-related protein 5 ( Lrp5 ) gene and alternations in human bone mass. The activation of Wnt/β-catenin signalling induces the expression of osterix, a transcription factor, which promotes osteoblast differentiation. Furthermore, this signalling induces the expression of osteoprotegerin, an osteoclast inhibitory factor in osteoblast-lineage cells to prevent bone resorption. Recent studies have also shown that Wnt5a, a typical non-canonical Wnt ligand, enhanced osteoclast formation. In contrast, Wnt16 inhibited osteoclast formation through β-catenin-independent signalling. In this review, we discussed the current understanding of the Wnt signalling molecules involved in bone formation and resorption.

Description: Parathyroid hormone-related protein (PTHrP) has two different targeting signals: an N-terminal signal peptide for the endoplasmic reticulum (ER) targeting and an internal nuclear localization signal. The protein not only functions as a secretory protein, but is also found in the nucleus and/or nucleolus under certain conditions. PTHrP signal peptide is less hydrophobic than most signal peptides mainly due to its evolutionarily well-conserved region (QQWS). The substitution of four tandem leucine residues for this conserved region resulted in a significant inhibition of the signal peptide cleavage. At the same time, proportion of nuclear and/or nucleolar localization decreased, probably due to tethering of the protein to the ER membrane by the uncleaved mutant signal peptide. Almost complete cleavage of the signal peptide accompanied by a lack of nuclear/nucleolar localization was achieved by combining the hydrophobic h-region and an optimized sequence of the cleavage site. In addition, mutational modifications of the distribution of charged residues in and around the signal peptide affect its cleavage and/or nuclear/nucleolar localization of the protein. These results indicate that the well-conserved region in the signal peptide plays an essential role in the dual localization of PTHrP through ER targeting and/or the membrane translocation.

Description: Distribution of the isoelectric point (pI) was calculated for the hypervariable regions of Fab fragments of the antibody molecules, which structure is annotated in the structural antibody database SabDab. The distribution is consistent with the universal for all organisms dividing the proteome into two sets of acidic and basic proteins. It shows the additional fine structure in a form of the narrow-sized peaks of pI values. This is an explanation why a small change of the environmental pH can have a strong effect on the antibody-antigen affinity. To show this, a typical enzyme-linked immunospecific assay experiment for testing the reaction of goat anti-human IgA antibodies with human IgA immunoglobulins of saliva as antigens was modified in such a way that Fe 3 O 4 magnetic nanoparticles were added to PBS buffer. The magnetic nanoparticles were remotely heated by the radio frequency magnetic field providing the local change of temperature and pH. It was observed that short times of the heating were significantly increasing the antibody-antigen binding strength while it was not the case for a longer time. The finding discussed in the study can be useful for biopharmaceuticals using antibodies, the immunoassay techniques as well as for control over the use of hyperthermia.

Description: The 5'-AMP-activated protein kinase (AMPK) functions as a cellular energy sensor. 5-Aminoimidazole-4-carboxyamide-1-β-D-ribofranoside (AICAR) is a chemical activator of AMPK. In the liver, AICAR suppresses expression of the phosphoenolpyruvate carboxykinase ( PEPCK ) gene. The rat enhancer of split- and hairy-related protein-2 (SHARP-2) is an insulin-inducible transcriptional repressor and its target is the PEPCK gene. In this study, we examined an issue of whether the SHARP-2 gene expression is regulated by AICAR via the AMPK. AICAR increased the level of SHARP-2 mRNA in H4IIE cells. Whereas an AMPK inhibitor, compound-C, had no effects on the AICAR-induction, inhibitors for both phosphoinositide 3-kinase (PI 3-K) and protein kinase C (PKC) completely diminished the effects of AICAR. Western blot analyses showed that AICAR rapidly activated atypical PKC lambda (aPKC). In addition, when a dominant negative form of aPKC was expressed, the induction of SHARP-2 mRNA level by AICAR was inhibited. Calcium ion is not required for the activation of aPKC. A calcium ion-chelating reagent had no effects on the AICAR-induction. Furthermore, the AICAR-induction was inhibited by treatment with an RNA polymerase inhibitor or a protein synthesis inhibitor. Thus, we conclude that the AICAR-induction of the SHARP-2 gene is mediated at transcription level by a PI 3-K/aPKC pathway.