Search

Recent large-scale genome-wide association studies have identified common genetic variations that may contribute to the risk of amyotrophic lateral sclerosis (ALS). However, pinpointing the risk variants in noncoding regions and underlying biological mechanisms remains a major challenge. Here, we constructed a convolutional neural network model with a large-scale GWAS meta-analysis dataset to unravel functional noncoding variants associated with ALS based on their epigenetic features. After filtering and prioritizing of candidates, we fine-mapped two new risk variants, rs2370964 and rs3093720, on chromosome 3 and 17, respectively. Further analysis revealed that these polymorphisms are associated with the expression level of CX3CR1 and TNFAIP1, and affect the transcription factor binding sites for CTCF, NFATc1 and NR3C1. Our results may provide new insights for ALS pathogenesis, and the proposed research methodology can be applied for other complex diseases as well.

Although the incidence of acute coronary syndrome (ACS) has increased over the decades, the overall prognosis has improved with newer stents, tailored medication, and better intervention techniques. Atrial fibrillation (AF) and ventricular arrhythmia at the time of ACS diagnosis are known indicators of a poor acute prognosis. However, there is a lack of data regarding the long-term arrhythmic impact of ventricular tachyarrhythmia (VA) on mortality in ACS patients. This study sought to elucidate the impact of tachyarrhythmia on mortality during long-term follow-up in patients with a history of ACS. This retrospective study was conducted in a single university hospital, and it evaluated the clinical outcomes, especially regarding cardiovascular mortality and readmission. The enrolled patients underwent percutaneous coronary intervention (PCI) for ACS between February 2004 and March 2018. Clinical information was attained by a thorough chart review. We retrospectively analyzed 560 ACS patients. We reviewed all electrocardiograms (ECGs) before and immediately after PCI, during hospitalization, and within 3 months of the index PCI. Three months after the index PCI procedure, any Holter monitoring or ECG was also reviewed for arrhythmia diagnosis. During follow-up, 91 patients were diagnosed with AF and 36 patients were diagnosed with VA. Overall mortality was related to the presence of anemia, low body mass index, low left ventricular ejection fraction after PCI, late-diagnosed AF, and any VA during follow-up. Readmission was higher in patients with chronic kidney disease and newly diagnosed AF during the follow-up. Diagnosis of late tachyarrhythmia during follow-up was associated with increased mortality in post-ACS patients.

Background Systematic in vitro loss-of-function screens provide valuable resources that can facilitate the discovery of drugs targeting cancer vulnerabilities. Results We develop a deep learning-based method to predict tumor-specific vulnerabilities in patient samples by leveraging a wealth of in vitro screening data. Acquired dependencies of tumors are inferred in cases in which one allele is disrupted by inactivating mutations or in association with oncogenic mutations. Nucleocytoplasmic transport by Ran GTPase is identified as a common vulnerability in Her2-positive breast cancers. Vulnerability to loss of Ku70/80 is predicted for tumors that are defective in homologous recombination and rely on nonhomologous end joining for DNA repair. Our experimental validation for Ran, Ku70/80, and a proteasome subunit using patient-derived cells shows that they can be targeted specifically in particular tumors that are predicted to be dependent on them. Conclusion This approach can be applied to facilitate the development of precision therapeutic targets for different tumors.

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in Drosophila melanogaster brains. Genetic mutants of astray (aay), a fly homolog of the rate-limiting phosphoserine phosphatase in serine biosynthesis, displayed reduced starvation-induced sleep suppression. In contrast, a hypomorphic mutation in a serine/threonine-metabolizing enzyme, serine/threonine dehydratase (stdh), exaggerated starvation-induced sleep suppression. Analyses of double mutants indicated that aay and stdh act on the same genetic pathway to titrate serine levels in the head as well as to adjust starvation-induced sleep behaviors. RNA interference-mediated depletion of aay expression in neurons, using cholinergic Gal4 drivers, phenocopied aay mutants, while a nicotinic acetylcholine receptor antagonist selectively rescued the exaggerated starvation-induced sleep suppression in stdh mutants. Taken together, these data demonstrate that neural serine metabolism controls sleep during starvation, possibly via cholinergic signaling. We propose that animals have evolved a sleep-regulatory mechanism that reprograms amino acid metabolism for adaptive sleep behaviors in response to metabolic needs.

The process of direct cell reprogramming, also named transdifferentiation, permits for the conversion of one mature cell type directly into another, without returning to a dedifferentiated state. This makes direct reprogramming a promising approach for the development of several cellular and tissue engineering therapies. To achieve the change in the cell identity, direct reprogramming requires an arsenal of tools that combine experimental and computational techniques. In the recent years, several methods of transdifferentiation have been developed. In this review, we will introduce the concept of direct cell reprogramming and its background, and cover the recent developments in the experimental and computational prediction techniques with their applications. We also discuss the challenges of translating this technology to clinical setting, accompanied with potential solutions.

Major progress in disease genetics has been made through genome-wide association studies (GWASs). One of the key tasks for post-GWAS analyses is to identify causal noncoding variants with regulatory function. Here, on the basis of > 2,000 functional features, we developed a convolutional neural network framework for combinatorial, nonlinear modeling of complex patterns shared by risk variants scattered among multiple associated loci. When applied for major psychiatric disorders and autoimmune diseases, neural and immune features, respectively, exhibited high explanatory power while reflecting the pathophysiology of the relevant disease. The predicted causal variants were concentrated in active regulatory regions of relevant cell types and tended to be in physical contact with transcription factors while residing in evolutionarily conserved regions and resulting in expression changes of genes related to the given disease. We demonstrate some examples of novel candidate causal variants and associated genes. Our method is expected to contribute to the identification and functional interpretation of causal noncoding variants in post-GWAS analyses.

Sleep and metabolism are physiologically and behaviorally intertwined; however, the molecular basis for their interaction remains poorly understood. Here, we identified a serine metabolic pathway as a key mediator for starvation-induced sleep suppression. Transcriptome analyses revealed that enzymes involved in serine biosynthesis were induced upon starvation in

The tumor necrosis factor-receptor 1 (TNFR1)-associated death domain protein (TRADD) contains an N-terminal TRAF binding domain and a C-terminal death domain. TRADD is known to interact directly with TNF receptor 2 (TNFR2) and the Fas-associated death domain protein (FADD), which are signal transducers that activate NF- and induce apoptosis, respectively. To date, there has been no structural information on the TRADD and FADD death domain (DDs) complex. In this study, the death domains of TRADD and FADD were co-expressed and purified from Escherichia coli for structural characterization. We found that human TRADD (hTRADD) interacted strongly with mouse FADD (mFADD) via their DDs and interacted weakly with human FADD (hFADD)-DD. Moreover, the structures of the TRADD-DD:FADD-DD complexes were separately modeled from predicted structures in the protein data bank (PDB). The results of this study will have important applications in human diseases such as cancer, AIDS, degenerative and autoimmune diseases, and infectious diseases.

Overproduced yeast ribosomal protein (RP) Rpl26 fails to assemble into ribosomes and is degraded in the nucleus/nucleolus by a ubiquitin-proteasome system quality control pathway comprising the E2 enzymes Ubc4/Ubc5 and the ubiquitin ligase Tom1. tom1 cells show reduced ubiquitination of multiple RPs, exceptional accumulation of detergent-insoluble proteins including multiple RPs, and hypersensitivity to imbalances in production of RPs and rRNA, indicative of a profound perturbation to proteostasis. Tom1 directly ubiquitinates unassembled RPs primarily via residues that are concealed in mature ribosomes. Together, these data point to an important role for Tom1 in normal physiology and prompt us to refer to this pathway as ERISQ, for excess ribosomal protein quality control. A similar pathway, mediated by the Tom1 homolog Huwe1, restricts accumulation of overexpressed hRpl26 in human cells. We propose that ERISQ is a key element of the quality control machinery that sustains protein homeostasis and cellular fitness in eukaryotes. DOI: http://dx.doi.org/10.7554/eLife.19105.001, eLife digest Ribosomes are the molecular machines in cells that produce proteins. The ribosomes themselves are composed of almost 80 different proteins that are held together by scaffolds made from molecules of RNA. Each protein is present in one copy, and so equal numbers of all proteins are needed to assemble a ribosome. However, because it takes many steps to produce a protein and biological processes are inherently imprecise, it is essentially impossible for a cell to produce exactly the same number of copies of all the proteins in a ribosome. Much research suggests that, to overcome these issues, a cell will make more of certain ribosomal proteins than it needs, and then degrade the leftovers that are not used. However, it was not clear how this happens, nor was it known what are the consequences of failing to degrade the leftovers. Now, Sung et al. show that yeast cells use an enzyme named Tom1 to attach a protein-marker called ubiquitin to ribosomal proteins that are made in excess and not assembled into ribosomes. The ubiquitin serves as a tag that marks proteins for degradation, and yeast cell that lack Tom1 fail to degrade any excess ribosomal proteins. Consequently, the mutant yeast become sensitive to any factors that alter the balance of the protein and RNA building blocks used to assemble ribosomes. The human equivalent of Tom1 is known as Huwe1, and the data of Sung et al. suggest that this enzyme acts in a similar pathway. Further experiments are now needed to explore the role of Huwe1 in greater depth, and investigate if problems with this enzyme are associated with any human diseases. Finally, working out the exactly how Tom1 recognizes unassembled ribosomal proteins will be another important challenge for future studies. DOI: http://dx.doi.org/10.7554/eLife.19105.002

Background Examples of heterozygote advantage in humans are scarce and limited to protein-coding sequences. Here, we attempt a genome-wide functional inference of advantageous heterozygosity at cis-regulatory regions. Results The single-nucleotide polymorphisms bearing the signatures of balancing selection are enriched in active cis-regulatory regions of immune cells and epithelial cells, the latter of which provide barrier function and innate immunity. Examples associated with ancient trans-specific balancing selection are also discovered. Allelic imbalance in chromatin accessibility and divergence in transcription factor motif sequences indicate that these balanced polymorphisms cause distinct regulatory variation. However, a majority of these variants show no association with the expression level of the target gene. Instead, single-cell experimental data for gene expression and chromatin accessibility demonstrate that heterozygous sequences can lower cell-to-cell variability in proportion to selection strengths. This negative correlation is more pronounced for highly expressed genes and consistently observed when using different data and methods. Based on mathematical modeling, we hypothesize that extrinsic noise from fluctuations in transcription factor activity may be amplified in homozygotes, whereas it is buffered in heterozygotes. While high expression levels are coupled with intrinsic noise reduction, regulatory heterozygosity can contribute to the suppression of extrinsic noise. Conclusions This mechanism may confer a selective advantage by increasing cell population homogeneity and thereby enhancing the collective action of the cells, especially of those involved in the defense systems in humans. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1027-8) contains supplementary material, which is available to authorized users.

Background One of the greatest challenges in cancer genomics is to distinguish driver mutations from passenger mutations. Whereas recurrence is a hallmark of driver mutations, it is difficult to observe recurring noncoding mutations owing to a limited amount of whole-genome sequenced samples. Hence, it is required to develop a method to predict potentially recurrent mutations. Results In this work, we developed a random forest classifier that predicts regulatory mutations that may recur based on the features of the mutations repeatedly appearing in a given cohort. With breast cancer as a model, we profiled 35 quantitative features describing genetic and epigenetic signals at the mutation site, transcription factors whose binding motif was disrupted by the mutation, and genes targeted by long-range chromatin interactions. A true set of mutations for machine learning was generated by interrogating publicly available pan-cancer genomes based on our statistical model of mutation recurrence. The performance of our random forest classifier was evaluated by cross validations. The variable importance of each feature in the classification of mutations was investigated. Our statistical recurrence model for the random forest classifier showed an area under the curve (AUC) of ~0.78 in predicting recurrent mutations. Chromatin accessibility at the mutation sites, the distance from the mutations to known cancer risk loci, and the role of the target genes in the regulatory or protein interaction network were among the most important variables. Conclusions Our methods enable to characterize recurrent regulatory mutations using a limited number of whole-genome samples, and based on the characterization, to predict potential driver mutations whose recurrence is not found in the given samples but likely to be observed with additional samples. Electronic supplementary material The online version of this article (doi:10.1186/s12859-016-1385-y) contains supplementary material, which is available to authorized users.

In eukaryotic cells, ribosomal DNA (rDNA) forms the basis of the nucleolus. In Saccharomyces cerevisiae, 100–200 copies of a 9.1-kb rDNA repeat exist as a tandem array on chromosome XII. The stability of this highly repetitive array is maintained through silencing. However, the precise mechanisms that regulate rDNA silencing are poorly understood. Here, we report that S. cerevisiae Ydr026c, which we name NTS1 silencing protein 1 (Nsi1), plays a significant role in rDNA silencing. By studying the subcellular localization of 159 nucleolar proteins, we identified 11 proteins whose localization pattern is similar to that of Net1, a well-established rDNA silencing factor. Among these proteins is Nsi1, which is associated with the NTS1 region of rDNA and is required for rDNA silencing at NTS1. In addition, Nsi1 physically interacts with the known rDNA silencing factors Net1, Sir2 and Fob1. The loss of Nsi1 decreases the association of Sir2 with NTS1 and increases histone acetylation at NTS1. Furthermore, Nsi1 contributes to the longevity of yeast cells. Taken together, our findings suggest that Nsi1 is a new rDNA silencing factor that contributes to rDNA stability and lifespan extension in S. cerevisiae.

E-mail: sbjang@pusan.ac.krReceived September 26, 2011, Accepted November 21, 2011Galectins are generally believed to be potential candidates for use in the development of novel anti-inflammatory agents or as selective modulators of the immune response. In particular, galectin-9 exhibits someof the extracellular functions, including cell aggregation, adhesion, chemoattraction, activation, and apoptosis.Tl-galectin (Tl-gal, galectin-9 homologue gene) was isolated from an adult worm of the Toxascaris leonina.The full-length Tl-gal gene, which was incorporated into pET-28a, was overexpressed in E. coli and purifiedby nickel affinity and gel filtration chromatographies. The purified Tl-gal was crystallized using the hanging-drop vapor-diffusion method. The crystal belonged to the tetragonal space group P4

Adrenergic α2C receptor (ADRA2C) is an inhibitory modulator of the sympathetic nervous system. Knockout mice for this gene show physiological and behavioural alterations that are associated with the fight-or-flight response. There is evidence of positive selection on the regulation of this gene during chicken domestication. Here, we find that the neuronal expression of ADRA2C is lower in human and chimpanzee than in other primates. On the basis of three-dimensional chromatin structure, we identified a cis-regulatory region whose DNA sequences have been significantly accelerated in human and chimpanzee. Active histone modification marks this region in rhesus macaque but not in human and chimpanzee; instead, repressive marks are enriched in various human brain samples. This region contains two neuron-restrictive silencer factor (NRSF) binding motifs, each of which harbours a polymorphism. Our genotyping and analysis of population genome data indicate that at both polymorphic sites, the derived allele has reached fixation in humans and chimpanzees but not in bonobos, whereas only the ancestral allele is present among macaques. Our CRISPR/Cas9 genome editing and reporter assays show that both derived nucleotides repress ADRA2C, most likely by increasing NRSF binding. In addition, we detected signatures of recent positive selection for lower neuronal ADRA2C expression in humans. Our findings indicate that there has been selective pressure for enhanced sympathetic nervous activity in the evolution of humans and chimpanzees., Author summary Adrenergic α2C receptor (ADRA2C) is a regulator of the fight-or-flight response. It has been shown in mice that repression of this gene can result in relevant physiological and behavioral alterations. A strong selection signature in the genomes of domesticated chickens has been reported for this gene, suggesting that less aggression toward humans has been positively selected during chicken domestication. In this work, we analyze the genomes, transcriptomes, and epigenomes of a large number of humans and chimpanzees along with those of other primates to propose that repression of this gene has been positively selected in the evolution of humans and chimpanzees.

The bimolecular fluorescence complementation (BiFC) assay has been widely accepted for studying in vivo detection of protein-protein interactions in several organisms. To facilitate the application of the BiFC assay to yeast research, we have created a series of plasmids that allow single-step, PCR-based C- or N-terminal tagging of yeast proteins with yellow fluorescent protein fragments for BiFC assay. By examination of several interacting proteins (Sis1-Sis1, Net1-Sir2, Cet1-Cet1 and Pho2-Pho4), we demonstrate that the BiFC assay can be used to reliably analyse the occurrence and subcellular localization of protein-protein interactions in living yeast cells. The sequences for the described plasmids were submitted to the GenBank under Accession Nos: EF210802, pFA6a-VN-His3MX6; EF210803, pFA6a-VC-His3MX6; EF210804, pFA6a-VN-TRP1; EF210807, pFA6a-VC-TRP1; EF210808, pFA6a-VN-kanMX6; EF210809, pFA6a-VC-kanMX6; EF210810, pFA6a-His3MX6-P(GAL1)-VN; EF210805, pFA6a-His3MX6-P(GAL1)-VC; EF210806, pFA6a-TRP1-P(GAL1)-VN; EF210811, pFA6a-TRP1-P(GAL1)-VC; EF210812, pFA6a-kanMX6-P(GAL1)-VN; EF210813, pFA6a-kanMX6-P(GAL1)-VC; EF521883, pFA6a-His3MX6-P(CET1)-VN; EF521884, pFA6a-His3MX6-P(CET1)-VC; EF521885, pFA6a-TRP1-P(CET1)-VN; EF521886, pFA6a-TRP1-P(CET1)-VC; EF521887, pFA6a-kanMX6-P(CET1)-VN; EF521888, pFA6a-kanMX6-P(CET1)-VC.

Multidrug resistance (MDR) of cancer cells is, at least in part, associated with the overexpression of P-glycoprotein (P-gp). Many studies have demonstrated that natural compounds obtained from fruits, vegetables, teas and medicinal plants may modulate P-gp activity. The objective of the present investigation was to examine the effect of seven natural compounds on the P-gp activity in human uterine sarcoma cell line, MES-SA/DX5. Daunomycin uptake was significantly increased by biochanin A and silymarin (p value of daunomycin (p

An HPLC method was employed for the determination of ethambutol in human plasma. After addition of internal standard (IS, octylamine, ) and alkalinization of the plasma with 5 M sodium hydroxide, the drug and IS were extracted into the mixture of chloroform and diethyl ether (40:60, v/v). Following a 15-min vortex-mixing and a 10min centrifugation, the organic phase was spiked with of phenylethylisocyanate for chemical derivatization, mixed for 5 min and evaporated to dryness under a stream of nitrogen. The residue was reconstituted with of mobile phase and was injected into C18 column with a mobile phase consisting of methanol:water (70:30, v/v). The samples were detected utilizing an ultraviolet detector at 200 nm. The method was specific and validated with a limit of . Intra- and inter-day precision and accuracy were acceptable for all quality control samples including the lower limit of quantification. The applicability of this method was demonstrated by analysis of human plasma after oral administration of a single 1200-mg dose to 20 healthy subjects. From the plasma ethambutol concentration vs. time curves, the mean AUC was and Cmax of reached 2.73 hr after administration. The mean biological half-life of ethambutol was hr. Based on the results, this simple and validated assay could readily be used in any pharmacokinetic studies using humans.

Protein location and function can change dynamically depending on many factors, including environmental stress, disease state, age, developmental stage, and cell type. Here, we describe an integrative computational framework, called the conditional function predictor (CoFP; http://nbm.ajou.ac.kr/cofp/), for predicting changes in subcellular location and function on a proteome-wide scale. The essence of the CoFP approach is to cross-reference general knowledge about a protein and its known network of physical interactions, which typically pool measurements from diverse environments, against gene expression profiles that have been measured under specific conditions of interest. Using CoFP, we predict condition-specific subcellular locations, biological processes, and molecular functions of the yeast proteome under 18 specified conditions. In addition to highly accurate retrieval of previously known gold standard protein locations and functions, CoFP predicts previously unidentified condition-dependent locations and functions for nearly all yeast proteins. Many of these predictions can be confirmed using high-resolution cellular imaging. We show that, under DNA-damaging conditions, Tsr1, Caf120, Dip5, Skg6, Lte1, and Nnf2 change subcellular location and RNA polymerase I subunit A43, Ino2, and Ids2 show changes in DNA binding. Beyond specific predictions, this work reveals a global landscape of changing protein location and function, highlighting a surprising number of proteins that translocate from the mitochondria to the nucleus or from endoplasmic reticulum to Golgi apparatus under stress.

In inner ear development, phosphatase and tensin homolog (PTEN) is necessary for neuronal maintenance, such as neuronal survival and accurate nerve innervations of hair cells. We previously reported that Pten conditional knockout (cKO) mice exhibited disorganized fasciculus with neuronal apoptosis in spiral ganglion neurons (SGNs). To better understand the genes and signaling networks related to auditory neuron maintenance, we compared the profiles of differentially expressed genes (DEGs) using microarray analysis of the inner ear in E14.5 Pten cKO and wild-type mice. We identified 46 statistically significant transcripts using significance analysis of microarrays, with the false-discovery rate set at 0%. Among the DEGs, expression levels of candidate genes and expression domains were validated by quantitative real-time RT-PCR and in situ hybridization, respectively. Ingenuity pathway analysis using DEGs identified significant signaling networks associated with apoptosis, cellular movement, and axon guidance (i.e., secreted phosphoprotein 1 (Spp1)-mediated cellular movement and regulator of G-protein signaling 4 (Rgs4)-mediated axon guidance). This result was consistent with the phenotypic defects of SGNs in Pten cKO mice (e.g., neuronal apoptosis, abnormal migration, and irregular nerve fiber patterns of SGNs). From this study, we suggest two key regulatory signaling networks mediated by Spp1 and Rgs4, which may play potential roles in neuronal differentiation of developing auditory neurons.

The definition of protein–protein interactions (PPIs) in the natural cellular context is essential for properly understanding various biological processes. So far, however, most large-scale PPI analyses have not been performed in the natural cellular context. Here, we describe the construction of a Saccharomyces cerevisiae fusion library in which each endogenous gene is C-terminally tagged with the N-terminal fragment of Venus (VN) for a genome-wide bimolecular fluorescence complementation assay, a powerful technique for identifying PPIs in living cells. We illustrate the utility of the VN fusion library by systematically analyzing the interactome of the small ubiquitin-related modifier (SUMO) and provide previously unavailable information on the subcellular localization, types, and protease dependence of SUMO interactions. Our data set is highly complementary to the existing data sets and represents a useful resource for expanding the understanding of the physiological roles of SUMO. In addition, the VN fusion library provides a useful research tool that makes it feasible to systematically analyze PPIs in the natural cellular context.

A skyline of a d-dimensional dataset contains the points that are not dominated by any other point on all dimensions. Due to its usefulness, a skyline query has recently received a considerable attention in several applications. However, as the number of dimensions increases, the probability of one point dominating another point becomes very low. In consequence, the number of points in the skyline becomes tremendous. To remedy this disadvantage, the k-dominant skyline has been introduced, which relaxes the domination relationship. Although the number of k-dominant skyline points is smaller than the number of skyline points, some important points in the dataset may be excluded from the result of a k-dominant skyline query due to the cyclic dominance relationship. With this problem in mind, we introduce a novel types of skyline queries, called the scored k-dominant skyline query. A scored k-dominant skyline is computed from skyline points by utilizing the notions of (i) k-dominance relationship and (ii) k-dominant score. We also present the search algorithm for the scored k-dominant skyline. Finally, we demonstrate the effectiveness of the scored k-dominant skyline through a set of simulations by using both real dataset and synthetic dataset.

DNA Damage Pathways Revealed Despite the dynamic nature of cellular responses, the genetic networks that govern these responses have been mapped primarily as static snapshots. Bandyopadhyay et al. (p. 1385 ; see the Perspective by Friedman and Schuldiner ) report a comparison of large genetic interactomes measured among all yeast kinases, phosphatases, and transcription factors, as the cell responded to DNA damage. The interactomes revealed were highly dynamic structures that changed dramatically with changing conditions. These dynamic interactions reveal genetic relationships that can be more effective than classical “static” interactions (for example, synthetic lethals and epistasis maps) in identifying pathways of interest.

Most of the biological processes are carried out and regulated by dynamic networks of protein-protein interactions. In this study, we demonstrate the feasibility of the bimolecular fluorescence complementation (BiFC) assay for in vivo quantitative analysis of protein-protein interactions in Saccharomyces cerevisiae. We show that the BiFC assay can be used to quantify not only the amount but also the cell-to-cell variation of protein-protein interactions in S. cerevisiae. In addition, we show that protein sumoylation and condition-specific protein-protein interactions can be quantitatively analyzed by using the BiFC assay. Taken together, our results validate that the BiFC assay is a very effective method for quantitative analysis of protein-protein interactions in living yeast cells and has a great potential as a versatile tool for the study of protein function.

Recently, wireless mobile computing has been realized in the industry, where mobile clients communicate by using their handheld devices. Meanwhile, data broadcasting is an effective way for data dissemination due to its beneficial characteristics such as bandwidth efficiency, energy-efficiency, and scalability. In this paper, we propose an XML stream optimization method for time critical data, which is highly dependant to the time. To end this, we utilize structural index to integrate the elements of same path into one node. Furthermore, we propose an attribute summarization strategy to minimize the size of the XML steam by classifying attribute names and values. Experimental results show that our method outperforms the previous wireless XML broadcast methods in terms of energy and latency-efficiency.

Five phenylbutenoid derivatives from the rhizomes of Zingiber cassumunar Roxb. (Zingiberaceae) were evaluated for their P-glycoprotein (P-gp) inhibitory effects in a P-gp over-expressing multidrug resistant (MDR) human breast cancer cell line, MCF-7/ADR. As a result, a phenylbutenoid dimer, (±)-trans-3-(3,4-dimethoxyphenyl)-4-[(E)-3,4-dimethoxystyryl]cyclohex-1-ene (1), exhibited highly potent P-gp inhibitory activity, decreasing the IC50 value of daunomycin (DNM) to 4.31 ± 0.40 µm in the cells (DNM IC50 = 37.1 ± 0.59 µm). The positive control, verapamil decreased the IC50 value of DNM to 6.94 ± 0.40 µm. Three phenylbutenoid monomers, 2–4 from this plant, also resulted in a significant decrease in the IC50 values of DNM compared with the control. In particular, compound 1 markedly enhanced [3H]-DNM accumulation and significantly reduced [3H]-DNM efflux compared with the control, and this effect was more potent than that of verapamil, a well-known P-gp inhibitor. These results suggest that compound 1 of Z. cassumunar can be developed as a potent chemo-sensitizing agent that reverses P-gp-mediated MDR in human cancer chemotherapy. Copyright © 2008 John Wiley & Sons, Ltd.

To examine whether the epigenetic status of the human Oct4 promoter is similarly regulated in mouse cells, we engineered a human bacterial artificial chromosome to express green fluorescent protein under the control of the hOct4 promoter and stably integrated it into mouse embryonic stem cells (mESCs), NIH3T3, and 293T cells. The hOct4 promoter is unmethylated in mESCs and it undergoes methylation during retinoic acid-induced differentiation. However, the hOct4 promoter is demethylated in NIH3T3 cells even though it is fully methylated in 293T cells. Methylation status of the hOct4 promoter is associated with green fluorescent protein expression at transcription level. Our findings indicate that the hOct4 promoter is differently regulated in mouse cells.

In Saccharomyces cerevisiae, one-step PCR-mediated modification of chromosomal genes allows fast and efficient tagging of yeast proteins with various epitopes at the C- or N-terminus. For many purposes, C-terminal tagging is advantageous in that the expression pattern of epitope tag is comparable to that of the authentic protein and the possibility for the tag to affect normal folding of polypeptide chain during translation is minimized. As experiments are getting complicated, it is often necessary to construct several fusion proteins tagged with various kinds of epitopes. Here, we describe development of a series of plasmids that allow efficient and economical switching of C-terminally tagged epitopes, using just one set of universal oligonucleotide primers. Containing a variety of epitopes (GFP, TAP, GST, Myc, HA and FLAG tag) and Kluyveromyces lactis URA3 gene as a selectable marker, the plasmids can be used to replace any MX6 module-based C-terminal epitope tag with one of the six epitopes. Furthermore, the plasmids also allow additional C-terminal epitope tagging of proteins in yeast cells that already carry MX6 module-based gene deletion or C-terminal epitope tag.

(-)-Syringaresinol and tricin, isolated from the AcOEt-soluble extract of the whole plants of Sasa borealis (Gramineae), showed inhibitory effects on the P-glycoprotein in adriamycin-resistant human breast cancer cells, MCF-7/ADR.