Recent Submissions

  • Geometry-Based Self-Assembly of Histone–DNA Nanostructures at Single-Nucleotide Resolution

    Serag, Maged F.; Aikeremu, Aimaiti; Tsukamoto, Ryoko; Piwonski, Hubert Marek; Abadi, Maram; Kaji, Noritada; Dwyer, Jason R.; Baba, Yoshinobu; Habuchi, Satoshi (ACS Nano, American Chemical Society (ACS), 2019-06-25) [Article]
    Histones are basic protein monomers capable of interacting with DNA, providing the mechanism of DNA compaction inside the cell nucleus. The well-ordered assembly process of histone and DNA is a potential candidate as the approach for building DNA–protein nanostructures. Here, utilizing the sequence-independent histone–DNA interaction, we present an approach to self-assemble histones and single-stranded DNA (ssDNA) to form well-defined histone–DNA (sHD) nanoparticles and their multidimensional cross-linked complexes (cHD). By using various molecular biology and microscopy techniques, we elucidate the structure of these complexes, and we show that they are formed at carefully controlled conditions of temperature, ionic strength, concentration, and incubation time. We also demonstrate using a set of ssDNA molecular rulers and a geometric accommodation model that the assembly of sHD and cHD particles proceeds with precise geometry so that the number of ssDNA in these particles can be programmed by the length of ssDNA. We further show that the formation of cHD amplifies the effect of the length of ssDNA on the self-assembly, allowing for distinguishing ssDNA of different lengths at single nucleotide resolution. We envision that our geometry-directed approach of self-assembling histone–DNA nanostructures and the fundamental insights can serve as a structural platform to advance building precisely ordered DNA–protein nanostructures.
  • Inhibition of autotransporter biogenesis by small molecules

    Steenhuis, Maurice; Abdallah, Abdallah; de Munnik, Sabrina M; Kuhne, Sebastiaan; Sterk, Geert-Jan; van der Berg van Saparoea, Bart; Westerhausen, Sibel; Wagner, Samuel; van der Wel, Nicole N; Wijtmans, Maikel; van Ulsen, Peter; Jong, Wouter S; Luirink, Joen (Molecular Microbiology, Wiley, 2019-04-15) [Article]
    Disarming pathogens by targeting virulence factors is a promising alternative to classic antibiotics. Many virulence factors in Gram-negative bacteria are secreted via the autotransporter (AT) pathway, also known as Type 5 secretion. These factors are secreted with the assistance of two membrane-based protein complexes: Sec and Bam. To identify inhibitors of the AT pathway we used transcriptomics analysis to develop a fluorescence-based high-throughput assay that reports on the stress induced by the model AT hemoglobin protease (Hbp) when its secretion across the outer membrane is inhibited. Screening a library of 1600 fragments yielded the compound VUF15259 that provokes cell envelope stress and secretion inhibition of the ATs Hbp and Antigen-43. VUF15259 also impairs β-barrel folding activity of various outer membrane proteins. Furthermore, we found that mutants that are compromised in outer membrane protein biogenesis are more susceptible to VUF15259. Finally, VUF15259 induces the release of vesicles that appear to assemble in short chains. Taken together, VUF15259 is the first reported compound that inhibits AT secretion and our data are mostly consistent with VUF15259 interfering with the Bam-complex as potential mode of action. The validation of the presented assay incites its use to screen larger compound libraries with drug-like compounds. This article is protected by copyright. All rights reserved.
  • Quantitative Phosphoproteomic Using Titanium Dioxide Micro-Columns and Label-Free Quantitation

    Barrios-Llerena, Martin; Le Bihan, Thierry (Mass Spectrometry of Proteins, Springer Nature, 2019-04-12) [Book Chapter]
    Phosphorylation events are important during cellular function. Analysis of phosphorylation in complex samples has been extensively studied using large-scale phosphopeptide enrichment methods. Quantitative analysis of the enriched phosphopeptides is subsequently performed using label-based methodologies (e.g., SILAC, iTRAQ, and others). Here we describe the protocol for the quantitative analysis of phosphopeptides, enriched with titanium dioxide micro-column, using an intensity-based label-free quantitation.
  • Changes in the Arabidopsis RNA-binding proteome reveal novel stress response mechanisms

    Marondedze, Claudius; Thomas, Ludivine; Gehring, Christoph A; Lilley, Kathryn S. (BMC Plant Biology, Springer Nature, 2019-04-11) [Article]
    BACKGROUND:RNA-binding proteins (RBPs) are increasingly recognized as regulatory component of post-transcriptional gene expression. RBPs interact with mRNAs via RNA-binding domains and these interactions affect RNA availability for translation, RNA stability and turn-over thus affecting both RNA and protein expression essential for developmental and stimulus specific responses. Here we investigate the effect of severe drought stress on the RNA-binding proteome to gain insights into the mechanisms that govern drought stress responses at the systems level. RESULTS:Label-free mass spectrometry enabled the identification 567 proteins of which 150 significantly responded to the drought-induced treatment. A gene ontology analysis revealed enrichment in the
  • Integrated transcriptomic and proteomic analysis of pathogenic mycobacteria and their esx-1 mutants reveal secretion-dependent regulation of ESX-1 substrates and WhiB6 as a transcriptional regulator

    Abdallah, Abdallah; Weerdenburg, Eveline M.; Guan, Qingtian; Ummels, Roy; Borggreve, Stephanie; Adroub, Sabir; Malas, Tareq Majed Yasin; Naeem, Raeece; Zhang, Huoming; Otto, Thomas D.; Bitter, Wilbert; Pain, Arnab (PLOS ONE, Public Library of Science (PLoS), 2019-01-23) [Article]
    The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.
  • Composite epigenetic biomarkers for accurate screening, diagnosis and prognosis of colorectal cancer

    Bajic, Vladimir B.; Incitti, Roberto; Mansour, Hicham (2018-11-22) [Patent]
    The present disclosure concerns particular biomarkers for screening, diagnosing and/or prognosticating colorectal cancer, in particular in an accurate manner. The methods and compositions concern analysis of methylation patterns of one or more of 176 methylatable genomic DNA regions identified as described herein. In particular embodiments there are methods of detecting methylatable regions in genomic sequences.
  • Diagnostic nuclear markers for hybrid Nemos in Kimbe Bay, PNG-Amphiprion chrysopterus x Amphiprion sandaracinos hybrids

    He, Song; Planes, Serge; Sinclair-Taylor, Tane; Berumen, Michael L. (Marine Biodiversity, Springer Nature, 2018-07-09) [Article]
    Hybridization among clownfish (genus Amphiprion) is common in the aquarium trade and has also been reported in natural environments. Putative hybrids between Amphiprion chrysopterus and Amphiprion sandaracinos have been identified in a previous genetic study. However, convenient diagnostic tools for hybrid detection for this case are still missing. During the present study, several potential nuclear markers were tested on both parental species and their hybrids collected from Kimbe Bay, Papua New Guinea. Two diagnostic nuclear markers were found that can confidently discriminate the parent species and their hybrids. A haploweb analysis based on those two diagnostic nuclear markers indicated that one backcrossed hybrid was misclassified as one of the purebred parent species. Identification results from these two diagnostic markers were supported by results from microsatellites, mitochondrial gene markers, and morphological traits analysis.
  • Insights into Brevibacillus borstelensis AK1 through Whole Genome Sequencing: A Thermophilic Bacterium Isolated from a Hot Spring in Saudi Arabia

    Khalil, Amjad B.; Neelamegam, Sivakumar; Arslan, Muhammad; Saleem, Hamna; Alqarawi, Sami (BioMed Research International, Hindawi Limited, 2018-05-24) [Article]
    Brevibacillus borstelensis AK1 is a thermophile which grows between the temperatures of 45°C and 70°C. The present study is an extended genome report of B. borstelensis AK1 along with the morphological characterization. The strain is isolated from a hot spring in Saudi Arabia (southeast of the city Gazan). It is observed that the strain AK1 is rod-shaped, motile, and strictly aerobic bacterium. The whole genome sequence resulted in 29 contigs with a total length of 5,155,092 bp. In total, 3,946 protein-coding genes and 139 RNA genes were identified. Comparison with the previously submitted strains of B. borstelensis strains illustrates that strain AK1 has a small genome size but high GC content. The strain possesses putative genes for degradation of a wide range of substrates including polyethylene (plastic) and long-chain hydrocarbons. These genomic features may be useful for future environmental/biotechnological applications.
  • WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates in pathogenic mycobacteria.

    Abdallah, Abdallah; Weerdenburg, Eveline; Guan, Qingtian; Ummels, Roy; Borggreve, S; Adroub, Sabir; Malas, Tareq Majed Yasin; Naeem, Raeece; Zhang, Huoming; Otto, Thomas; Bitter, Wilbert; Pain, Arnab (Cold Spring Harbor Laboratory, 2018-04-09) [Preprint]
    The mycobacterial type VII secretion system ESX-1 is responsible for the secretion of a number of proteins that play important roles during host infection. The regulation of the expression of secreted proteins is often essential to establish successful infection. Using transcriptome sequencing, we found that the abrogation of ESX-1 function in Mycobacterium marinum leads to a pronounced increase in gene expression levels of the espA operon during the infection of macrophages, suggesting an important role in ESX-1-mediated virulence during the early phase of infection. In addition, the disruption of ESX-1-mediated protein secretion also leads to a specific down-regulation of the ESX-1 substrates, but not of the structural components of this system, during growth in culture medium. This effect is observed in both M. marinum and M. tuberculosis. We established that down-regulation of ESX-1 substrates is the result of a regulatory process that is influenced by the putative transcriptional regulator whib6, which is located adjacent to the esx-1 locus. In addition, the overexpression of the ESX-1-associated PE35/PPE68 protein pair resulted in a significantly increased secretion of the ESX-1 substrate EsxA, demonstrating a functional link between these proteins. Taken together, these data show that WhiB6 is required for the secretion-dependent regulation of ESX-1 substrates and that ESX-1 substrates are regulated independently from the structural components, both during infection and as a result of active secretion.
  • Genome-wide analysis of multi- and extensively drug-resistant Mycobacterium tuberculosis

    Coll, Francesc; Phelan, Jody; Hill-Cawthorne, Grant A.; Nair, Mridul; Mallard, Kim; Ali, Shahjahan; Abdallah, Abdallah; Alghamdi, Saad; Alsomali, Mona; Ahmed, Abdallah O.; Portelli, Stephanie; Oppong, Yaa; Alves, Adriana; Bessa, Theolis Barbosa; Campino, Susana; Caws, Maxine; Chatterjee, Anirvan; Crampin, Amelia C.; Dheda, Keertan; Furnham, Nicholas; Glynn, Judith R.; Grandjean, Louis; Minh Ha, Dang; Hasan, Rumina; Hasan, Zahra; Hibberd, Martin L.; Joloba, Moses; Jones-López, Edward C.; Matsumoto, Tomoshige; Miranda, Anabela; Moore, David J.; Mocillo, Nora; Panaiotov, Stefan; Parkhill, Julian; Penha, Carlos; Perdigão, João; Portugal, Isabel; Rchiad, ‍Zineb; Robledo, Jaime; Sheen, Patricia; Shesha, Nashwa Talaat; Sirgel, Frik A.; Sola, Christophe; Oliveira Sousa, Erivelton; Streicher, Elizabeth M.; Helden, Paul Van; Viveiros, Miguel; Warren, Robert M.; McNerney, Ruth; Pain, Arnab; Clark, Taane G. (Nature Genetics, Springer Nature, 2018-01-16) [Article]
    To characterize the genetic determinants of resistance to antituberculosis drugs, we performed a genome-wide association study (GWAS) of 6,465 Mycobacterium tuberculosis clinical isolates from more than 30 countries. A GWAS approach within a mixed-regression framework was followed by a phylogenetics-based test for independent mutations. In addition to mutations in established and recently described resistance-associated genes, novel mutations were discovered for resistance to cycloserine, ethionamide and para-aminosalicylic acid. The capacity to detect mutations associated with resistance to ethionamide, pyrazinamide, capreomycin, cycloserine and para-aminosalicylic acid was enhanced by inclusion of insertions and deletions. Odds ratios for mutations within candidate genes were found to reflect levels of resistance. New epistatic relationships between candidate drug-resistance-associated genes were identified. Findings also suggest the involvement of efflux pumps (drrA and Rv2688c) in the emergence of resistance. This study will inform the design of new diagnostic tests and expedite the investigation of resistance and compensatory epistatic mechanisms.
  • Methods of analyzing carbon nanostructures, methods of preparation of analytes from carbon nanostructures, and systems for analyzing carbon nanostructures

    COSTA, Pedro Miquel Ferreira Joaquim DA; Patole, Shashikant P.; Yapici, Tahir; Warsama, Bashir H.; Simoes, Filipa Fernandes (2018-01-10) [Patent]
    Provided herein is a method determining the concentration of impurities in a carbon material, comprising: mixing a flux and a carbon material to form a mixture, wherein the carbon material is selected from the group consisting of graphene, carbon nanotubes, fullerene, carbon onions, graphite, carbon fibers, and a combination thereof; heating the mixture using microwave energy to form fused materials; dissolution of the fused materials in an acid mixture; and measuring the concentration of one or more impurities.
  • Evidence for miRNA-mediated modulation of the host transcriptome in cnidarian-dinoflagellate symbiosis

    Baumgarten, Sebastian; Cziesielski, Maha Joana; Thomas, Ludivine; Michell, Craig; Esherick, Lisl Y.; Pringle, John R.; Aranda, Manuel; Voolstra, Christian R. (Molecular Ecology, Wiley, 2017-12-08) [Article]
    Reef-building corals and other cnidarians living in symbiotic relationships with intracellular, photosynthetic dinoflagellates in the genus Symbiodinium undergo transcriptomic changes during infection with the algae and maintenance of the endosymbiont population. However, the precise regulatory mechanisms modulating the host transcriptome are unknown. Here we report apparent post-transcriptional gene regulation by miRNAs in the sea anemone Aiptasia, a model system for cnidarian-dinoflagellate endosymbiosis. Aiptasia encodes mainly species-specific miRNAs, and there appears to have been recent differentiation within the Aiptasia genome of miRNAs that are commonly conserved among anthozoan cnidarians. Analysis of miRNA expression showed that both conserved and species-specific miRNAs are differentially expressed in response to endosymbiont infection. Using cross-linking immunoprecipitation of Argonaute, the central protein of the miRNA-induced silencing complex, we identified miRNA binding sites on a transcriptome-wide scale and found that the targets of the miRNAs regulated in response to symbiosis include genes previously implicated in biological processes related to Symbiodinium infection. Our study shows that cnidarian miRNAs recognize their mRNA targets via high-complementarity target binding and suggests that miRNA-mediated modulations of genes and pathways are important during the onset and maintenance of cnidarian-dinoflagellate endosymbiosis. This article is protected by copyright. All rights reserved.
  • Engineering a promiscuous pyrrolysyl-tRNA synthetase by a high throughput FACS screen

    Hohl, Adrian; Karan, Ram; Gespers (Akal), Anastassja; Renn, Dominik; Liu, Xuechao; Dharmarajnadar, Alaguraj; Ghoprade, Seema Arun; Groll, Michael; Rueping, Magnus; Eppinger, Jörg (Cold Spring Harbor Laboratory, 2017-12-06) [Preprint]
    The Pyrrolysyl-tRNA synthetase (PylRS) and its cognate tRNAPyl are used to facilitate the incorporation of non-canonical amino acids (ncAAs) into the genetic code of bacterial and eukaryotic cells by orthogonally reassigning the amber codon. Currently, the incorporation of new ncAAs requires a cumbersome engineering process composed of several positive and negative selection rounds to select the appropriate PylRS/tRNAPyl pair. Our fast and sensitive engineering approach required only a single FACS selection round to identify 110 orthogonal PylRS variants for the aminoacylation of 20 ncAAs. Pocket-substrate relationship from these variants led to the design of a highly promiscuous PylRS (HpRS), which catalyzed the aminoacylation of 31 structurally diverse lysine derivatives bearing clickable, fluorinated, fluorescent, and biotinylated entities. The high speed and sensitivity of our approach provides a competitive alternative to existing screening methodologies, and delivers insights into the complex PylRS-substrate interactions to facilitate the generation of additional promiscuous variants.
  • Evolution and Strain Variation in BCG

    Abdallah, Abdallah; Behr, Marcel A. (Strain Variation in the Mycobacterium tuberculosis Complex: Its Role in Biology, Epidemiology and Control, Springer Nature, 2017-11-07) [Book Chapter]
    BCG vaccines were derived by in vitro passage, during the years 1908–1921, at the Pasteur Institute of Lille. Following the distribution of stocks of BCG to vaccine production laboratories around the world, it was only a few decades before different BCG producers recognized that there were variants of BCG, likely due to different passaging conditions in the different laboratories. This ultimately led to the lyophilization of stable BCG products in the 1950s and 1960s, but not before considerable evolution of the different BCG strains had taken place. The application of contemporary research methodologies has now revealed genomic, transcriptomic and proteomic differences between BCG strains. These molecular differences in part account for phenotypic differences in vitro between BCG strains, such as their variable secretion of antigenic proteins. Yet, the relevance of BCG variability for immunization policy remains elusive. In this chapter we present an overview of what is known about BCG evolution and its resulting strain variability, and provide some speculation as to the potential relevance for a vaccine given to over 100 million newborns each year.
  • Comparative proteomics and codon substitution analysis reveal mechanisms of differential resistance to hypoxia in congeneric snails

    Mu, Huawei; Sun, Jin; Cheung, Siu Gin; Fang, Ling; Zhou, Haiyun; Luan, Tiangang; Zhang, Huoming; Wong, Chris K.C.; Qiu, Jian-Wen (Journal of Proteomics, Elsevier BV, 2017-11-06) [Article]
    Although high-throughput proteomics has been widely applied to study mechanisms of environmental adaptation, the conclusions from studies that are based on one species can be confounded by phylogeny. We compare the freshwater snail Pomacea canaliculata (a notorious invasive species) and its congener Pomacea diffusa (a non-invasive species) to understand the molecular mechanisms of their differential resistance to hypoxia. A 72-h acute exposure experiment showed that P. canaliculata is more tolerant to hypoxia than P. diffusa. The two species were then exposed to three levels of dissolved oxygen (6.7, 2.0 and 1.0mgL−1) for 8h, and their gill proteins were analyzed using iTRAQ-coupled LC-MS/MS. The two species showed striking differences in protein expression profiles, with the more hypoxia tolerant P. canaliculata having more up-regulated proteins in signal transduction and down-regulated proteins in glycolysis and the tricarboxylic acid cycle. Evolutionary analysis revealed five orthologous genes encoding differentially expressed proteins having clear signal of positive selection, indicating selection has acted on some of the hypoxia responsive genes. Our case study has highlighted the potential of integrated proteomics and comparative evolutionary analysis for understanding the genetic basis of adaptation to global environmental change in non-model species. SignificanceRapid globalization in recent decades has greatly facilitated species introduction around the world. Successfully established introduced species, so-called invasive species, have threatened the invaded ecosystems. There has been substantial interest in studying how invasive species respond to extreme environmental conditions because the results can help not only predict their range of expansion and manage their impact, but also may reveal the adaptive mechanisms underlying their invasiveness. Our study has adopted a comparative approach to study the differential physiological and proteomic responses of two congeneric snails to various hypoxic conditions, as well as codon substitution analysis at transcriptomic level to detect signals of positive selection in hypoxia-responsive genes. The integrated physiological, proteomic and transcriptomic approach can be applied in other non-model species to understand the molecular mechanisms of adaptation to global environmental change.
  • Hollow Co2P nanoflowers organized by nanorods for ultralong cycle-life supercapacitors

    Cheung, Ming Sin; Fan, Hongsheng; Xu, Yingying; Wang, Rongming; Zhang, Xixiang (Nanoscale, Royal Society of Chemistry (RSC), 2017-08-24) [Article]
    Hollow Co2P nanoflowers (Co2P HNF) are successfully prepared via a one-step, template-free method. Microstructure analysis reveals that Co2P HNF is assembled by nanorods, possesses abundant mesopores and a amorphous carbon shell. Density functional theory calculation and electrochemical measurements demonstrate the high electrical conductivity of Co2P. Benefiting from the unique nanostructures, when employed as electrode material for supercapacitors, Co2P HNF exhibits a high specific capacitance, an outstanding rate capability, and an ultralong cycle stability. Furthermore,. the constructed Co2P HNF//AC ASC yields a high energy density of 30.5 Wh kg-1 at a power density of 850 W kg-1, along with an superior cycling performance (108.0% specific capacitance retained after 10000 cycles at 5 A g-1). These impressive results make Co2P HNF a promising candidate for supercapacitor applications.
  • Comparative proteome analysis between C . briggsae embryos and larvae reveals a role of chromatin modification proteins in embryonic cell division

    An, Xiaomeng; Shao, Jiaofang; Zhang, Huoming; Ren, Xiaoliang; Ho, Vincy Wing Sze; Li, Runsheng; Wong, Ming-Kin; Zhao, Zhongying (Scientific Reports, Springer Nature, 2017-06-21) [Article]
    Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.
  • Novel Anoxybacillus flavithermus AK1: A Thermophile Isolated from a Hot Spring in Saudi Arabia

    Khalil, Amjad B.; Neelamegam, Sivakumar; Arslan, Muhammad; Alqarawi, Sami (Arabian Journal for Science and Engineering, Springer Nature, 2017-06-14) [Article]
    Anoxybacillus flavithermus AK1 is a thermophilic bacterium that is able to survive at temperatures ranging from 55 to 60∘C. The AK1 strain was isolated from the hot spring “Al-Ain Alhara” located at a distance of 50 km southeast of the city of Gazan, Saudi Arabia. This study presents the morphological characterization of A. flavithermus AK1, including a detailed description of its complete genome sequence. A total of 50 contigs were used to produce a genome sequence of 2,630,664 bp that includes 2724 protein-coding genes and 75 RNA genes, 18 of which are rRNA genes. A comparison of this genome sequence with those of Anoxybacillus flavithermus strains that were previously submitted to NCBI revealed that the AK1 strain has the smallest genome size with the highest GC content. The strain can therefore be exploited for several biotechnological applications based on its high thermophilic potential.
  • Role of G3BP1 in glucocorticoid receptor-mediated microRNA-15b and microRNA-23a biogenesis in endothelial cells

    Kwok, Hoi-Hin; Poon, Po-Ying; Mak, Kylie Hin-Man; Zhang, Lin-Yao; Liu, Pei-Nian; Zhang, Huoming; Mak, Nai-Ki; Yue, Patrick Ying-Kit; Wong, Ricky Ngok-Shun (Cellular and Molecular Life Sciences, Springer Nature, 2017-05-18) [Article]
    MicroRNAs (miRNAs) are a family of non-coding RNAs that play crucial roles in regulating various normal cellular responses. Recent studies revealed that the canonical miRNA biogenesis pathway is subject to sophisticated regulation. Hormonal control of miRNA biogenesis by androgen and estrogen has been demonstrated, but the direct effects of the glucocorticoid receptor (GR) on miRNA biogenesis are unknown. This study revealed the role of GR in miRNA maturation. We showed that two GR agonists, dexamethasone and ginsenoside-Rg1 rapidly suppressed the expression of mature miR-15b, miR-23a, and miR-214 in human endothelial cells. RNA pulldown coupled with proteomic analysis identified GTPase-activating protein (SH3 domain) binding protein 1 (G3BP1) as one of the RNA-binding proteins mediating GR-regulated miRNA maturation. Activated GR induced phosphorylation of v-AKT Murine Thymoma Viral Oncogene Homologue (AKT) kinase, which in turn phosphorylated and promoted nuclear translocation of G3BP1. The nuclear G3BP1 bound to the G3BP1 consensus sequence located on primary miR-15b~16-2 and miR-23a~27a~24-2 to inhibit their maturation. The findings from this study have advanced our understanding of the non-genomic effects of GR in the vascular system.
  • Characterization of Recombinant Thermococcus kodakaraensis (KOD) DNA Polymerases Produced Using Silkworm-Baculovirus Expression Vector System

    Yamashita, Mami; Xu, Jian; Morokuma, Daisuke; Hirata, Kazuma; Hino, Masato; Mon, Hiroaki; Takahashi, Masateru; Hamdan, Samir; Sakashita, Kosuke; Iiyama, Kazuhiro; Banno, Yutaka; Kusakabe, Takahiro; Lee, Jae Man (Molecular Biotechnology, Springer Nature, 2017-05-08) [Article]
    The KOD DNA polymerase from Thermococcus kodakarensis (Tkod-Pol) has been preferred for PCR due to its rapid elongation rate, extreme thermostability and outstanding fidelity. Here in this study, we utilized silkworm-baculovirus expression vector system (silkworm-BEVS) to express the recombinant Tkod-Pol (rKOD) with N-terminal (rKOD-N) or C-terminal (rKOD-C) tandem fusion tags. By using BEVS, we produced functional rKODs with satisfactory yields, about 1.1 mg/larva for rKOD-N and 0.25 mg/larva for rKOD-C, respectively. Interestingly, we found that rKOD-C shows higher thermostability at 95 °C than that of rKOD-N, while that rKOD-N is significantly unstable after exposing to long period of heat-shock. We also assessed the polymerase activity as well as the fidelity of purified rKODs under various conditions. Compared with commercially available rKOD, which is expressed in E. coli expression system, rKOD-C exhibited almost the same PCR performance as the commercial rKOD did, while rKOD-N did lower performance. Taken together, our results suggested that silkworm-BEVS can be used to express and purify efficient rKOD in a commercial way.

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