Recent Submissions

  • Arginine citrullination of proteins as a specific response mechanism in Arabidopsis thaliana

    Marondedze, Claudius; Elia, Giuliano; Thomas, Ludivine; Wong, Aloysius; Gehring, Christoph A (Cold Spring Harbor Laboratory, 2020-09-13) [Preprint]
    Arginine citrullination, also referred to as arginine deimination, is a post-translational modification involved in an increasing number of physiological processes in animals, including histone modifications and transcriptional regulation, and in severe diseases such as rheumatoid arthritis and neurodegenerative conditions. It occurs when arginine side chains are deiminated and converted into side chains of the amino acid citrulline, a process catalysed by a family of Ca2+-dependent peptidyl arginine deiminases (PADs). PADs have been discovered in several mammalian species and in other vertebrates, like birds and fish, but have not been observed in bacteria, lower eukaryotes or higher plants. Here we show, firstly, that the Arabidopsis thaliana proteome does contain citrullinated proteins; secondly and importantly, that the citrullination signature changes in response to cold stress. Among the citrullinated proteins are DNA- or RNA-binding proteins thus implying a role for it the control of the transcriptional programming in plant cells. Thirdly, through sequence and structural analysis, we identify one arabidopsis protein, currently annotated as agmatine deiminase (At5g08170), as a candidate protein arginine deiminase. Finally, we show biochemical evidence that AT5G08170 can citrullinate peptides from LHP1-interacting factor 2 (AT4G00830) an RNA-binding protein that has been identified as citrullinated in cell suspension cultures of Arabidopsis thaliana roots. In addition, we show that, in vitro, agmatine deiminase can undergo auto-citrullination. In conclusion, our work established the presence of protein arginine citrullination in higher plants and assigns it a role in post-translational modifications during abiotic stress responses.
  • What is the right sequencing approach? Solo VS extended family analysis in consanguineous populations.

    Alfares, Ahmed; Alsubaie, Lamia; Aloraini, Taghrid; Alaskar, Aljoharah; Althagafi, Azza Th.; Alahmad, Ahmed; Rashid, Mamoon; Alswaid, Abdulrahman; Alothaim, Ali; Eyaid, Wafaa; Ababneh, Faroug; Albalwi, Mohammed; Alotaibi, Raniah; Almutairi, Mashael; Altharawi, Nouf; Alsamer, Alhanouf; Abdelhakim, Marwa; Kafkas, Senay; Mineta, Katsuhiko; Cheung, Nicole; Abdallah, Abdallah; Büchmann-Møller, Stine; Fukasawa, Yoshinori; Zhao, Xiang; Rajan, Issaac; Hoehndorf, Robert; Al Mutairi, Fuad; Gojobori, Takashi; Alfadhel, Majid (BMC medical genomics, Springer Science and Business Media LLC, 2020-07-19) [Article]
    BACKGROUND:Testing strategies is crucial for genetics clinics and testing laboratories. In this study, we tried to compare the hit rate between solo and trio and trio plus testing and between trio and sibship testing. Finally, we studied the impact of extended family analysis, mainly in complex and unsolved cases. METHODS:Three cohorts were used for this analysis: one cohort to assess the hit rate between solo, trio and trio plus testing, another cohort to examine the impact of the testing strategy of sibship genome vs trio-based analysis, and a third cohort to test the impact of an extended family analysis of up to eight family members to lower the number of candidate variants. RESULTS:The hit rates in solo, trio and trio plus testing were 39, 40, and 41%, respectively. The total number of candidate variants in the sibship testing strategy was 117 variants compared to 59 variants in the trio-based analysis. We noticed that the average number of coding candidate variants in trio-based analysis was 1192 variants and 26,454 noncoding variants, and this number was lowered by 50-75% after adding additional family members, with up to two coding and 66 noncoding homozygous variants only, in families with eight family members. CONCLUSION:There was no difference in the hit rate between solo and extended family members. Trio-based analysis was a better approach than sibship testing, even in a consanguineous population. Finally, each additional family member helped to narrow down the number of variants by 50-75%. Our findings could help clinicians, researchers and testing laboratories select the most cost-effective and appropriate sequencing approach for their patients. Furthermore, using extended family analysis is a very useful tool for complex cases with novel genes.
  • LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data.

    Fukasawa, Yoshinori; Ermini, Luca; Wang, Hai; Carty, Karen; Cheung, Ming Sin (G3 (Bethesda, Md.), Genetics Society of America, 2020-02-12) [Article]
    We propose LongQC as an easy and automated quality control tool for genomic datasets generated by third generation sequencing (TGS) technologies such as Oxford Nanopore technologies (ONT) and SMRT sequencing from Pacific Bioscience (PacBio). Key statistics were optimized for long read data, and LongQC covers all major TGS platforms. LongQC processes and visualizes those statistics automatically and quickly.
  • A landscape of genomic alterations at the root of a near-untreatable tuberculosis epidemic.

    Klopper, Marisa; Heupink, Tim Hermanus; Hill-Cawthorne, Grant; Streicher, Elizabeth Maria; Dippenaar, Anzaan; De Vos, Margaretha; Abdallah, Abdallah; Limberis, Jason; Merker, Matthias; Burns, Scott; Niemann, Stefan; Dheda, Keertan; Posey, James; Pain, Arnab; Warren, Robin Mark (BMC medicine, Springer Science and Business Media LLC, 2020-02-05) [Article]
    BACKGROUND:Atypical Beijing genotype Mycobacterium tuberculosis strains are widespread in South Africa and have acquired resistance to up to 13 drugs on multiple occasions. It is puzzling that these strains have retained fitness and transmissibility despite the potential fitness cost associated with drug resistance mutations. METHODS:We conducted Illumina sequencing of 211 Beijing genotype M. tuberculosis isolates to facilitate the detection of genomic features that may promote acquisition of drug resistance and restore fitness in highly resistant atypical Beijing forms. Phylogenetic and comparative genomic analysis was done to determine changes that are unique to the resistant strains that also transmit well. Minimum inhibitory concentration (MIC) determination for streptomycin and bedaquiline was done for a limited number of isolates to demonstrate a difference in MIC between isolates with and without certain variants. RESULTS:Phylogenetic analysis confirmed that two clades of atypical Beijing strains have independently developed resistance to virtually all the potent drugs included in standard (pre-bedaquiline) drug-resistant TB treatment regimens. We show that undetected drug resistance in a progenitor strain was likely instrumental in this resistance acquisition. In this cohort, ethionamide (ethA A381P) resistance would be missed in first-line drug-susceptible isolates, and streptomycin (gidB L79S) resistance may be missed due to an MIC close to the critical concentration. Subsequent inadequate treatment historically led to amplification of resistance and facilitated spread of the strains. Bedaquiline resistance was found in a small number of isolates, despite lack of exposure to the drug. The highly resistant clades also carry inhA promoter mutations, which arose after ethA and katG mutations. In these isolates, inhA promoter mutations do not alter drug resistance, suggesting a possible alternative role. CONCLUSION:The presence of the ethA mutation in otherwise susceptible isolates from ethionamide-naïve patients demonstrates that known exposure is not an adequate indicator of drug susceptibility. Similarly, it is demonstrated that bedaquiline resistance can occur without exposure to the drug. Inappropriate treatment regimens, due to missed resistance, leads to amplification of resistance, and transmission. We put these results into the context of current WHO treatment regimens, underscoring the risks of treatment without knowledge of the full drug resistance profile.
  • Drought Stress Causes Specific Changes to the Spliceosome and Stress Granule Components

    Marondedze, Claudius; Thomas, Ludivine; Lilley, Kathryn S.; Gehring, Christoph A (Frontiers in Molecular Biosciences, Frontiers Media SA, 2020-01-21) [Article]
    The spliceosome processes RNAs from a pre-RNA state to a mature mRNA thereby influencing RNA availability for translation, localization, and turnover. It consists of complex structures containing RNA-binding proteins (RBPs) essential for post-transcriptional gene expression control. Here we investigate the dynamic modifications of spliceosomal RBPs under stress and in particular drought stress. We do so by mRNA interactome capture in Arabidopsis thaliana using label free quantitation. This approach identified 44 proteins associated with the spliceosome and further 32 proteins associated with stress granules. We noted a high enrichment in the motifs RDRR and RSRSRS that are characteristic of RNA interacting proteins. Identification of splicing factors reflect direct and/or indirect stress induced splicing events that have a direct effect on transcriptome and proteome changes under stress. Furthermore, detection of stress granule components is consistent with transcriptional arrest. Identification of drought induced stress granule components is critical in determining common abiotic stress-induced foci that can have biotechnological applications. This study may therefore open ways to modify plant stress responses at a systems level through the modification of key spliceosome components.
  • Mutagenesis-Based Characterization and Improvement of a Novel Inclusion Body Tag

    Jong, Wouter S.P.; ten Hagen-Jongman, Corinne M.; Vikström, David; Dontje, Wendy; Abdallah, Abdallah; de Gier, Jan Willem; Bitter, Wilbert; Luirink, Joen (Frontiers in Bioengineering and Biotechnology, Frontiers Media SA, 2020-01-10) [Article]
    Whereas, bacterial inclusion bodies (IBs) for long were regarded as undesirable aggregates emerging during recombinant protein production, they currently receive attention as promising nanoparticulate biomaterials with diverse applications in biotechnology and biomedicine. We previously identified ssTorA, a signal sequence that normally directs protein export via the Tat pathway in E. coli, as a tag that induces the accumulation of fused proteins into IBs under overexpression conditions. Here, we used targeted mutagenesis to identify features and motifs being either critical or dispensable for IB formation. We found that IB formation is neither related to the function of ssTorA as a Tat-signal sequence nor is it a general feature of this family of signal sequences. IB formation was inhibited by co-overexpression of ssTorA binding chaperones TorD and DnaK and by amino acid substitutions that affect the propensity of ssTorA to form an α-helix. Systematic deletion experiments identified a minimal region of ssTorA required for IB formation in the center of the signal sequence. Unbiased genetic screening of a library of randomly mutagenized ssTorA sequences for reduced aggregation properties allowed us to pinpoint residues that are critical to sustain insoluble expression. Together, the data point to possible mechanisms for the aggregation of ssTorA fusions. Additionally, they led to the design of a tag with superior IB-formation properties compared to the original ssTorA sequence.
  • What is the right sequencing approach? Solo VS extended family analysis in consanguineous populations

    Alfares, Ahmed; Alsubaie, Lamia; Aloraini, Taghrid; Alaskar, Aljoharah; Althagafi, Azza Th.; Alahmad, Ahmed; Rashid, Mamoon; Alswaid, Abdulrahman; Alothaim, Ali; Eyaid, Wafaa; Ababneh, Faroug; Albalwi, Mohammed; Alotaibi, Raniah; Almutairi, Mashael; Altharawi, Nouf; Alsamer, Alhanouf; Abdelhakim, Marwa; Kafkas, Senay; Mineta, Katsuhiko; Cheung, Nicole; Abdallah, Abdallah; Büchmann-Møller, Stine; Fukasawa, Yoshinori; Zhao, Xiang; Rajan, Issaac; Hoehndorf, Robert; Al Mutairi, Fuad; Gojobori, Takashi; Alfadhel, Majid (figshare, 2020) [Dataset]
    Abstract Background Testing strategies is crucial for genetics clinics and testing laboratories. In this study, we tried to compare the hit rate between solo and trio and trio plus testing and between trio and sibship testing. Finally, we studied the impact of extended family analysis, mainly in complex and unsolved cases. Methods Three cohorts were used for this analysis: one cohort to assess the hit rate between solo, trio and trio plus testing, another cohort to examine the impact of the testing strategy of sibship genome vs trio-based analysis, and a third cohort to test the impact of an extended family analysis of up to eight family members to lower the number of candidate variants. Results The hit rates in solo, trio and trio plus testing were 39, 40, and 41%, respectively. The total number of candidate variants in the sibship testing strategy was 117 variants compared to 59 variants in the trio-based analysis. We noticed that the average number of coding candidate variants in trio-based analysis was 1192 variants and 26,454 noncoding variants, and this number was lowered by 50–75% after adding additional family members, with up to two coding and 66 noncoding homozygous variants only, in families with eight family members. Conclusion There was no difference in the hit rate between solo and extended family members. Trio-based analysis was a better approach than sibship testing, even in a consanguineous population. Finally, each additional family member helped to narrow down the number of variants by 50–75%. Our findings could help clinicians, researchers and testing laboratories select the most cost-effective and appropriate sequencing approach for their patients. Furthermore, using extended family analysis is a very useful tool for complex cases with novel genes.
  • Supplemental Material for LongQC: A quality control tool for third generation sequencing long read data, 2020

    Fukasawa, Yoshinori; Ermini, Luca; Wang, Hai; Carty, Karen; Cheung, Ming Sin (GSA Journals, 2020) [Dataset]
    This file is the supplementary information of the article titled "LongQC: A quality control tool for third generation sequencing long read data".Supplementary Materials and Methods, Supplementary Figures and Tables, and example plots of the software with brief explanations are all included in this file.
  • 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, 2019-05-03) [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, 2019-04-13) [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.
  • Changes in the Arabidopsis RNA-binding proteome reveal novel stress response mechanisms

    Marondedze, Claudius; Thomas, Ludivine; Gehring, Christoph A; Lilley, Kathryn S. (figshare, 2019) [Dataset]
    Abstract 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 “RNA binding” and “RNA processing” categories as well as biological processes such as “response to abscisic acid” and “response to water deprivation”. Importantly, a large number of the stress responsive proteins have not previously been identified as RBPs and include proteins in carbohydrate metabolism and in the glycolytic and citric acid pathways in particular. This suggests that RBPs have hitherto unknown roles in processes that govern metabolic changes during stress responses. Furthermore, a comparative analysis of RBP domain architectures shows both, plant specific and common domain architectures between plants and animals. The latter could be an indication that RBPs are part of an ancient stress response. Conclusion This study establishes mRNA interactome capture technique as an approach to study stress signal responses implicated in environmental changes. Our findings denote RBP changes in the proteome as critical components in plant adaptation to changing environments and in particular drought stress protein-dependent changes in RNA metabolism.
  • 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, 2018-01-22) [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.

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