Recent Submissions

  • Investigations of crude-oil emulsions at the micro-to-nano scales

    Ravaux, Florent; Medina, Sandra Constanza; Behzad, Ali Reza; Zafar, Humaira; George, Abraham; Morin, Stephane; Ghaffour, NorEddine; Anjum, Dalaver H. (Fuel, Elsevier BV, 2021-12) [Article]
    The removal of the micro droplets of emulsified water from crude oil causes high cost and energy. In this paper we show that cryo electron microscopy (cryo-EM) imaging of micro and nano emulsions prepared from United Arab Emirate based crude oil provides critical information on their stability. Specifically, the cryoSEM imaging analysis applied to emulsion of murban-2019, and upper zakum-2019 crude-oils allowed determining naturally occurring surfactants in these crude-oils. Moreover, the applied method also turned out to be an efficient way to qualitatively investigate the effect of synthetic surfactant on the stability of the emulsions. The high resolution cryoTEM imaging analysis of emulsions from upper zakum-2019 sample enabled visualizing “bilayer” of naturally occurring surfactants, presumably the asphaltene. The cryoTEM analysis further allowed estimating the volume-fraction of emulsified water in the crude-oil at nanoscales and turned out to be about 1% for the upper zakum-2019 samples.
  • CDKL5 kinase controls transcription-coupled responses to DNA damage

    Khanam, Taran; Muñoz, Ivan; Weiland, Florian; Carroll, Thomas; Morgan, Michael; Borsos, Barbara N.; Pantazi, Vasiliki; Slean, Meghan; Novak, Miroslav; Toth, Rachel; Appleton, Paul; Pankotai, Tibor; Zhou, Houjiang; Rouse, John (The EMBO Journal, EMBO, 2021-10-04) [Article]
    Mutations in the gene encoding the CDKL5 kinase are among the most common genetic causes of childhood epilepsy and can also give rise to the severe neurodevelopmental condition CDD (CDKL5 deficiency disorder). Despite its importance for human health, the phosphorylation targets and cellular roles of CDKL5 are poorly understood, especially in the cell nucleus. Here, we report that CDKL5 is recruited to sites of DNA damage in actively transcribed regions of the nucleus. A quantitative phosphoproteomic screen for nuclear CDKL5 substrates reveals a network of transcriptional regulators including Elongin A (ELOA), phosphorylated on a specific CDKL5 consensus motif. Recruitment of CDKL5 and ELOA to damaged DNA, and subsequent phosphorylation of ELOA, requires both active transcription and the synthesis of poly(ADP-ribose) (PAR), to which CDKL5 can bind. Critically, CDKL5 kinase activity is essential for the transcriptional silencing of genes induced by DNA double-strand breaks. Thus, CDKL5 is a DNA damage-sensing, PAR-controlled transcriptional modulator, a finding with implications for understanding the molecular basis of CDKL5-related diseases.
  • LeafGo: Leaf to Genome, a quick workflow to produce high-quality de novo plant genomes using long-read sequencing technology.

    Driguez, Patrick; Bougouffa, Salim; Carty, Karen; Putra, Alexander; Jabbari, Kamel; Reddy, Muppala P.; Soppe, Richard Willem Otto; Cheung, Ming Sin; Fukasawa, Yoshinori; Ermini, Luca (Genome biology, Springer Science and Business Media LLC, 2021-09-03) [Article]
    Currently, different sequencing platforms are used to generate plant genomes and no workflow has been properly developed to optimize time, cost, and assembly quality. We present LeafGo, a complete de novo plant genome workflow, that starts from tissue and produces genomes with modest laboratory and bioinformatic resources in approximately 7 days and using one long-read sequencing technology. LeafGo is optimized with ten different plant species, three of which are used to generate high-quality chromosome-level assemblies without any scaffolding technologies. Finally, we report the diploid genomes of Eucalyptus rudis and E. camaldulensis and the allotetraploid genome of Arachis hypogaea.
  • Microscopy techniques applied to submicron characterization of oilfield produced water

    Medina, Sandra Constanza; Anjum, Dalaver H.; Behzad, Ali Reza; Vilagines, Regis D.; Tabatabai, S. Assiyeh Alizadeh; Leiknes, TorOve (Journal of Petroleum Science and Engineering, Elsevier BV, 2021-05-26) [Article]
    Produced water (PW) and formation water are complex mixtures of hydrocarbons and water produced at oil and gas upstream facilities. Submicron oil droplets represent a multitude of issues affecting the performance of downstream advanced water treatment processes, such as micro and ultra-filtration processes. Conventional de-oiling technologies do not efficiently remove submicron oil droplets in PW. An accurate characterization of submicron oil droplets and contaminants is required to improve PW treatment technology. In this study, a methodology for visualization and quantification of submicron oil droplets size distribution (DSD), using optical and electron microscopy techniques, was developed. Various microscopy techniques were evaluated, including epifluorescence microscopy (EpiFM), confocal laser scanning microscopy (CLSM), cryogenic scanning and transmission electron microscopy (cryo-SEM and cryo-TEM, respectively). Synthetic PW was used to improve and standardize the sample preparation and characterization methodology. The improved methodology was then tested with two PW samples from different oilfields in the Middle East region. Two methods were developed for the determination of DSD in oilfield PW samples. The first method is suitable for highly polydisperse PW samples with oil droplets larger than 250 nm. This method is based on using low-temperature agarose to immobilize the samples, avoiding coalescence, and allowing clear visualization of the oil droplets at high magnification in EpiFM. The second method is suitable for concentrated PW samples and oil droplets as small as 20 nm in size. This method is based on cryo-TEM with plunge freezing and without the use of agarose for sample immobilization. The agarose-immobilization technique was also applied for sample preparation in cryo-SEM. Cryo-SEM fixation by high-pressure freezing (HPF) preserved the morphology of oil droplets in synthetic oil-concentrated samples and allowed its visualization in a wide range of sizes from 50 nm up to 20 μm.
  • Giant clam inspired high-speed photo-conversion for ultraviolet optical wireless communication

    Subedi, Ram Chandra; Rossbach, Susann; Kang, Chun Hong; Alkhazragi, Omar; Sun, Xiaobin; Holguin Lerma, Jorge Alberto; Mitra, Somak; Roqan, Iman S.; Behzad, Ali Reza; Sougrat, Rachid; Ng, Tien Khee; Bradley, Donal; Duarte, Carlos M.; Ooi, Boon S. (Optical Materials Express, The Optical Society, 2021-04-22) [Article]
    Organisms have evolved the ability to manipulate light for vision, as a means to capture its energy, to protect themselves from damage, especially against ultraviolet (UV) and other high flux radiation, and for display purposes. The makeup of the structural elements used for this manipulation often discloses novel pathways for man-made photonic devices. Iridocytes in the mantle of giant clams in the Tridacninae subfamily manipulate light in many ways, e.g., as reflectors, scattering centers, and diffusers. There is, however, a void in understanding the absorption and photoluminescence (PL) emission dynamics of these cells. In turn, a profound understanding of iridocytes’ photophysics can offer the prospect for a new generation of advanced optoelectronic materials and devices. Here, the structural and optical properties of the iridocytes embedded in the mantle tissue of the Tridacna maxima are investigated and their use as a high-speed color convertor for UV photodetection, well-suited to application in UV optical wireless communication, is demonstrated.
  • Citrullination of Proteins as a Specific Response Mechanism in Plants.

    Marondedze, Claudius; Elia, Giuliano; Thomas, Ludivine; Wong, Aloysius; Gehring, Christoph A (Frontiers in plant science, Frontiers Media SA, 2021-04-08) [Article]
    Arginine deimination, also referred to as citrullination of proteins by L-arginine deiminases, is a post-translational modification affecting histone modifications, epigenetic transcriptional regulation, and proteolysis in animals but has not been reported in higher plants. Here we report, firstly, that Arabidopsis thaliana proteome contains proteins with a specific citrullination signature and that many of the citrullinated proteins have nucleotide-binding regulatory functions. Secondly, we show that changes in the citrullinome occur in response to cold stress, and thirdly, we identify an A. thaliana protein with peptidyl arginine deiminase activity that was shown to be calcium-dependent for many peptide substrates. Taken together, these findings establish this post-translational modification as a hitherto neglected component of cellular reprogramming during stress responses.
  • LeafGo - Eucalyptus and Peanut genome sequencing

    Driguez, Patrick; Bougouffa, Salim; Carty, Karen; Putra, Alexander; Jabbari, Kamel; Reddy, Muppala P.; Soppe, Richard Willem Otto; Cheung, Ming Sin; Fukasawa, Yoshinori; Ermini, Luca (NCBI, 2020-11-05) [Bioproject, Dataset]
    The sequencing and the assembly of chromosome-level genomes following the LeafGo protocol using a single long read technology.
  • 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 Nature, 2020-07-17) [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.
  • Immunomodulatory and antineoplastic efficacy of common spices and their connection with phenolic antioxidants

    Xu, Baojun; Ganesan, Kumar; Mickymaray, Suresh; Alfaiz3, Faiz Abdulaziz; Thatchinamoorthi, Rajarajan; Aboody, Mohammed Saleh Al (Bioactive Compounds in Health and Disease, Functional Food Center, 2020-02-28) [Article]
    Background: Spices have generally offered a conventional way to avert and heal various communicable and non-communicable diseases due to their efficacy and safety and their noteworthy contribution towards understanding targeted drug action and drug delivery systems. Hence, the current investigation is designed to evaluate the immunomodulatory and antineoplastic efficacy of 15 spices that connect with the flavonoid and total polyphenol ingredients. This study includes the 15 adopted spices and their total flavonoid and polyphenol contents, cell viability assay (MTT), immunomodulatory efficacy (NO, TNF-α), and antineoplastic efficacy (using six cancer cell lines).Methods: The quantification of the flavonoid and phenolic content of methanolic extracts of 15 spices was performed by colorimetric assay. The immunomodulatory efficacy was studied according to their capacity to prevent NO and TNF-α synthesis in LPS stimulated RAW 264.7 macrophages. Cell viability was observed using MTT colorimetric assay. Antineoplastic efficacy was determined with six cancer cell lines, namely liver (HepG2), colon (HT29), breast (MCF7), pancreas (MIA PaCa2), lung (A549) and blood (Raji). Results: The outcome of significant immunomodulatory efficacy of the spices was noted in the following sequences: Acorus calamus L.(Inhibition of NO-49.32 ± 4.29 µg/mL and TNF-α 96.35 ± 8.23 µg/mL)> Alpinia galanga Wild (Inhibition of NO-55.69 ± 5.89µg/mL and TNF-α 102.36 ± 8.96 µg/mL)> Armoracia rusticana Gaerth (Inhibition of NO-82.44 ± 5.98 µg/mL and TNF-α 115.69 ± 7.59)> Capparis spinosa L. (Inhibition of NO-127.59 ± 5.68 µg/mL and TNF-α 123.58 ± 8.56 µg/mL) > Aframomum melegueta K. Schum (Inhibition of NO-169.89 ± 6.89 µg/mL and TNF-α 144.59 ± 7.89 µg/mL). The remaining spices considerably inhibited the generation of NO and TNF-α. All spices studied exhibited highly significant antineoplastic effects against all six cell lines. Noteworthy biological activities were observed in A. calamus, A. galanga, A. rusticana, C. spinose, and A. melegueta which have bulk quantities of polyphenols.Conclusion: Based on the present findings, spices are possible candidates for novel antioxidant, anti-inflammatory, and antineoplastic agents.Keywords: Spices; cancer cell lines; immunomodulatory; antineoplastic; total polyphenol contents
  • 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 Nature, 2020-02-21) [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.
  • LongQC: A Quality Control Tool for Third Generation Sequencing Long Read Data.

    Fukasawa, Yoshinori; Ermini, Luca; Wang, Hai; Carty, Karen; Cheung, Ming Sin (G3 Genes|Genomes|Genetics, Genetics Society of America, 2020-02-10) [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.
  • 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

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