- A molecular biologist with electrical and electronic engineering beginningedit
It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational... more
It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with ~90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite, and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a smal...
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The formation of viroplasms is a well-conserved step in the rotavirus (RV) life cycle. In these structures, both virus genome replication and progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are... more
The formation of viroplasms is a well-conserved step in the rotavirus (RV) life cycle. In these structures, both virus genome replication and progeny assembly take place. A stabilized microtubule cytoskeleton and lipid droplets are required for the viroplasm formation, which involves several virus proteins. The viral spike protein VP4 has not previously been shown to have a direct role in viroplasm formation. However, it is involved with virus-cell attachment, endocytic internalization, and virion morphogenesis. Moreover, VP4 interacts with actin cytoskeleton components, mainly in processes involving virus entrance and egress, and thereby may have an indirect role in viroplasm formation. In this study, we used reverse genetics to construct a recombinant RV, rRV/VP4-BAP, which contains a biotin acceptor peptide (BAP) in the K145-G150 loop of the VP4 lectin domain, permitting live monitoring. The recombinant virus was replication competent but showed a reduced fitness. We demonstrate ...
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Additional file 4: Figure S1. (a) Schematic representation of transcript isoforms of VEGFR1. (UniProtKB-P17948). In red are marked identical N-terminus coding sequences, while in yellow the C-terminus unique sequences belonging to... more
Additional file 4: Figure S1. (a) Schematic representation of transcript isoforms of VEGFR1. (UniProtKB-P17948). In red are marked identical N-terminus coding sequences, while in yellow the C-terminus unique sequences belonging to splicing isoforms. Specific primers (depicted in dotted lines) were designed in the unique regions to discriminate the different isoforms by qRT-PCR. (b) Agarose gel shows PCR products of VEGFR1 isoforms analysed in HUVEC; GAPDH was used as housekeeping gene. PCR product sizes were expressed in base pair (bp). (c) In human degenerated IVD tissues (n = 11) and (d) monolayer disc cell cultures (n = 11) mVEGFR1 and sVEGFR1 were similarly expressed. Soluble variants isoform 3 and isoform 4 were not detected by qRT-PCR. Box plots represent relative mRNA expression normalized on GAPDH in annulus fibrosus (AF) and nucleus pulposus (NP). The line across the box indicates the median, bubbles indicate outliers, stars indicate extreme values. No statistical significa...
Additional file 3: Table S3. Microarray samples information.
Additional file 2: Table S2. Human primers used in qRT-PCR.
Additional file 1: Table S1. Demographic details of Intervertebral disc donors.
Additional file 6: Table S5. Expression profiles of genes of abnormal angiogenesis signalling pathway.
Additional file 5: Table S4. Expression profiles of genes of classic angiogenesis signalling pathway.
Trypanosoma brucei undergoes major biochemical and morphological changes during its development from the bloodstream form in the mammalian host to the procyclic form in the midgut of its insect host. The underlying regulation of gene... more
Trypanosoma brucei undergoes major biochemical and morphological changes during its development from the bloodstream form in the mammalian host to the procyclic form in the midgut of its insect host. The underlying regulation of gene expression, however, is poorly understood. More than 60% of the predicted genes remain annotated as hypothetical, and the 5' and 3' untranslated regions important for regulation of gene expression are unknown for >90% of the genes. In this review, we compare the data from four recently published high-throughput RNA sequencing studies in light of the different experimental setups and discuss how these data can enhance genome annotation and give insights into the regulation of gene expression in T. brucei.
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Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the... more
Trans-splicing of leader sequences onto the 5′ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5′splice sites and analyze the corresponding gene expression profile that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5′ splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.
Research Interests: Microbiology, Immunology, Medical Microbiology, Biology, Medicine, and 11 moreGene expression, Alternative splicing, Trypanosoma brucei, Trypanosoma brucei brucei, Alternative Splicing, Base Sequence, Gene expression profiling, Differential expression, Molecular Sequence Data, PLOs, and reverse transcriptase polymerase chain reaction
It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational... more
It has long been known that trypanosomes regulate mitochondrial biogenesis during the life cycle of the parasite; however, the mitochondrial protein inventory (MitoCarta) and its regulation remain unknown. We present a novel computational method for genome-wide prediction of mitochondrial proteins using a support vector machine-based classifier with ∼90% prediction accuracy. Using this method, we predicted the mitochondrial localization of 468 proteins with high confidence and have experimentally verified the localization of a subset of these proteins. We then applied a recently developed parallel sequencing technology to determine the expression profiles and the splicing patterns of a total of 1065 predicted MitoCarta transcripts during the development of the parasite and showed that 435 of the transcripts significantly changed their expressions while 630 remain unchanged in any of the three life stages analyzed. Furthermore, we identified 298 alternatively splicing events, a small subset of which could lead to dual localization of the corresponding proteins.
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Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional... more
Unlike other eukaryotes, the protein-coding genes of Trypanosoma cruzi are arranged in large polycistronic gene clusters transcribed by polymerase II (Pol II). Thus, it is thought that trypanosomes rely solely on posttranscriptional processes to regulate gene expression. Here, we show that the glucosylated thymine DNA base (β- d -glucosyl-hydroxymethyluracil or base J) is present within sequences flanking the polycistronic units (PTUs) in T. cruzi. The loss of base J at sites of transcription initiation, via deletion of the two enzymes that regulate base J synthesis (JBP1 and JBP2), correlates with an increased rate of Pol II transcription and subsequent genome-wide increase in gene expression. The affected genes include virulence genes, and the resulting parasites are defective in host cell invasion and egress. These studies indicate that base J is an epigenetic factor regulating Pol II transcription initiation in kinetoplastids and provides the first biological role of the only hy...
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Background Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology. Results Using Stable... more
Background Trypanosoma brucei is the causative agent of human African sleeping sickness and Nagana in cattle. In addition to being an important pathogen T. brucei has developed into a model system in cell biology. Results Using Stable Isotope Labelling of Amino acids in Cell culture (SILAC) in combination with mass spectrometry we determined the abundance of >1600 proteins in the long slender (LS), short stumpy (SS) mammalian bloodstream form stages relative to the procyclic (PC) insect-form stage. In total we identified 2645 proteins, corresponding to ~30% of the total proteome and for the first time present a comprehensive overview of relative protein levels in three life stages of the parasite. Conclusions We can show the extent of pre-adaptation in the SS cells, especially at the level of the mitochondrial proteome. The comparison to a previously published report on monomorphic in vitro grown bloodstream and procyclic T. brucei indicates a loss of stringent regulation particu...
Research Interests: Computational Biology, Principal Component Analysis, Mass Spectrometry, Western blotting, Biological Sciences, and 10 moreAnimals, Alternative splicing, Amino Acids, Trypanosoma brucei brucei, Alternative Splicing, Proteome, Isotope Labeling, Gene expression profiling, Mitochondrial Proteins, and Medical and Health Sciences
ObjectiveDuring degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular... more
ObjectiveDuring degeneration of the intervertebral disc ingrowth of blood vessels and nerves into the disc are associated with back pain. Vascular endothelial growth factors promote vasculogenesis by binding to the membrane vascular endothelial growth factor receptor 1, while shorter soluble forms of this receptor can inhibit vascularization. We hypothesized that membrane and soluble receptor forms might change between stages of intervertebral disc degeneration.ResultsExpression of soluble and membrane forms of vascular endothelial growth factor receptor 1 in human degenerated intervertebral discs and healthy bovine caudal discs was assessed by qRT-PCR and immunoblot. Comparative microarray meta-analysis across disc degeneration grades showed that membrane and soluble forms of this receptor, together with other components of classic vascularization pathways, are constitutively expressed across human disc degeneration stages. Contrary to our hypothesis, we observed that expression of...
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While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts... more
While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation.
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The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the... more
The rotavirus (RV) VP4 spike protrudes as a trimeric structure from the five-fold axes of the virion triple-layer. Infectious RV particles need to be proteolytically cleaved in VP4 into two subunits, VP8* and VP5*, constituting both the distal part and central body of the virus spike. Modification of VP4 has been challenging as it is involved in biological processes such as the interaction with sialic acid and integrins, cell tropism and hemagglutinin activity. Using RV reverse genetics, four loops in the lectin domain of the VP8* subunit were engineered independently to harbor a small biotin acceptor peptide (BAP) tag and then tested for their ability to rescue virus. Only a single recombinant virus, rRV/VP4-BAP, harboring VP4 with a modified loop at position K145-G150 was rescued. This rRV/VP4-BAP internalizes, replicates, and generates virus progeny, demonstrating that the VP4 spike of RV particles can be genetically manipulated by the incorporation of at least 15 exogenous amino...
Induction of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) is essential for macrophage differentiation into osteoclasts (OCs), but... more
Induction of nuclear factor of activated T cell cytoplasmic 1 (NFATc1) by macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL) is essential for macrophage differentiation into osteoclasts (OCs), but the underlying mechanisms remain unclear. The ability of poly(ADP-ribose) polymerase 1 (PARP1) to poly-ADP-ribosylate NFATc1 in T cells prompted us to investigate the PARP1 and NFATc1 interaction during osteoclastogenesis. However, extensive studies failed to directly link PARP1 to NFATc1. A combination of transcriptomics and proteomics studies was then used to identify PARP1 targets under these conditions. These unbiased approaches in conjunction with site-directed mutagenesis studies revealed that PARP1 inhibited NFATc1 expression and OC formation by ADP-ribosylating histone H2B at serine 7 and decreasing the occupancy of this histone variant at the NFATc1 promoter. The anti-osteoclastogenic function of PARP1 was confirmed in vivo in several mouse models of PARP1 loss-of-function or gain-of-function, including a novel model in which PARP1 was conditionally ablated in myeloid cells. Thus, PARP1 ADP-ribosylates H2B to negatively regulate NFATc1 expression and OC differentiation. © 2019 American Society for Bone and Mineral Research.
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Mice deficient for ADP-ribosyltransferase diphteria toxin-like 1 (ARTD1) are protected against microbially induced inflammation. To address the contribution of ARTD1 to inflammation specifically in myeloid cells, we generated an... more
Mice deficient for ADP-ribosyltransferase diphteria toxin-like 1 (ARTD1) are protected against microbially induced inflammation. To address the contribution of ARTD1 to inflammation specifically in myeloid cells, we generated an Artd1ΔMyel mouse strain with conditional ARTD1 deficiency in myeloid lineages and examined the strain in three disease models. We found that ARTD1, but not its enzymatic activity, enhanced the transcriptional activation of distinct LPS-induced genes that included IL-12, TNF-α, and IL-6 in primary bone marrow-derived macrophages and LPS-induced IL-12/18-IFN-γ signaling in Artd1ΔMyel mice. The loss of Artd1 in myeloid cells also reduced the TH1 response to Helicobacter pylori and impaired immune control of the bacteria. Furthermore, Artd1ΔMyel mice failed to control tumor growth in a s.c. MC-38 model of colon cancer, which could be attributed to reduced TH1 and CD8 responses. Together, these data provide strong evidence for a cell-intrinsic role of ARTD1 in myeloid cells that is independent of its enzymatic activity and promotes type I immunity by promoting IL-12/18 expression.
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Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep... more
Despite recent mass spectrometry (MS)‐based breakthroughs, comprehensive ADP‐ribose (ADPr)‐acceptor amino acid identification and ADPr‐site localization remain challenging. Here, we report the establishment of an unbiased, multistep ADP‐ribosylome data analysis workflow that led to the identification of tyrosine as a novel ARTD1/PARP1‐dependent in vivo ADPr‐acceptor amino acid. MS analyses of in vitro ADP‐ribosylated proteins confirmed tyrosine as an ADPr‐acceptor amino acid in RPS3A (Y155) and HPF1 (Y238) and demonstrated that trans‐modification of RPS3A is dependent on HPF1. We provide an ADPr‐site Localization Spectra Database (ADPr‐LSD), which contains 288 high‐quality ADPr‐modified peptide spectra, to serve as ADPr spectral references for correct ADPr‐site localizations.
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Four recent publications used next generation sequencing to analyse the transcriptome of Trypanosoma brucei. These have produced a wealth of data that have refined and, to some extent, redefined our concept of the genome. At the same... more
Four recent publications used next generation sequencing to analyse the transcriptome of Trypanosoma brucei. These have produced a wealth of data that have refined and, to some extent, redefined our concept of the genome. At the same time, they provide a welcome opportunity to re-examine older publications and place them in a post-genomic context.
TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding... more
TbRRM1 of Trypanosoma brucei is a nucleoprotein that was previously identified in a search for splicing factors in T. brucei. We show that TbRRM1 associates with mRNAs and with the auxiliary splicing factor polypyrimidine tract-binding protein 2, but not with components of the core spliceosome. TbRRM1 also interacts with several retrotransposon hot spot (RHS) proteins and histones. RNA immunoprecipitation of a tagged form of TbRRM1 from procyclic (insect) form trypanosomes identified ca. 1,500 transcripts that were enriched and 3,000 transcripts that were underrepresented compared to cellular mRNA. Enriched transcripts encoded RNA-binding proteins, including TbRRM1 itself, several RHS transcripts, mRNAs with long coding regions, and a high proportion of stage-regulated mRNAs that are more highly expressed in bloodstream forms. Transcripts encoding ribosomal proteins, other factors involved in translation, and procyclic-specific transcripts were underrepresented. Knockdown of TbRRM1 ...
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Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering... more
Sterols are an essential class of lipids in eukaryotes, where they serve as structural components of membranes and play important roles as signaling molecules. Sterols are also of high pharmacological significance: cholesterol-lowering drugs are blockbusters in human health, and inhibitors of ergosterol biosynthesis are widely used as antifungals. Inhibitors of ergosterol synthesis are also being developed for Chagas's disease, caused by Trypanosoma cruzi. Here we develop an in silico pipeline to globally evaluate sterol metabolism and perform comparative genomics. We generate a library of hidden Markov model-based profiles for 42 sterol biosynthetic enzymes, which allows expressing the genomic makeup of a given species as a numerical vector. Hierarchical clustering of these vectors functionally groups eukaryote proteomes and reveals convergent evolution, in particular metabolic reduction in obligate endoparasites. We experimentally explore sterol metabolism by testing a set of sterol biosynthesis inhibitors against trypanosomatids, Plasmodium falciparum, Giardia, and mammalian cells, and by quantifying the expression levels of sterol biosynthetic genes during the different life stages of T. cruzi and Trypanosoma brucei. The phenotypic data correlate with genomic makeup for simvastatin, which showed activity against trypanosomatids. Other findings, such as the activity of terbinafine against Giardia, are not in agreement with the genotypic profile.
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The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early... more
The protozoan pathogen Trypanosoma brucei is transmitted between mammals by tsetse flies. The first compartment colonised by trypanosomes after a blood meal is the fly midgut lumen. Trypanosomes present in the lumen—designated as early procyclic forms—express the stage-specific surface glycoproteins EP and GPEET procyclin. When the trypanosomes establish a mature infection and colonise the ectoperitrophic space, GPEET is down-regulated, and EP becomes the major surface protein of late procyclic forms. A few years ago, it was discovered that procyclic form trypanosomes exhibit social motility (SoMo) when inoculated on a semi-solid surface. We demonstrate that SoMo is a feature of early procyclic forms, and that late procyclic forms are invariably SoMo-negative. In addition, we show that, apart from GPEET, other markers are differentially expressed in these two life-cycle stages, both in culture and in tsetse flies, indicating that they have different biological properties and should ...