Publications by authors named "Thomas Dandekar"

235 Publications

Modeling Immune Dynamics in Plants Using JIMENA-Package.

Methods Mol Biol 2021 ;2328:183-189

Department of Bioinformatics, Biocenter, University of Wuerzburg, Am Hubland, Wuerzburg, Germany.

Plant immunity is a highly dynamic process and requires dynamic modeling to capture the events of complexity mediated by the interaction between plant host and the attacking pathogen. The events of recognition are invoked by pathogen-based epitopes, while the subversion of host defenses are orchestrated by pathogen-originated effector molecules. The pathogen constitutes an immune signaling network inside the host cells. We model plant immune dynamics by using JIMENA-package, which is a java-based genetic regulatory network (GRN) simulation framework. It can efficiently compute network behavior and system states mediated by pathogenic perturbations. Here, we describe a step-by-step protocol to introduce the application of JIMENA-package to quantify immune dynamics in plant-pathogen interaction networks.
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http://dx.doi.org/10.1007/978-1-0716-1534-8_11DOI Listing
January 2021

Computational Enzyme Engineering Pipelines for Optimized Production of Renewable Chemicals.

Front Bioeng Biotechnol 2021 15;9:673005. Epub 2021 Jun 15.

Department of Bioinformatics, Julius-Maximilians University of Würzburg, Würzburg, Germany.

To enable a sustainable supply of chemicals, novel biotechnological solutions are required that replace the reliance on fossil resources. One potential solution is to utilize tailored biosynthetic modules for the metabolic conversion of CO or organic waste to chemicals and fuel by microorganisms. Currently, it is challenging to commercialize biotechnological processes for renewable chemical biomanufacturing because of a lack of highly active and specific biocatalysts. As experimental methods to engineer biocatalysts are time- and cost-intensive, it is important to establish efficient and reliable computational tools that can speed up the identification or optimization of selective, highly active, and stable enzyme variants for utilization in the biotechnological industry. Here, we review and suggest combinations of effective state-of-the-art software and online tools available for computational enzyme engineering pipelines to optimize metabolic pathways for the biosynthesis of renewable chemicals. Using examples relevant for biotechnology, we explain the underlying principles of enzyme engineering and design and illuminate future directions for automated optimization of biocatalysts for the assembly of synthetic metabolic pathways.
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http://dx.doi.org/10.3389/fbioe.2021.673005DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8239229PMC
June 2021

Rapid proliferation due to better metabolic adaptation results in full virulence of a filament-deficient Candida albicans strain.

Nat Commun 2021 06 23;12(1):3899. Epub 2021 Jun 23.

Research Group Microbial Immunology, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute, Beutenbergstraße 11a, Jena, Germany.

The ability of the fungal pathogen Candida albicans to undergo a yeast-to-hypha transition is believed to be a key virulence factor, as filaments mediate tissue damage. Here, we show that virulence is not necessarily reduced in filament-deficient strains, and the results depend on the infection model used. We generate a filament-deficient strain by deletion or repression of EED1 (known to be required for maintenance of hyphal growth). Consistent with previous studies, the strain is attenuated in damaging epithelial cells and macrophages in vitro and in a mouse model of intraperitoneal infection. However, in a mouse model of systemic infection, the strain is as virulent as the wild type when mice are challenged with intermediate infectious doses, and even more virulent when using low infectious doses. Retained virulence is associated with rapid yeast proliferation, likely the result of metabolic adaptation and improved fitness, leading to high organ fungal loads. Analyses of cytokine responses in vitro and in vivo, as well as systemic infections in immunosuppressed mice, suggest that differences in immunopathology contribute to some extent to retained virulence of the filament-deficient mutant. Our findings challenge the long-standing hypothesis that hyphae are essential for pathogenesis of systemic candidiasis by C. albicans.
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http://dx.doi.org/10.1038/s41467-021-24095-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8222383PMC
June 2021

Design, Synthesis, and Anticancer Screening for Repurposed Pyrazolo[3,4-d]pyrimidine Derivatives on Four Mammalian Cancer Cell Lines.

Molecules 2021 May 16;26(10). Epub 2021 May 16.

Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Alexandria University, Alexandria 21500, Egypt.

The present study reports the synthesis of new purine bioisosteres comprising a pyrazolo[3,4-d]pyrimidine scaffold linked to mono-, di-, and trimethoxy benzylidene moieties through hydrazine linkages. First, in silico docking experiments of the synthesized compounds against Bax, Bcl-2, Caspase-3, Ki67, p21, and p53 were performed in a trial to rationalize the observed cytotoxic activity for the tested compounds. The anticancer activity of these compounds was evaluated in vitro against Caco-2, A549, HT1080, and Hela cell lines. Results revealed that two ( and ) of the three synthesized compounds (, , and ) showed high cytotoxic activity against all tested cell lines with IC values in the micro molar concentration. Our in vitro results show that there is no significant apoptotic effect for the treatment with the experimental compounds on the viability of cells against A549 cells. Ki67 expression was found to decrease significantly following the treatment of cells with the most promising candidate: drug . The overall results indicate that these pyrazolopyrimidine derivatives possess anticancer activity at varying doses. The suggested mechanism of action involves the inhibition of the proliferation of cancer cells.
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http://dx.doi.org/10.3390/molecules26102961DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8156061PMC
May 2021

RhoA/Cdc42 signaling drives cytoplasmic maturation but not endomitosis in megakaryocytes.

Cell Rep 2021 May;35(6):109102

Institute of Experimental Biomedicine, University Hospital, University of Würzburg, 97080 Würzburg, Germany; Rudolf Virchow Center, University of Würzburg, 97080 Würzburg, Germany. Electronic address:

Megakaryocytes (MKs), the precursors of blood platelets, are large, polyploid cells residing mainly in the bone marrow. We have previously shown that balanced signaling of the Rho GTPases RhoA and Cdc42 is critical for correct MK localization at bone marrow sinusoids in vivo. Using conditional RhoA/Cdc42 double-knockout (DKO) mice, we reveal here that RhoA/Cdc42 signaling is dispensable for the process of polyploidization in MKs but essential for cytoplasmic MK maturation. Proplatelet formation is virtually abrogated in the absence of RhoA/Cdc42 and leads to severe macrothrombocytopenia in DKO animals. The MK maturation defect is associated with downregulation of myosin light chain 2 (MLC2) and β1-tubulin, as well as an upregulation of LIM kinase 1 and cofilin-1 at both the mRNA and protein level and can be linked to impaired MKL1/SRF signaling. Our findings demonstrate that MK endomitosis and cytoplasmic maturation are separately regulated processes, and the latter is critically controlled by RhoA/Cdc42.
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http://dx.doi.org/10.1016/j.celrep.2021.109102DOI Listing
May 2021

Modeling of stringent-response reflects nutrient stress induced growth impairment and essential amino acids in different Staphylococcus aureus mutants.

Sci Rep 2021 May 6;11(1):9651. Epub 2021 May 6.

Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, 97074, Würzburg, Germany.

Stapylococcus aureus colonises the nose of healthy individuals but can also cause a wide range of infections. Amino acid (AA) synthesis and their availability is crucial to adapt to conditions encountered in vivo. Most S. aureus genomes comprise all genes required for AA biosynthesis. Nevertheless, different strains require specific sets of AAs for growth. In this study we show that regulation inactivates pathways under certain conditions which result in these observed auxotrophies. We analyzed in vitro and modeled in silico in a Boolean semiquantitative model (195 nodes, 320 edges) the regulatory impact of stringent response (SR) on AA requirement in S. aureus HG001 (wild-type) and in mutant strains lacking the metabolic regulators RSH, CodY and CcpA, respectively. Growth in medium lacking single AAs was analyzed. Results correlated qualitatively to the in silico predictions of the final model in 92% and quantitatively in 81%. Remaining gaps in our knowledge are evaluated and discussed. This in silico model is made fully available and explains how integration of different inputs is achieved in SR and AA metabolism of S. aureus. The in vitro data and in silico modeling stress the role of SR and central regulators such as CodY for AA metabolisms in S. aureus.
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http://dx.doi.org/10.1038/s41598-021-88646-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8102509PMC
May 2021

Population-Predicted MHC Class II Epitope Presentation of SARS-CoV-2 Structural Proteins Correlates to the Case Fatality Rates of COVID-19 in Different Countries.

Int J Mol Sci 2021 Mar 5;22(5). Epub 2021 Mar 5.

Department of Bioinformatics, Biocenter, Am Hubland, University of Würzburg, 97074 Würzburg, Germany.

We observed substantial differences in predicted Major Histocompatibility Complex II (MHCII) epitope presentation of SARS-CoV-2 proteins for different populations but only minor differences in predicted MHCI epitope presentation. A comparison of this predicted epitope MHC-coverage revealed for the early phase of infection spread (till day 15 after reaching 128 observed infection cases) highly significant negative correlations with the case fatality rate. Specifically, this was observed in different populations for MHC class II presentation of the viral spike protein (-value: 0.0733 for linear regression), the envelope protein (-value: 0.023), and the membrane protein (-value: 0.00053), indicating that the high case fatality rates of COVID-19 observed in some countries seem to be related with poor MHC class II presentation and hence weak adaptive immune response against these viral envelope proteins. Our results highlight the general importance of the SARS-CoV-2 structural proteins in immunological control in early infection spread looking at a global census in various countries and taking case fatality rate into account. Other factors such as health system and control measures become more important after the early spread. Our study should encourage further studies on MHCII alleles as potential risk factors in COVID-19 including assessment of local populations and specific allele distributions.
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http://dx.doi.org/10.3390/ijms22052630DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7961590PMC
March 2021

In vitro skin culture media influence the viability and inflammatory response of primary macrophages.

Sci Rep 2021 Mar 29;11(1):7070. Epub 2021 Mar 29.

Laboratory for Biointerfaces, Empa - Swiss Federal Laboratories for Materials Science and Technology, St. Gallen, Switzerland.

The replacement of animal models for investigation of inflammation and wound healing has been advancing by means of in vitro skin equivalents with increasing levels of complexity. However, the current in vitro skin models still have a limited pre-clinical relevance due to their lack of immune cells. So far, few steps have been made towards the incorporation of immune cells into in vitro skin and the requirements for immunocompetent co-cultures remain unexplored. To establish suitable conditions for incorporating macrophages into skin models, we evaluated the effects of different media on primary keratinocytes, fibroblasts and macrophages. Skin maturation was affected by culture in macrophage medium, while macrophages showed reduced viability, altered cell morphology and decreased response to pro- and anti-inflammatory stimuli in skin differentiation media, both in 2D and 3D. The results indicate that immunocompetent skin models have specific, complex requirements for supporting an accurate detection of immune responses, which point at the identification of a suitable culture medium as a crucial pre-requisite for the development of physiologically relevant models.
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http://dx.doi.org/10.1038/s41598-021-86486-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8007571PMC
March 2021

Modulatory and Toxicological Perspectives on the Effects of the Small Molecule Kinetin.

Molecules 2021 Jan 28;26(3). Epub 2021 Jan 28.

Department of Life and Environmental Sciences, College of Natural and Health Sciences, Zayed University, Abu Dhabi 144534, UAE.

Plant hormones are small regulatory molecules that exert pharmacological actions in mammalian cells such as anti-oxidative and pro-metabolic effects. Kinetin belongs to the group of plant hormones cytokinin and has been associated with modulatory functions in mammalian cells. The mammalian adenosine receptor (A2a-R) is known to modulate multiple physiological responses in animal cells. Here, we describe that kinetin binds to the adenosine receptor (A2a-R) through the Asn253 residue in an adenosine dependent manner. To harness the beneficial effects of kinetin for future human use, we assess its acute toxicity by analyzing different biochemical and histological markers in rats. Kinetin at a dose below 1 mg/kg had no adverse effects on the serum level of glucose or on the activity of serum alanine transaminase (ALT) or aspartate aminotransferase (AST) enzymes in the kinetin treated rats. Whereas, creatinine levels increased after a kinetin treatment at a dose of 0.5 mg/kg. Furthermore, 5 mg/kg treated kinetin rats showed normal renal corpuscles, but a mild degeneration was observed in the renal glomeruli and renal tubules, as well as few degenerated hepatocytes were also observed in the liver. Kinetin doses below 5 mg/kg did not show any localized toxicity in the liver and kidney tissues. In addition to unraveling the binding interaction between kinetin and A2a-R, our findings suggest safe dose limits for the future use of kinetin as a therapeutic and modulatory agent against various pathophysiological conditions.
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http://dx.doi.org/10.3390/molecules26030670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7865834PMC
January 2021

An optimized genetically encoded dual reporter for simultaneous ratio imaging of Ca and H reveals new insights into ion signaling in plants.

New Phytol 2021 06 18;230(6):2292-2310. Epub 2021 Feb 18.

Department of Botany I, Julius-Von-Sachs Institute for Biosciences, University of Wuerzburg, Wuerzburg, 97082, Germany.

Whereas the role of calcium ions (Ca ) in plant signaling is well studied, the physiological significance of pH-changes remains largely undefined. Here we developed CapHensor, an optimized dual-reporter for simultaneous Ca and pH ratio-imaging and studied signaling events in pollen tubes (PTs), guard cells (GCs), and mesophyll cells (MCs). Monitoring spatio-temporal relationships between membrane voltage, Ca - and pH-dynamics revealed interconnections previously not described. In tobacco PTs, we demonstrated Ca -dynamics lag behind pH-dynamics during oscillatory growth, and pH correlates more with growth than Ca . In GCs, we demonstrated abscisic acid (ABA) to initiate stomatal closure via rapid cytosolic alkalization followed by Ca elevation. Preventing the alkalization blocked GC ABA-responses and even opened stomata in the presence of ABA, disclosing an important pH-dependent GC signaling node. In MCs, a flg22-induced membrane depolarization preceded Ca -increases and cytosolic acidification by c. 2 min, suggesting a Ca /pH-independent early pathogen signaling step. Imaging Ca and pH resolved similar cytosol and nuclear signals and demonstrated flg22, but not ABA and hydrogen peroxide to initiate rapid membrane voltage-, Ca - and pH-responses. We propose close interrelation in Ca - and pH-signaling that is cell type- and stimulus-specific and the pH having crucial roles in regulating PT growth and stomata movement.
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http://dx.doi.org/10.1111/nph.17202DOI Listing
June 2021

Comparison of the central human and mouse platelet signaling cascade by systems biological analysis.

BMC Genomics 2020 Dec 22;21(1):897. Epub 2020 Dec 22.

Functional Genomics and Systems Biology Group, Department of Bioinformatics, Biocenter, Am Hubland, University of Würzburg, D-97074, Würzburg, Germany.

Background: Understanding the molecular mechanisms of platelet activation and aggregation is of high interest for basic and clinical hemostasis and thrombosis research. The central platelet protein interaction network is involved in major responses to exogenous factors. This is defined by systemsbiological pathway analysis as the central regulating signaling cascade of platelets (CC).

Results: The CC is systematically compared here between mouse and human and major differences were found. Genetic differences were analysed comparing orthologous human and mouse genes. We next analyzed different expression levels of mRNAs. Considering 4 mouse and 7 human high-quality proteome data sets, we identified then those major mRNA expression differences (81%) which were supported by proteome data. CC is conserved regarding genetic completeness, but we observed major differences in mRNA and protein levels between both species. Looking at central interactors, human PLCB2, MMP9, BDNF, ITPR3 and SLC25A6 (always Entrez notation) show absence in all murine datasets. CC interactors GNG12, PRKCE and ADCY9 occur only in mice. Looking at the common proteins, TLN1, CALM3, PRKCB, APP, SOD2 and TIMP1 are higher abundant in human, whereas RASGRP2, ITGB2, MYL9, EIF4EBP1, ADAM17, ARRB2, CD9 and ZYX are higher abundant in mouse. Pivotal kinase SRC shows different regulation on mRNA and protein level as well as ADP receptor P2RY12.

Conclusions: Our results highlight species-specific differences in platelet signaling and points of specific fine-tuning in human platelets as well as murine-specific signaling differences.
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http://dx.doi.org/10.1186/s12864-020-07215-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7756956PMC
December 2020

Intracellular Staphylococcus aureus Perturbs the Host Cell Ca Homeostasis To Promote Cell Death.

mBio 2020 12 15;11(6). Epub 2020 Dec 15.

Chair of Microbiology, University of Würzburg, Würzburg, Germany

The opportunistic human pathogen causes serious infectious diseases that range from superficial skin and soft tissue infections to necrotizing pneumonia and sepsis. While classically regarded as an extracellular pathogen, is able to invade and survive within human cells. Host cell exit is associated with cell death, tissue destruction, and the spread of infection. The exact molecular mechanism employed by to escape the host cell is still unclear. In this study, we performed a genome-wide small hairpin RNA (shRNA) screen and identified the calcium signaling pathway as being involved in intracellular infection. induced a massive cytosolic Ca increase in epithelial host cells after invasion and intracellular replication of the pathogen. This was paralleled by a decrease in endoplasmic reticulum Ca concentration. Additionally, calcium ions from the extracellular space contributed to the cytosolic Ca increase. As a consequence, we observed that the cytoplasmic Ca rise led to an increase in mitochondrial Ca concentration, the activation of calpains and caspases, and eventually to cell lysis of -infected cells. Our study therefore suggests that intracellular disturbs the host cell Ca homeostasis and induces cytoplasmic Ca overload, which results in both apoptotic and necrotic cell death in parallel or succession. Despite being regarded as an extracellular bacterium, the pathogen can invade and survive within human cells. The intracellular niche is considered a hideout from the host immune system and antibiotic treatment and allows bacterial proliferation. Subsequently, the intracellular bacterium induces host cell death, which may facilitate the spread of infection and tissue destruction. So far, host cell factors exploited by intracellular to promote cell death are only poorly characterized. We performed a genome-wide screen and found the calcium signaling pathway to play a role in invasion and cytotoxicity. The intracellular bacterium induces a cytoplasmic and mitochondrial Ca overload, which results in host cell death. Thus, this study first showed how an intracellular bacterium perturbs the host cell Ca homeostasis.
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http://dx.doi.org/10.1128/mBio.02250-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7773986PMC
December 2020

Modular micro-physiological human tumor/tissue models based on decellularized tissue for improved preclinical testing

ALTEX 2020 12 11;38(2):289-306. Epub 2020 Dec 11.

Department Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Würzburg, Germany.

High attrition rates associated with drug testing in 2D cell culture and animal models stress the need for improved modeling of human tumor tissues. In previous studies, our 3D models on a decellularized tissue matrix have shown better predictivity and higher chemoresistance. A single porcine intestine yields material for 150 3D models of breast, lung, colorectal cancer (CRC) or leukemia. The uniquely preserved structure of the basement membrane enables physiological anchorage of endothelial cells and epithelial-derived carcinoma cells. The matrix provides different niches for cell growth: on top as monolayer, in crypts as aggregates, and within deeper layers. Dynamic culture in bioreactors enhances cell growth. Comparing gene expression between 2D and 3D cultures, we observed changes related to proliferation, apoptosis and stemness. For drug target predictions, we utilize tumor-specific sequencing data in our in silico model, finding an additive effect of metformin and gefitinib treatment for lung cancer in silico, validated in vitro. To analyze mode-of-action, immune therapies such as trispecific T-cell engagers in leukemia or toxicity on non-cancer cells, the model can be modularly enriched with human endothelial cells (hECs), immune cells and fibroblasts. Upon addition of hECs, transmigration of immune cells through the endothelial barrier can be investigated. In an allogenic CRC model, we observe a lower basic apoptosis rate after applying PBMCs in 3D compared to 2D, which offers new options to mirror antigen-specific immunotherapies in vitro. In conclusion, we present modular human 3D tumor models with tissue-like features for preclinical testing to reduce animal experiments.
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http://dx.doi.org/10.14573/altex.2008141DOI Listing
December 2020

Novel Approach for Characterizing Propofol Binding Affinities to Serum Albumins from Different Species.

ACS Omega 2020 Oct 30;5(40):25543-25551. Epub 2020 Sep 30.

Department of Anesthesia and Critical Care, Würzburg University Hospital, Würzburg 97080, Germany.

The interaction between the main carrier (serum albumin, SA) of endogenous and exogenous compounds in the bloodstream of different species (human, bovine, canine, rat, rabbit, and sheep) and a general anesthetic agent (propofol, PR) was investigated using an experimental technique (high-performance liquid chromatography) and computational methods (molecular docking, molecular dynamics, sequence, and phylogenetic analyses). The obtained results revealed the differences in the PR binding affinity to various homologous forms of this protein with reliable statistics ( = 0.9 and -value < 0.005), correlating with the evolutionary relationships among SAs from different species. Additionally, the protein conformational changes (root-mean-square deviation ≈ 1.0 Å) and amino acid conservation of binding sites in protein domains were detected, contributing to the SA-PR binding modes. Overall, the outcomes from this study might provide a novel methodology to assess protein-ligand interactions and to gain some interesting insights into drug pharmacokinetics and pharmacodynamics to explain its variations among different species.
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http://dx.doi.org/10.1021/acsomega.0c01295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7557242PMC
October 2020

A systems-biology model of the tumor necrosis factor (TNF) interactions with TNF receptor 1 and 2.

Bioinformatics 2021 05;37(5):669-676

Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Würzburg, Würzburg 97080, Germany.

Motivation: Clustering enables TNF receptors to stimulate intracellular signaling. The differential soluble ligand-induced clustering behavior of TNF receptor 1 (TNFR1) and TNFR2 was modeled. A structured, rule-based model implemented ligand-independent pre-ligand binding assembly domain (PLAD)-mediated homotypic low affinity interactions of unliganded and liganded TNF receptors.

Results: Soluble TNF initiates TNFR1 signaling but not TNFR2 signaling despite receptor binding unless it is secondarily oligomerized. We consider high affinity binding of TNF to signaling-incompetent pre-assembled dimeric TNFR1 and TNFR2 molecules and secondary clustering of liganded dimers to signaling competent ligand-receptor clusters. Published receptor numbers, affinities and measured different activities of clustered receptors validated model simulations for a large range of receptor and ligand concentrations. Different PLAD-PLAD affinities and different activities of receptor clusters explain the observed differences in the TNF receptor stimulating activities of soluble TNF.

Availability And Implementation: All scripts and data are in manuscript and supplement at Bioinformatics online.

Supplementary Information: Supplementary data are available at Bioinformatics online.
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http://dx.doi.org/10.1093/bioinformatics/btaa844DOI Listing
May 2021

Integrated Framework of the Immune-Defense Transcriptional Signatures in the Arabidopsis Shoot Apical Meristem.

Int J Mol Sci 2020 Aug 11;21(16). Epub 2020 Aug 11.

Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, D-97074 Würzburg, Germany.

The growing tips of plants grow sterile; therefore, disease-free plants can be generated from them. How plants safeguard growing apices from pathogen infection is still a mystery. The shoot apical meristem (SAM) is one of the three stem cells niches that give rise to the above ground plant organs. This is very well explored; however, how signaling networks orchestrate immune responses against pathogen infections in the SAM remains unclear. To reconstruct a transcriptional framework of the differentially expressed genes (DEGs) pertaining to various SAM cellular populations, we acquired large-scale transcriptome datasets from the public repository Gene Expression Omnibus (GEO). We identify here distinct sets of genes for various SAM cellular populations that are enriched in immune functions, such as immune defense, pathogen infection, biotic stress, and response to salicylic acid and jasmonic acid and their biosynthetic pathways in the SAM. We further linked those immune genes to their respective proteins and identify interactions among them by mapping a transcriptome-guided SAM-interactome. Furthermore, we compared stem-cells regulated transcriptome with innate immune responses in plants showing transcriptional separation among their DEGs in . Besides unleashing a repertoire of immune-related genes in the SAM, our analysis provides a SAM-interactome that will help the community in designing functional experiments to study the specific defense dynamics of the SAM-cellular populations. Moreover, our study promotes the essence of large-scale omics data re-analysis, allowing a fresh look at the SAM-cellular transcriptome repurposing data-sets for new questions.
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http://dx.doi.org/10.3390/ijms21165745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7460820PMC
August 2020

Integrated structural and functional analysis of the protective effects of kinetin against oxidative stress in mammalian cellular systems.

Sci Rep 2020 08 7;10(1):13330. Epub 2020 Aug 7.

Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, Wuerzburg, Germany.

Metabolism and signaling of cytokinins was first established in plants, followed by cytokinin discoveries in all kingdoms of life. However, understanding of their role in mammalian cells is still scarce. Kinetin is a cytokinin that mitigates the effects of oxidative stress in mammalian cells. The effective concentrations of exogenously applied kinetin in invoking various cellular responses are not well standardized. Likewise, the metabolism of kinetin and its cellular targets within the mammalian cells are still not well studied. Applying vitality tests as well as comet assays under normal and hyper-oxidative states, our analysis suggests that kinetin concentrations of 500 nM and above cause cytotoxicity as well as genotoxicity in various cell types. However, concentrations below 100 nM do not cause any toxicity, rather in this range kinetin counteracts oxidative burst and cytotoxicity. We focus here on these effects. To get insights into the cellular targets of kinetin mediating these pro-survival functions and protective effects we applied structural and computational approaches on two previously testified targets for these effects. Our analysis deciphers vital residues in adenine phosphoribosyltransferase (APRT) and adenosine receptor (A2A-R) that facilitate the binding of kinetin to these two important human cellular proteins. We finally discuss how the therapeutic potential of kinetin against oxidative stress helps in various pathophysiological conditions.
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http://dx.doi.org/10.1038/s41598-020-70253-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414151PMC
August 2020

Chagas Disease: Detection of by a New, High-Specific Real Time PCR.

J Clin Med 2020 May 18;9(5). Epub 2020 May 18.

Julius-Maximilians University, Department of Bioinformatics, Biocenter, Functional Genomics and Systems Biology Group, 97070 Wuerzburg, Germany.

Background: Chagas disease (CD) is a major burden in Latin America, expanding also to non-endemic countries. A gold standard to detect the CD causing pathogen is currently not available. Existing real time polymerase chain reactions (RT-PCRs) lack sensitivity and/or specificity. We present a new, highly specific RT-PCR for the diagnosis and monitoring of CD.

Material And Methods: We analyzed 352 serum samples from Indigenous people living in high endemic CD areas of Colombia using three leading RT-PCRs (k-DNA-, TCZ-, 18S rRNA-PCR), the newly developed one (NDO-PCR), a Rapid Test/enzyme-linked immuno sorbent assay (ELISA), and immunofluorescence. Eighty-seven PCR-products were verified by sequence analysis after plasmid vector preparation.

Results: The NDO-PCR showed the highest sensitivity (92.3%), specificity (100%), and accuracy (94.3%) for detection in the 87 sequenced samples. Sensitivities and specificities of the kDNA-PCR were 89.2%/22.7%, 20.5%/100% for TCZ-PCR, and 1.5%/100% for the 18S rRNA-PCR. The kDNA-PCR revealed a 77.3% false positive rate, mostly due to cross-reactions with (NDO-PCR 0%). TCZ- and 18S rRNA-PCR showed a false negative rate of 79.5% and 98.5% (NDO-PCR 7.7%), respectively.

Conclusions: The NDO-PCR demonstrated the highest specificity, sensitivity, and accuracy compared to leading PCRs. Together with serologic tests, it can be considered as a reliable tool for CD detection and can improve CD management significantly.
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http://dx.doi.org/10.3390/jcm9051517DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7291166PMC
May 2020

Integrative functional genomics decodes herpes simplex virus 1.

Nat Commun 2020 04 27;11(1):2038. Epub 2020 Apr 27.

Institute for Virology and Immunobiology, Julius-Maximilians-University Würzburg, Versbacher Straße 7, 97078, Würzburg, Germany.

The predicted 80 open reading frames (ORFs) of herpes simplex virus 1 (HSV-1) have been intensively studied for decades. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identify a total of 201 transcripts and 284 ORFs including all known and 46 novel large ORFs. This includes a so far unknown ORF in the locus deleted in the FDA-approved oncolytic virus Imlygic. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of ORFs as well as N-terminal extensions (NTEs) and truncations. We show that NTEs with non-canonical start codons govern the subcellular protein localization and packaging of key viral regulators and structural proteins. We extend the current nomenclature to include all viral gene products and provide a genome browser that visualizes all the obtained data from whole genome to single-nucleotide resolution.
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http://dx.doi.org/10.1038/s41467-020-15992-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7184758PMC
April 2020

Modeling of shotgun sequencing of DNA plasmids using experimental and theoretical approaches.

BMC Bioinformatics 2020 Apr 3;21(1):132. Epub 2020 Apr 3.

Department of Bioinformatics, University of Würzburg, 97074, Würzburg, Germany.

Background: Processing and analysis of DNA sequences obtained from next-generation sequencing (NGS) face some difficulties in terms of the correct prediction of DNA sequencing outcomes without the implementation of bioinformatics approaches. However, algorithms based on NGS perform inefficiently due to the generation of long DNA fragments, the difficulty of assembling them and the complexity of the used genomes. On the other hand, the Sanger DNA sequencing method is still considered to be the most reliable; it is a reliable choice for virtual modeling to build all possible consensus sequences from smaller DNA fragments.

Results: In silico and in vitro experiments were conducted: (1) to implement and test our novel sequencing algorithm, using the standard cloning vectors of different length and (2) to validate experimentally virtual shotgun sequencing using the PCR technique with the number of cycles from 1 to 9 for each reaction.

Conclusions: We applied a novel algorithm based on Sanger methodology to correctly predict and emphasize the performance of DNA sequencing techniques as well as in de novo DNA sequencing and its further application in synthetic biology. We demonstrate the statistical significance of our results.
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http://dx.doi.org/10.1186/s12859-020-3461-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7126183PMC
April 2020

Rational Drug Design of Axl Tyrosine Kinase Type I Inhibitors as Promising Candidates Against Cancer.

Front Chem 2019 4;7:920. Epub 2020 Feb 4.

Department of Bioinformatics, Biocenter, University of Würzburg, Würzburg, Germany.

The high level of Axl tyrosine kinase expression in various cancer cell lines makes it an attractive target for the development of anti-cancer drugs. In this study, we carried out several sets of screening for the ATP-competitive Axl kinase inhibitors based on different molecular docking protocols. The best drug-like candidates were identified, after parental structure modifications, by their highest affinity to the target protein. We found that our newly designed compound R5, a derivative of the R428 patented analog, is the most promising inhibitor of the Axl kinase according to the three molecular docking algorithms applied in the study. The molecular docking results are in agreement with the molecular dynamics simulations using the MM-PBSA/GBSA implicit solvation models, which confirm the high affinity of R5 toward the protein receptor. Additionally, the selectivity test against other kinases also reveals a high affinity of R5 toward ABL1 and Tyro3 kinases, emphasizing its promising potential for the treatment of malignant tumors.
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http://dx.doi.org/10.3389/fchem.2019.00920DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7010640PMC
February 2020

Delaying memory decline: different options and emerging solutions.

Transl Psychiatry 2020 01 21;10(1):13. Epub 2020 Jan 21.

Department of Bioinformatics, Biocenter, University of Würzburg, 97074, Würzburg, Germany.

Memory decline can be a devastating disease and increases in aging Western populations. Memory enhancement technologies hold promise for this and other conditions. Approaches include stem cell transplantation, which improved memory in several animal studies as well as vaccination against Alzheimer´s disease (AD) by β-amyloid antibodies. For a positive clinical effect, the vaccine should probably be administered over a long period of time and before amyloid pathologies manifest in the brain. Different drugs, such as erythropoietin or antiplatelet therapy, improve memory in neuropsychiatric diseases or AD or at least in animal studies. Omega-3 polyunsaturated fatty acid-rich diets improve memory through the gut-brain axis by altering the gut flora through probiotics. Sports, dancing, and memory techniques (e.g., Method of Loci) utilize behavioral approaches for memory enhancement, and were effective in several studies. Augmented reality (AR) is an auspicious way for enhancing memory in real time. Future approaches may include memory prosthesis for head-injured patients and light therapy for restoring memory in AD. Memory enhancement in humans in health and disease holds big promises for the future. Memory training helps only in mild or no impairment. Clinical application requires further investigation.
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http://dx.doi.org/10.1038/s41398-020-0697-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026464PMC
January 2020

Genome-wide inference of the Camponotus floridanus protein-protein interaction network using homologous mapping and interacting domain profile pairs.

Sci Rep 2020 02 11;10(1):2334. Epub 2020 Feb 11.

Functional Genomics and Systems Biology Group, Department of Bioinformatics, Biocenter, Am Hubland, D-97074, Würzburg, Germany.

Apart from some model organisms, the interactome of most organisms is largely unidentified. High-throughput experimental techniques to determine protein-protein interactions (PPIs) are resource intensive and highly susceptible to noise. Computational methods of PPI determination can accelerate biological discovery by identifying the most promising interacting pairs of proteins and by assessing the reliability of identified PPIs. Here we present a first in-depth study describing a global view of the ant Camponotus floridanus interactome. Although several ant genomes have been sequenced in the last eight years, studies exploring and investigating PPIs in ants are lacking. Our study attempts to fill this gap and the presented interactome will also serve as a template for determining PPIs in other ants in future. Our C. floridanus interactome covers 51,866 non-redundant PPIs among 6,274 proteins, including 20,544 interactions supported by domain-domain interactions (DDIs), 13,640 interactions supported by DDIs and subcellular localization, and 10,834 high confidence interactions mediated by 3,289 proteins. These interactions involve and cover 30.6% of the entire C. floridanus proteome.
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http://dx.doi.org/10.1038/s41598-020-59344-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7012867PMC
February 2020

Platelet glycoprotein VI promotes metastasis through interaction with cancer cell-derived galectin-3.

Blood 2020 04;135(14):1146-1160

Institute of Experimental Biomedicine, University Hospital Würzburg, Würzburg, Germany.

Increasing evidence suggests that platelets play a predominant role in colon and breast cancer metastasis, but the underlying molecular mechanisms remain elusive. Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin that triggers platelet activation through immunoreceptor tyrosine-based activation motif (ITAM) signaling and thereby regulates diverse functions, including platelet adhesion, aggregation, and procoagulant activity. GPVI has been proposed as a safe antithrombotic target, because its inhibition is protective in models of arterial thrombosis, with only minor effects on hemostasis. In this study, the genetic deficiency of platelet GPVI in mice decreased experimental and spontaneous metastasis of colon and breast cancer cells. Similar results were obtained with mice lacking the spleen-tyrosine kinase Syk in platelets, an essential component of the ITAM-signaling cascade. In vitro and in vivo analyses supported that mouse, as well as human GPVI, had platelet adhesion to colon and breast cancer cells. Using a CRISPR/Cas9-based gene knockout approach, we identified galectin-3 as the major counterreceptor of GPVI on tumor cells. In vivo studies demonstrated that the interplay between platelet GPVI and tumor cell-expressed galectin-3 uses ITAM-signaling components in platelets and favors the extravasation of tumor cells. Finally, we showed that JAQ1 F(ab')2-mediated inhibition of GPVI efficiently impairs platelet-tumor cell interaction and tumor metastasis. Our study revealed a new mechanism by which platelets promote the metastasis of colon and breast cancer cells and suggests that GPVI represents a promising target for antimetastatic therapies.
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http://dx.doi.org/10.1182/blood.2019002649DOI Listing
April 2020

Synthetic Rewiring of Plant CO Sequestration Galvanizes Plant Biomass Production.

Trends Biotechnol 2020 04 17;38(4):354-359. Epub 2020 Jan 17.

Functional Genomics and Systems Biology Group, Department of Bioinformatics, Biocenter, University of Würzburg, Am Hubland, Würzburg, Germany. Electronic address:

Synthetically designed alternative photorespiratory pathways in tobacco and rice plants have paved the way to enhanced plant biomass production. Likewise, some in vitro- and in vivo-tested carbon-concentrating cycles hold promise to increase plant biomass. We hypothesize a further increase in plant productivity if photorespiratory bypasses are integrated with carbon-concentrating cycles in plants.
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http://dx.doi.org/10.1016/j.tibtech.2019.12.019DOI Listing
April 2020

Connecting Cancer Pathways to Tumor Engines: A Stratification Tool for Colorectal Cancer Combining Human In Vitro Tissue Models with Boolean In Silico Models.

Cancers (Basel) 2019 Dec 20;12(1). Epub 2019 Dec 20.

Chair of Tissue Engineering and Regenerative Medicine, University Hospital Würzburg, Röntgenring 11, 97070 Würzburg, Germany.

To improve and focus preclinical testing, we combine tumor models based on a decellularized tissue matrix with bioinformatics to stratify tumors according to stage-specific mutations that are linked to central cancer pathways. We generated tissue models with -mutant colorectal cancer (CRC) cells (HROC24 and HROC87) and compared treatment responses to two-dimensional (2D) cultures and xenografts. As the BRAF inhibitor vemurafenib is-in contrast to melanoma-not effective in CRC, we combined it with the EGFR inhibitor gefitinib. In general, our 3D models showed higher chemoresistance and in contrast to 2D a more active HGFR after gefitinib and combination-therapy. In xenograft models murine HGF could not activate the human HGFR, stressing the importance of the human microenvironment. In order to stratify patient groups for targeted treatment options in CRC, an in silico topology with different stages including mutations and changes in common signaling pathways was developed. We applied the established topology for in silico simulations to predict new therapeutic options for BRAF-mutated CRC patients in advanced stages. Our in silico tool connects genome information with a deeper understanding of tumor engines in clinically relevant signaling networks which goes beyond the consideration of single drivers to improve CRC patient stratification.
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http://dx.doi.org/10.3390/cancers12010028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7017315PMC
December 2019

Mapping a Transcriptome-Guided Arabidopsis SAM Interactome.

Methods Mol Biol 2020 ;2094:113-118

Department of Bioinformatics, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

The advent of multi-OMICS approaches has a significant impact on the investigation of biological processes occurring in plants. RNA-SEQ, cellular proteomics, and metabolomics have added a considerable ease in studying the dynamics of stem cell niches. New cell sorting approaches coupled with the labeling of stem cell population specific marker genes are highly instrumental in enriching distinct cellular populations for various types of analysis. One more promising field of OMICS is the mapping of cellular interactomes. The plant stem cells research is barely profited from this newly emerging field of OMICS. Generation of stem cell/niche-specific interactome is a time-consuming and labor-intensive task. Here, we describe a method on how to assemble a SAM-based interactome after using the available generic Arabidopsis interactomes. To define the context of SAM in a generic interactome, we used SAM cell population transcriptome datasets. Our step-by-step protocol can easily be adopted for other stem cell niches such as RAM and lateral meristems keeping in view the availability of transcriptome datasets for cellular populations of these niches.
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http://dx.doi.org/10.1007/978-1-0716-0183-9_12DOI Listing
February 2021

Molecular Modeling of the Interaction Between Stem Cell Peptide and Immune Receptor in Plants.

Methods Mol Biol 2020 ;2094:67-77

Department of Bioinformatics, Biocenter, University of Wuerzburg, Wuerzburg, Germany.

Molecular docking enables comprehensive exploration of interactions between chemical moieties and proteins. Modeling and docking approaches are useful to determine the three-dimensional (3D) structure of experimentally uncrystallized proteins and subsequently their interactions with various inhibitors and activators or peptides. Here, we describe a protocol for carrying out molecular modeling and docking of stem cell peptide CLV3p on plant innate immune receptor FLS2.
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http://dx.doi.org/10.1007/978-1-0716-0183-9_8DOI Listing
February 2021

Bacterial Shoot Apical Meristem Inoculation Assay.

Methods Mol Biol 2020 ;2094:17-22

Department of Bioinformatics, Biocenter, Functional Genomics and Systems Biology Group, University of Würzburg, Würzburg, Germany.

By virtue of their sessile nature, plants may not show the fight-and-flight response, but they are not devoid of protecting themselves from disease-causing agents, attack by herbivores, and damages that are caused by other environmental factors. Plants differentially protect their life-sustaining organs such as plant apexes from the attack by microbial pathogens. There are well-established methods to inoculate/infect various plant parts such as leaves, roots, and stems with various different pathogens. The plant shoot apical meristems (SAM) are a high-value plant target that provides niche to stem cell populations. These stem cells are instrumental in maintaining future plant progenies by giving birth to cells that culminate in flowers, leaves, and stems. There are hardly few protocols available that allow us to study immune dynamics of the plant stem cells as they are hindered by various layers of the SAM cell populations. Here, we describe a step-by-step method on how to inoculate the Arabidopsis SAM with model plant pathogen Pseudomonas syringae pv. tomato DC3000.
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http://dx.doi.org/10.1007/978-1-0716-0183-9_2DOI Listing
February 2021

The New Genetic Landscape of Cushing's Disease: Deubiquitinases in the Spotlight.

Cancers (Basel) 2019 Nov 8;11(11). Epub 2019 Nov 8.

Department of Internal Medicine I, Division of Endocrinology and Diabetes, University Hospital, University of Würzburg, D-97080 Würzburg, Germany.

Cushing's disease (CD) is a rare condition caused by adrenocorticotropic hormone (ACTH)-producing adenomas of the pituitary, which lead to hypercortisolism that is associated with high morbidity and mortality. Treatment options in case of persistent or recurrent disease are limited, but new insights into the pathogenesis of CD are raising hope for new therapeutic avenues. Here, we have performed a meta-analysis of the available sequencing data in CD to create a comprehensive picture of CD's genetics. Our analyses clearly indicate that somatic mutations in the deubiquitinases are the key drivers in CD, namely USP8 (36.5%) and USP48 (13.3%). While in USP48 only Met415 is affected by mutations, in USP8 there are 26 different mutations described. However, these different mutations are clustering in the same hotspot region (affecting in 94.5% of cases Ser718 and Pro720). In contrast, pathogenic variants classically associated with tumorigenesis in genes like TP53 and BRAF are also present in CD but with low incidence (12.5% and 7%). Importantly, several of these mutations might have therapeutic potential as there are drugs already investigated in preclinical and clinical setting for other diseases. Furthermore, network and pathway analyses of all somatic mutations in CD suggest a rather unified picture hinting towards converging oncogenic pathways.
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http://dx.doi.org/10.3390/cancers11111761DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6895825PMC
November 2019