235 results on '"Vladimír Havlíček"'
Search Results
2. The Expanding Mycovirome of Aspergilli
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Josephine L. Battersby, David A. Stevens, Robert H. A. Coutts, Vladimír Havlíček, Joe L. Hsu, Gabriele Sass, and Ioly Kotta-Loizou
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mycovirus ,Aspergillus ,Polymycovirus ,RNA sequencing ,hypovirulence ,oxidative stress ,Biology (General) ,QH301-705.5 - Abstract
Mycoviruses are viruses that infect fungi and are widespread across all major fungal taxa, exhibiting great biological diversity. Since their discovery in the 1960s, researchers have observed a myriad of fungal phenotypes altered due to mycoviral infection. In this review, we examine the nuanced world of mycoviruses in the context of the medically and agriculturally important fungal genus, Aspergillus. The advent of RNA sequencing has revealed a previous underestimate of viral prevalence in fungi, in particular linear single-stranded RNA viruses, and here we outline the diverse viral families known to date that contain mycoviruses infecting Aspergillus. Furthermore, we describe these novel mycoviruses, highlighting those with peculiar genome structures, such as a split RNA dependent RNA polymerase gene. Next, we delineate notable mycovirus-mediated phenotypes in Aspergillus, in particular reporting on observations of mycoviruses that affect their fungal host’s virulence and explore how this may relate to virus-mediated decreased stress tolerance. Furthermore, mycovirus effects on microbial competition and antifungal resistance are discussed. The factors that influence the manifestation of these phenotypes, such as temperature, fungal life stage, and infection with multiple viruses, among others, are also evaluated. In addition, we attempt to elucidate the molecular mechanisms that underpin these phenotypes, examining how mycoviruses can be targets, triggers, and even suppressors of RNA silencing and how this can affect fungal gene expression and phenotypes. Finally, we highlight the potential therapeutic applications of mycoviruses and how, in an approach analogous to bacteriophage therapy, their ability to produce hypovirulence in Aspergillus might be used to attenuate invasive aspergillosis infections in humans.
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- 2024
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3. Current and Future Pathways in Aspergillus Diagnosis
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Radim Dobiáš, David A. Stevens, and Vladimír Havlíček
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aspergillosis ,galactomannan ,β-d-glucan ,PCR ,metagenomic next-generation sequencing ,lateral flow ,Therapeutics. Pharmacology ,RM1-950 - Abstract
Aspergillus fumigatus has been designated by the World Health Organization as a critical priority fungal pathogen. Some commercially available diagnostics for many forms of aspergillosis rely on fungal metabolites. These encompass intracellular molecules, cell wall components, and extracellular secretomes. This review summarizes the shortcomings of antibody tests compared to tests of fungal products in body fluids and highlights the application of β-d-glucan, galactomannan, and pentraxin 3 in bronchoalveolar lavage fluids. We also discuss the detection of nucleic acids and next-generation sequencing, along with newer studies on Aspergillus metallophores.
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- 2023
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4. Diagnosis of Aspergillosis in Horses
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Radim Dobiáš, Petr Jahn, Katarina Tóthová, Olga Dobešová, Denisa Višňovská, Rutuja Patil, Anton Škríba, Pavla Jaworská, Miša Škorič, Libor Podojil, Michaela Kantorová, Jakub Mrázek, Eva Krejčí, David A. Stevens, and Vladimír Havlíček
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equine aspergillosis ,invasive pulmonary aspergillosis ,bronchoalveolar lavage fluid ,tracheal wash ,serum ,galactomannan ,Biology (General) ,QH301-705.5 - Abstract
Invasive pulmonary aspergillosis (IPA) may be a rare cause of granulomatous pneumonia in horses. The mortality of IPA is almost 100%; direct diagnostic tools in horses are needed. Bronchoalveolar lavage fluid (BALF) and serum samples were collected from 18 horses, including individuals suffering from IPA (n = 1), equine asthma (EA, n = 12), and 5 healthy controls. Serum samples were collected from another 6 healthy controls. Samples of BALF (n = 18) were analyzed for Aspergillus spp. DNA, fungal galactomannan (GM), ferricrocin (Fc), triacetylfusarinin C (TafC), and gliotoxin (Gtx). Analysis of 24 serum samples for (1,3)-β-D-glucan (BDG) and GM was performed. Median serum BDG levels were 131 pg/mL in controls and 1142 pg/mL in IPA. Similar trends were observed in BALF samples for GM (Area under the Curve (AUC) = 0.941) and DNA (AUC = 0.941). The fungal secondary metabolite Gtx was detected in IPA BALF and lung tissue samples (86 ng/mL and 2.17 ng/mg, AUC = 1).
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- 2023
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5. Distinguishing Invasive from Chronic Pulmonary Infections: Host Pentraxin 3 and Fungal Siderophores in Bronchoalveolar Lavage Fluids
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Radim Dobiáš, Pavla Jaworská, Valeria Skopelidou, Jan Strakoš, Denisa Višňovská, Marcela Káňová, Anton Škríba, Pavlína Lysková, Tomáš Bartek, Ivana Janíčková, Radovan Kozel, Lucie Cwiková, Zbyněk Vrba, Milan Navrátil, Jan Martinek, Pavla Coufalová, Eva Krejčí, Vít Ulmann, Milan Raška, David A. Stevens, and Vladimír Havlíček
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invasive fungal disease ,pulmonary aspergillosis ,bronchoalveolar lavage fluid ,pentraxin-3 ,triacetylfusarinine C ,non-neutropenic ,Biology (General) ,QH301-705.5 - Abstract
The multiple forms of pulmonary aspergillosis caused by Aspergillus species are the most common respiratory mycoses. Although invasive, the analysis of diagnostic biomarkers in bronchoalveolar lavage fluid (BALF) is a clinical standard for diagnosing these conditions. The BALF samples from 22 patients with proven or probable aspergillosis were assayed for human pentraxin 3 (Ptx3), fungal ferricrocin (Fc), and triacetylfusarinine C (TafC) in a retrospective study. The infected group included patients with invasive pulmonary aspergillosis (IPA) and chronic aspergillosis (CPA). The BALF data were compared to a control cohort of 67 patients with invasive pulmonary mucormycosis (IPM), non-Aspergillus colonization, or bacterial infections. The median Ptx3 concentrations in patients with and without aspergillosis were 4545.5 and 242.0 pg/mL, respectively (95% CI, p < 0.05). The optimum Ptx3 cutoff for IPA was 2545 pg/mL, giving a sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of 100, 98, 95, and 100%, respectively. The median Ptx3 concentration for IPM was high at 4326 pg/mL. Pentraxin 3 assay alone can distinguish IPA from CPA and invasive fungal disease from colonization. Combining Ptx3 and TafC assays enabled the diagnostic discrimination of IPM and IPA, giving a specificity and PPV of 100%.
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- 2022
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6. Correction: Patil et al. Freeing Aspergillus fumigatus of Polymycovirus Infection Renders It More Resistant to Competition with Pseudomonas aeruginosa Due to Altered Iron-Acquiring Tactics. J. Fungi 2021, 7, 497
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Rutuja H. Patil, Ioly Kotta-Loizou, Andrea Palyzová, Tomáš Pluháček, Robert H. A. Coutts, David A. Stevens, and Vladimír Havlíček
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n/a ,Biology (General) ,QH301-705.5 - Abstract
In the original publication [...]
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- 2022
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7. Combined Use of Presepsin and (1,3)-β-D-glucan as Biomarkers for Diagnosing Candida Sepsis and Monitoring the Effectiveness of Treatment in Critically Ill Patients
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Radim Dobiáš, Marcela Káňová, Naděžda Petejová, Štefan Kis Pisti, Robert Bocek, Eva Krejčí, Helena Stružková, Michaela Cachová, Hana Tomášková, Petr Hamal, Vladimír Havlíček, and Milan Raška
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sepsis ,Candida ,bloodstream infections ,presepsin ,procalcitonin ,C-reactive protein ,Biology (General) ,QH301-705.5 - Abstract
New biomarker panel was developed and validated on 165 critically ill adult patients to enable a more accurate invasive candidiasis (IC) diagnosis. Serum levels of the panfungal biomarker (1,3)-β-D-glucan (BDG) and the inflammatory biomarkers C-reactive protein, presepsin (PSEP), and procalcitonin (PCT) were correlated with culture-confirmed candidemia or bacteremia in 58 and 107 patients, respectively. The diagnostic utility was evaluated in sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). BDG was the best marker for IC, achieving 96.6% sensitivity, 97.2% specificity, 94.9% PPV, and 98.1% NPV at a cut-off of 200 pg/mL (p ≤ 0.001). PSEP exhibited 100% sensitivity and 100% NPV at a cut-off of 700 pg/mL but had a lower PPV (36.5%) and low specificity (5.6%). Combined use of PSEP and BDG, thus, seems to be the most powerful laboratory approach for diagnosing IC. Furthermore, PSEP was more accurate for 28-day mortality prediction the area under the receiver operating characteristic curve (AUC = 0.74) than PCT (AUC = 0.31; PCT cut-off = 0.5 ng/mL). Finally, serum PSEP levels decreased significantly after only 14 days of echinocandin therapy (p = 0.0012). The probability of IC is almost 100% in critically ill adults with serum BDG and PSEP concentrations > 200 pg/mL and >700 pg/mL, respectively, defining a borderline between non-invasive superficial Candida colonization and IC.
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- 2022
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8. Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept
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Radim Dobiáš, Anton Škríba, Tomáš Pluháček, Miloš Petřík, Andrea Palyzová, Marcela Káňová, Eva Čubová, Jiří Houšť, Jiří Novák, David A. Stevens, Goran Mitulovič, Eva Krejčí, Petr Hubáček, and Vladimír Havlíček
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Aspergillus fumigatus ,Pseudomonas aeruginosa ,invasive infection ,noninvasive diagnosis ,coinfection ,virulence factor ,Biology (General) ,QH301-705.5 - Abstract
In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.
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- 2021
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9. Freeing Aspergillus fumigatus of Polymycovirus Infection Renders It More Resistant to Competition with Pseudomonas aeruginosa Due to Altered Iron-Acquiring Tactics
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Rutuja H. Patil, Ioly Kotta-Loizou, Andrea Palyzová, Tomáš Pluháček, Robert H. A. Coutts, David A. Stevens, and Vladimír Havlíček
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Aspergillus fumigatus ,intermicrobial competition ,polymycovirus ,Pseudomonas aeruginosa ,siderophore ,Biology (General) ,QH301-705.5 - Abstract
A virus-free (VF) A. fumigatus isolate has been shown to be resistant in competition with Pseudomonas as compared to the isogenic line infected with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1), and this phenotype was apparently related to alterations in iron metabolism. Here we investigated further the mechanisms underpinning this phenotype. The extracellular siderophore profiles of five isogenic VF and virus-infected (VI) strains were sampled at 24, 31, 48, 54, and 72 h in submerged cultures and quantitatively examined by liquid chromatography and mass spectrometry. Intracellular profiles of conidia and cultures at the stationary growth phase were defined. VF A. fumigatus demonstrated the best fitness represented by the fastest onset of its exponential growth when grown on an iron-limited mineral medium. The exponential phase and transitional production phase of the extracellular triacetylfusarinine C (TafC) were achieved at 24 and 31 h, respectively, contrary to VI strains, which acted more slowly. As a result, the TafC reservoir was consumed sooner in the VF strain. Additionally, the VF strain had lower ferricrocin and higher hydroxyferricrocin content in the pellet during the stationary phase. All of these differences were significant (Kruskal–Wallis, p < 0.01). In our study, the siderophore reservoir of a VF strain was consumed sooner, improving the fitness of the VF strain in competition with P. aeruginosa.
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- 2021
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10. Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs
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Tereza Juříková, Dominika Luptáková, Olga Kofroňová, Anton Škríba, Jiří Novák, Helena Marešová, Andrea Palyzová, Miloš Petřík, Vladimír Havlíček, and Oldřich Benada
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fixation ,scanning electron microscopy ,matrix-assisted laser desorption/ionization mass spectrometry imaging ,bacteria ,fungi ,rat lung tissue ,Biology (General) ,QH301-705.5 - Abstract
A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.
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- 2020
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11. Rhizoferrin Glycosylation in Rhizopus microsporus
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Anton Škríba, Rutuja Hiraji Patil, Petr Hubáček, Radim Dobiáš, Andrea Palyzová, Helena Marešová, Tomáš Pluháček, and Vladimír Havlíček
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Rhizopus microsporus ,glycoside ,rhizoferrin ,metabolite ,siderophore ,mass spectrometry ,Biology (General) ,QH301-705.5 - Abstract
Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.
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- 2020
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12. Antifungal Drugs
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Jiří Houšť, Jaroslav Spížek, and Vladimír Havlíček
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antifungal drugs ,amphotericin b ,flucytosine ,triazoles ,echinocandins ,invasive fungal infections ,resistance ,siderophores ,Microbiology ,QR1-502 - Abstract
We reviewed the licensed antifungal drugs and summarized their mechanisms of action, pharmacological profiles, and susceptibility to specific fungi. Approved antimycotics inhibit 1,3-β-d-glucan synthase, lanosterol 14-α-demethylase, protein, and deoxyribonucleic acid biosynthesis, or sequestrate ergosterol. Their most severe side effects are hepatotoxicity, nephrotoxicity, and myelotoxicity. Whereas triazoles exhibit the most significant drug−drug interactions, echinocandins exhibit almost none. The antifungal resistance may be developed across most pathogens and includes drug target overexpression, efflux pump activation, and amino acid substitution. The experimental antifungal drugs in clinical trials are also reviewed. Siderophores in the Trojan horse approach or the application of siderophore biosynthesis enzyme inhibitors represent the most promising emerging antifungal therapies.
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- 2020
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13. Visualizing spatial lipid distribution in porcine lens by MALDI imaging high-resolution mass spectrometry
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Veronika Vidová, Jaroslav Pól, Michael Volný, Petr Novák, Vladimír Havlíček, Susanne K. Wiedmer, and Juha M. Holopainen
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ocular lens ,sphingolipid ,matrix-assisted laser desorption/ionization ,Biochemistry ,QD415-436 - Abstract
The intraocular lens contains high levels of both cholesterol and sphingolipids, which are believed to be functionally important for normal lens physiology. The aim of this study was to explore the spatial distribution of sphingolipids in the ocular lens using mass spectrometry imaging (MSI). Matrix-assisted laser desorption/ionization (MALDI) imaging with ultra high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS) was used to visualize the lipid spatial distribution. Equatorially-cryosectioned, 12 μm thick slices of tissue were thaw-mounted to an indium-tin oxide (ITO) glass slide by soft-landing to an ethanol layer. This procedure maintained the tissue integrity. After the automated MALDI matrix deposition, the entire lens section was examined by MALDI MSI in a 150 μm raster. We obtained spatial- and concentration-dependent distributions of seven lens sphingomyelins (SM) and two ceramide-1-phosphates (CerP), which are important lipid second messengers. Glycosylated sphingolipids or sphingolipid breakdown products were not observed. Owing to ultra high resolution MS, all lipids were identified with high confidence, and distinct distribution patterns for each of them are presented. The distribution patterns of SMs provide an understanding of the physiological functioning of these lipids in clear lenses and offer a novel pathophysiological means for understanding diseases of the lens.
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- 2010
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14. Spatial distribution of glycerophospholipids in the ocular lens.
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Jaroslav Pól, Veronika Vidová, Tuulia Hyötyläinen, Michael Volný, Petr Novák, Martin Strohalm, Risto Kostiainen, Vladimír Havlíček, Susanne K Wiedmer, and Juha M Holopainen
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Medicine ,Science - Abstract
Knowledge of the spatial distribution of lipids in the intraocular lens is important for understanding the physiology and biochemistry of this unique tissue and for gaining a better insight into the mechanisms underlying diseases of the lens. Following our previous study showing the spatial distribution of sphingolipids in the porcine lens, the current study used ultra performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-QTOFMS) to provide the whole lipidome of porcine lens and these studies were supplemented by matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI) of the lens using ultra-high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) to determine the spatial distribution of glycerophospholipids. Altogether 172 lipid species were identified with high confidence and their concentration was determined. Sphingomyelins, phosphatidylcholines, and phosphatidylethanolamines were the most abundant lipid classes. We then determined the spatial and concentration-dependent distributions of 20 phosphatidylcholines, 6 phosphatidylethanolamines, and 4 phosphatidic acids. Based on the planar molecular images of the lipids, we report the organization of fiber cell membranes within the ocular lens and suggest roles for these lipids in normal and diseased lenses.
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- 2011
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15. Quantitation of small molecules from liquid chromatography—mass spectrometric accurate mass datasets using CycloBranch
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Jiří Novák, Kevin A Schug, and Vladimír Havlíček
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General Medicine ,Spectroscopy ,Atomic and Molecular Physics, and Optics - Abstract
Gaussian and exponentially modified Gaussian functions were incorporated into integrating algorithms used by an open-source, cross-platform tool called CycloBranch. The quantitation is demonstrated on bacterial pyoverdines separated by fine isotope features. Using our algorithm, we can separate the m/z values 694.25802 and 694.26731 (a 0.009 Da difference), where the former belongs to the most intense peak of pyoverdine D (PvdD), and the latter to the second most intense peak of pyoverdine E (PvdE) in the respective isotopic clusters of [M + Fe-H]2+ ions. The areas under chromatographic curves of standards were analyzed for the limit of detection (LOD), limit of quantitation (LOQ), and regression coefficient calculations. The quantitative module returned a LOD and LOQ of 1.4 and 4.3 ng/mL, respectively, for both PvdD and PvdE in human urine. If present and detected in mass spectra, the intensities of user-defined [M + H]+, [M + Na]+, [M + K]+, [M + Fe-H]2+, or other ion types, can be accumulated and used for quantitation. The quantitation result is returned by CycloBranch in seconds or minutes, contrary to an hours-long manual approach, prone to user-born errors originating from necessary copying among various software environments. Native Bruker, Waters, Thermo, txt, mgf, mzML, and mzXML data formats are supported in CycloBranch, which is freely available at https://ms.biomed.cas.cz/cyclobranch .
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- 2023
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16. Siderophore-Based Noninvasive Differentiation of Aspergillus fumigatus Colonization and Invasion in Pulmonary Aspergillosis
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Dominika Luptáková, Rutuja H. Patil, Radim Dobiáš, David A. Stevens, Tomáš Pluháček, Andrea Palyzová, Marcela Káňová, Milan Navrátil, Zbyněk Vrba, Petr Hubáček, and Vladimír Havlíček
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Microbiology (medical) ,Infectious Diseases ,General Immunology and Microbiology ,Ecology ,Physiology ,Genetics ,Cell Biology - Abstract
The importance of this research lies in the demonstration that siderophore analysis can distinguish between asymptomatic colonization and invasive pulmonary aspergillosis. We found clear associations between phases of fungal development, from conidial germination to the proliferative stage of invasive aspergillosis, and changes in secondary metabolite secretion.
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- 2023
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17. 68Ga-labelled desferrioxamine-B for bacterial infection imaging
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Vladislav Raclavsky, Miroslav Popper, Marian Hajduch, Clemens Decristoforo, Joachim Pfister, Andrea Palyzová, Zbynek Novy, Vladimír Havlíček, Eva Umlaufova, Milos Petrik, and Christian Mair
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0301 basic medicine ,Desferrioxamine-B ,Low protein ,Pseudomonas aeruginosa ,Chemistry ,Gallium-68 ,General Medicine ,medicine.disease_cause ,In vitro ,030218 nuclear medicine & medical imaging ,Microbiology ,Imaging ,03 medical and health sciences ,030104 developmental biology ,0302 clinical medicine ,PET ,Streptococcus agalactiae ,Pharmacokinetics ,Staphylococcus aureus ,In vivo ,medicine ,Radiology, Nuclear Medicine and imaging ,Original Article ,Infection ,Escherichia coli - Abstract
Purpose With the increase of especially hospital-acquired infections, timely and accurate diagnosis of bacterial infections is crucial for effective patient care. Molecular imaging has the potential for specific and sensitive detection of infections. Siderophores are iron-specific chelators recognized by specific bacterial transporters, representing one of few fundamental differences between bacterial and mammalian cells. Replacing iron by gallium-68 without loss of bioactivity is possible allowing molecular imaging by positron emission tomography (PET). Here, we report on the preclinical evaluation of the clinically used siderophore, desferrioxamine-B (Desferal®, DFO-B), radiolabelled with 68Ga for imaging of bacterial infections. Methods In vitro characterization of [68Ga]Ga-DFO-B included partition coefficient, protein binding and stability determination. Specific uptake of [68Ga]Ga-DFO-B was tested in vitro in different microbial cultures. In vivo biodistribution was studied in healthy mice and dosimetric estimation for human setting performed. PET/CT imaging was carried out in animal infection models, representing the most common pathogens. Results DFO-B was labelled with 68Ga with high radiochemical purity and displayed hydrophilic properties, low protein binding and high stability in human serum and PBS. The high in vitro uptake of [68Ga]Ga-DFO-B in selected strains of Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus agalactiae could be blocked with an excess of iron-DFO-B. [68Ga]Ga-DFO-B showed rapid renal excretion and minimal retention in blood and other organs in healthy mice. Estimated human absorbed dose was 0.02 mSv/MBq. PET/CT images of animal infection models displayed high and specific accumulation of [68Ga]Ga-DFO-B in both P. aeruginosa and S. aureus infections with excellent image contrast. No uptake was found in sterile inflammation, heat-inactivated P. aeruginosa or S. aureus and Escherichia coli lacking DFO-B transporters. Conclusion DFO-B can be easily radiolabelled with 68Ga and displayed suitable in vitro characteristics and excellent pharmacokinetics in mice. The high and specific uptake of [68Ga]Ga-DFO-B by P. aeruginosa and S. aureus was confirmed both in vitro and in vivo, proving the potential of [68Ga]Ga-DFO-B for specific imaging of bacterial infections. As DFO-B is used in clinic for many years and the estimated radiation dose is lower than for other 68Ga-labelled radiopharmaceuticals, we believe that [68Ga]Ga-DFO-B has a great potential for clinical translation.
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- 2020
18. CycloBranch 2: Molecular Formula Annotations Applied to imzML Data Sets in Bimodal Fusion and LC-MS Data Files
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Vladimír Havlíček, Jiří Novák, and Anton Škríba
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Biological Products ,Fusion ,Image fusion ,Databases, Factual ,business.industry ,Chemistry ,010401 analytical chemistry ,Pattern recognition ,010402 general chemistry ,Mass spectrometry ,01 natural sciences ,Mass Spectrometry ,Mass spectrometry imaging ,0104 chemical sciences ,Analytical Chemistry ,Molecular Weight ,Software ,Metabolomics ,Liquid chromatography–mass spectrometry ,Data file ,Artificial intelligence ,business ,Chromatography, Liquid - Abstract
Natural product chemistry, microbiology, and food, human, and plant metabolomics represent a few sources of complex metabolomics data generated by mass spectrometry. Among the medley of software tools used to handle these data sets, no universal tool can qualitatively, quantitatively, or statistically address major biological questions or tasks. CycloBranch 2, an open and platform-free software, at least now provides the de novo generation of molecular formulas of unknown compounds in both liquid chromatography/mass spectrometry and mass spectrometry imaging datafiles. For imaging files, this database-free approach was documented in the bimodal image fusion and characterization of three small molecules, including metallophores. The fine isotope ratio data filtering step distinguished 34S/13C2 and 41K/13C2 features. The standalone software package is implemented in C++ and can be downloaded from https://ms.biomed.cas.cz/cyclobranch/ and used under GNU General Public License.
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- 2020
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19. Bacteriocin ASM1 is an O / S ‐diglycosylated, plasmid‐encoded homologue of glycocin F
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Trevor S. Loo, Gillian E. Norris, Vladimír Havlíček, Patrick Main, Tomomi Hata, Petr Man, Mark L. Patchett, and Petr Novák
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Glycosylation ,Biophysics ,Biochemistry ,Genome ,03 medical and health sciences ,Plasmid ,Bacteriocins ,Bacteriocin ,Structural Biology ,Genetics ,Amino Acid Sequence ,Molecular Biology ,Gene ,IC50 ,Phylogeny ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Base Sequence ,biology ,030302 biochemistry & molecular biology ,Prokaryote ,Sequence Analysis, DNA ,Cell Biology ,biology.organism_classification ,Amino acid ,chemistry ,Genes, Bacterial ,Novobiocin ,Lactobacillus plantarum ,Plasmids - Abstract
Here, we report on the biochemical characterization of a new glycosylated bacteriocin (glycocin), ASM1, produced by Lactobacillus plantarum A-1 and analysis of the A-1 bacteriocinogenic genes. ASM1 is 43 amino acids in length with Ser18-O- and Cys43-S-linked N-acetylglucosamine moieties that are essential for its inhibitory activity. Its only close homologue, glycocin F (GccF), has five amino acid substitutions all residing in the flexible C-terminal 'tail' and a lower IC50 (0.9 nm) compared to that of ASM1 (1.5 nm). Asm/gcc genes share the same organization (asmH← →asmABCDE→F), and the asm genes reside on an 11 905-bp plasmid dedicated to ASM1 production. The A-1 genome also harbors a gene encoding a 'rare' bactofencin-type bacteriocin. As more examples of prokaryote S-GlcNAcylation are discovered, the functions of this modification may be understood.
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- 2020
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20. Infection metallomics for critical care in the post‐COVID era
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Vladimír Havlíček, Rutuja H. Patil, and Dominika Luptáková
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ved/biology ,medicine.drug_class ,Chemistry ,Critically ill ,ved/biology.organism_classification_rank.species ,Antibiotics ,Metallome ,Condensed Matter Physics ,General Biochemistry, Genetics and Molecular Biology ,Analytical Chemistry ,In vivo ,Immunology ,medicine ,Colonization ,Microbiome ,Model organism ,Pathogen ,Spectroscopy - Abstract
Infection metallomics is a mass spectrometry (MS) platform we established based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. Here we review the in vitro, in vivo, and clinical applications of metallophores from historical and functional perspectives, and identify under-studied and emerging application areas with high diagnostic potential for the post-COVID era. MS with isotope data filtering is fundamental to infection metallomics; it has been used to study the interplay between "frenemies" in hosts and to monitor the dynamic response of the microbiome to antibiotic and antimycotic therapies. During infection in critically ill patients, the hostile environment of the host's body activates secondary bacterial, mycobacterial, and fungal metabolism, leading to the production of metallophores that increase the pathogen's chance of survival in the host. MS can reveal the structures, stability, and threshold concentrations of these metal-containing microbial biomarkers of infection in humans and model organisms, and can discriminate invasive disease from benign colonization based on well-defined thresholds distinguishing proliferation from the colonization steady state.
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- 2021
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21. Presepsin and (1,3)-β-D-Glucan as a Tool for Predicting Candida Sepsis and Monitoring the Effectiveness of Treatment in Critically Ill Patients
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Hana Tomášková, Petr Hamal, Marcela Kanova, Milan Raska, Vladimír Havlíček, Stefan Kis Pisti, Nadezda Petejova, Eva Krejčí, Robert Bocek, Michaela Cachova, Helena Struzkova, and Radim Dobiáš
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Critically ill ,business.industry ,Immunology ,Medicine ,business ,Candida sepsis ,1 3 β d glucan - Abstract
Background: To improve the accuracy in invasive candidiasis diagnostics, a new biomarker panel has been developed and validated on a 165-patient cohort of critically ill adults.Methods: The serum levels of inflammatory biomarkers, C-reactive protein, presepsin (PSEP), procalcitonin (PCT), and of panfungal (1,3)-β-D-glucan (BDG), were correlated with culture-confirmed candidemia or bacteremia in 58 or 107 patients, respectively. The diagnostic effect of the host and pathogen biomarkers was expressed using sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV).Results: For invasive candidiasis, the best performing BDG exhibited 96.6% sensitivity, 97.2% specificity, 94.9% PPV and 98.1% NPV at a cut-off of 200 pg/mL (P ≤ 0.001). PSEP exhibited 100% sensitivity and 100% NPV at a cut-off of 700 pg/mL. Furthermore, PSEP was more accurate for 28-day mortality prediction (AUC = 0.74) than PCT (AUC = 0.31; PCT cut-off 0.5 ng/mL). Finally, PSEP showed a significant serum decrease as early as 14 days after echinocandin therapy initiation (P = 0.0012).Conclusions: At concentrations of BDG > 200 pg/mL and PSEP > 700 pg/mL, the probability of invasive candidiasis in critically ill adults is close to 100% defining a borderline between non-invasive superficial Candida colonization and invasive candidiasis.Trial registration: The study was approved by the University Hospital Ostrava Ethics Committee for Multicenter Clinical Trials (no. 448/2018) and registered in ClinicalTrials.gov (ID: NCT03584594), date of registration June 28,2018.
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- 2021
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22. [Treatment of chromoblastomycosis and phaeohyphomycosis]
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Radim, Dobiáš and Vladimír, Havlíček
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Phaeohyphomycosis ,Antifungal Agents ,Chromoblastomycosis ,Pyridines ,Nitriles ,Humans ,Triazoles - Abstract
Cases of chromoblastomycosis are frequent in certain parts of the world, especially in some developing countries. Clinical manifestations of chromoblastomycosis are typical. To a certain extent, pathogens causing chromoblastomycosis overlap with those causing phaeohyphomycosis. Although cases of phaeohyphomycosis are not very common, they may end fatally. Therefore early management of these life-threatening infections is rather important. Targeted antifungal therapy and surgery are effective in combating these infections. Recently, several triazole antifungals such as posaconazole and isavuconazole have been available to treat even the most severe cases. Prevention of the infection should be aimed at reducing the risk of subcutaneous trauma, particularly in persons in contact with potential sources of infection such as wood materials important from endemic areas.
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- 2021
23. [Chromoblastomycosis and phaeohyphomycosis - pathogenesis and laboratory diagnosis]
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Radim, Dobiáš and Vladimír, Havlíček
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Phaeohyphomycosis ,Antifungal Agents ,Chromoblastomycosis ,Clinical Laboratory Techniques ,Humans ,DNA, Fungal ,Polymerase Chain Reaction - Abstract
Chromoblastomycosis and phaeohyphomycosis are less common fungal infections caused by dark-pigmented fungi. Virulence factors play an important role in the pathogenesis of these diseases. One of these factors, muriform cells, are the most important element for differential diagnosis of chromoblastomycosis and phaeohyphomycosis using clinical samples and various staining techniques. Accurate identification of pathogens causing chromoblastomycosis and phaeohyphomycosis is very important for correct and early antifungal therapy. Therefore, species identification of the etiological agent should be confirmed by sequencing of DNA from the culture. Early diagnosis may be crucial, especially in case of invasive forms of these infections. The diagnosis may be guided by some immunohistochemistry methods and DNA detection using polymerase chain reaction directly from clinical samples seems to be useful for identification of pathogens causing these severe and life-threatening infections.
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- 2021
24. [Chromoblastomycosis and phaeohyphomycosis, overlooked fungal diseases]
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Radim, Dobiáš and Vladimír, Havlíček
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Adult ,Male ,Occupational Diseases ,Phaeohyphomycosis ,Antifungal Agents ,Chromoblastomycosis ,Humans ,Czech Republic - Abstract
Dark-pigmented microscopic fungi are worldwide-spread soil saprophytes often found on plant remnants. In chromoblastomycosis, infectious particles of these fungi enter the human body at the site of injury and may cause chronic infection, mainly in tropical and subtropical endemic areas. Chromoblastomycosis is almost exclusively diagnosed in patients with fully functioning immunity, with typically muriform cells present in infected tissue distinguishing this condition from phaeohyphomycosis. Phaeohyphomycosis, a less specific disease caused by dark-pigmented fungi, usually makes tissue necrotize rather than proliferate, involves a broader range of pathogens of the kingdom Fungi and is mainly associated with immune disorders. Chromoblastomycosis is usually a threat to male adults, globally considered an occupational disease affecting farmers, gardeners, loggers, agricultural commodity traders and other workers exposed to contaminated soil or handling materials of plant origin. In the Czech Republic, immunocompetent patients may be at risk of chromoblastomycosis as imported infection. In the past, however, the infection was also rarely documented as autochthonous in the country.
- Published
- 2021
25. Microbial siderophores: Markers of infectious diseases
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Vladimír Havlíček and Radim Dobiáš
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Siderophore ,Host (biology) ,Disease progression ,Host organism ,Colonization ,Biology ,Antimicrobial ,Pathogen ,Antimicrobial drug ,Microbiology - Abstract
Infectious diseases and constantly growing antimicrobial drug resistance claim millions of lives each year. Early, specific, and sensitive diagnostics, as well as methods for distinguishing between colonization and invasive infection, are essential for the clinical management of bacterial, mycobacterial, and fungal diseases. During infection, low-molecular-weight siderophores and microbial toxins are secreted into the host organism and serve as early biomarkers. These disease progression indicators also reflect the pathogen response to antimicrobial therapy in a host.
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- 2021
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26. Rhizoferrin Glycosylation in Rhizopus microsporus
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Petr Hubacek, Andrea Palyzová, Helena Marešová, Anton Škríba, Rutuja H. Patil, Tomáš Pluháček, Vladimír Havlíček, and Radim Dobiáš
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Microbiology (medical) ,glycoside ,posaconazole metabolism ,Rhizopus microsporus ,Posaconazole ,Glycosylation ,siderophore ,Metabolite ,metabolite ,rhizoferrin ,Plant Science ,law.invention ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Rhizopus ,Biotransformation ,law ,medicine ,liquid chromatography ,lcsh:QH301-705.5 ,Ecology, Evolution, Behavior and Systematics ,Polymerase chain reaction ,030304 developmental biology ,mass spectrometry ,0303 health sciences ,biology ,030306 microbiology ,Communication ,Mucormycosis ,human isolate ,biology.organism_classification ,medicine.disease ,chemistry ,lcsh:Biology (General) ,medicine.drug - Abstract
Rhizopus spp. are the most common etiological agents of mucormycosis, causing over 90% mortality in disseminated infections. The diagnosis relies on histopathology, culture, and/or polymerase chain reaction. For the first time, the glycosylation of rhizoferrin (RHF) was described in a Rhizopus microsporus clinical isolate by liquid chromatography and accurate tandem mass spectrometry. The fermentation broth lyophilizate contained 345.3 ± 13.5, 1.2 ± 0.03, and 0.03 ± 0.002 mg/g of RHF, imido-RHF, and bis-imido-RHF, respectively. Despite a considerable RHF secretion rate, we did not obtain conclusive RHF detection from a patient with disseminated mucormycosis caused by the same R. microsporus strain. We hypothesize that parallel antimycotic therapy, RHF biotransformation, and metabolism compromised the analysis. On the other hand, the full profile of posaconazole metabolites was retrieved by our in house software CycloBranch.
- Published
- 2020
27. CycloBranch: An open tool for fine isotope structures in conventional and product ion mass spectra
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Jiří Novák, Jakub Zápal, Marek Kuzma, Vladimír Havlíček, and Anton Škríba
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0301 basic medicine ,Isotope ,Chemistry ,010401 analytical chemistry ,Analytical chemistry ,Nuclear magnetic resonance spectroscopy ,Chromophore ,Mass spectrometry ,01 natural sciences ,Fourier transform ion cyclotron resonance ,0104 chemical sciences ,Ion ,03 medical and health sciences ,030104 developmental biology ,Product (mathematics) ,Mass spectrum ,Spectroscopy - Abstract
Within the growing community of Fourier transform mass spectrometry users, the identification of fine isotope structure has become an indispensable method for molecular formula determination. In this work, the fine isotope envelopes for accessing the mutual ratio of 2 closely related pyoverdines in a mixture were used. Bacterial siderophores pyoverdines D and E cannot be easily separated via liquid chromatography-mass spectrometry because their structures differ in (de)amidation at the respective chromophore parts only. Their mutual ratio was determined in a mixture via nuclear magnetic resonance spectroscopy and semiquantitative mass spectrometry using our open-source software CycloBranch, which represents a genuine free tool supporting the determination of fine isotope structures in both conventional and product ion mass spectra. Native Bruker, Thermo, and Waters data formats are supported in addition to XML and plain text formats.
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- 2018
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28. Scedosporium and Lomentospora: an updated overview of underrated opportunists
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Adela Martin-Vicente, Javier Pemán, Sandrine Giraud, Aitor Rementeria, José F. Cano-Lira, Ana Alastruey-Izquierdo, Mariana Ingrid Dutra da Silva Xisto, Jean-Philippe Bouchara, Jardel Vieira de Meirelles, Carsten Schwarz, Laszlo Irinyi, María Teresa Martín-Gómez, Anne Bernhardt, Rodrigo Rollin-Pinheiro, Kathrin Tintelnot, Aize Pellon, Fernando L. Hernando, Idoia Buldain, Stéphane Ranque, Leyre M. López-Soria, Andoni Ramirez-Garcia, Yohann Le Govic, Markus Nagl, Sharon C.-A. Chen, Eliana Barreto-Bergter, Wieland Meyer, Vladimír Havlíček, Michaela Lackner, Patrick Vandeputte, Johannes Rainer, Javier Capilla, Sybren de Hoog, Vecteurs - Infections tropicales et méditerranéennes (VITROME), Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut de Recherche Biomédicale des Armées (IRBA), Groupe d'Étude des Interactions Hôte-Pathogène (GEIHP), Université d'Angers (UA), Institute of Earth and Environmental Science [Potsdam], University of Potsdam, Robert Koch Institute [Berlin] (RKI), Universitat Rovira i Virgili, Innsbruck Medical University [Austria] (IMU), Centre for Infectious Disease and Microbiology (CIDM), The Westmead Institute for Medical Research, Institut de Recherche pour le Développement (IRD)-Aix Marseille Université (AMU)-Institut de Recherche Biomédicale des Armées [Brétigny-sur-Orge] (IRBA), University of Potsdam = Universität Potsdam, Innsbruck Medical University = Medizinische Universität Innsbruck (IMU), Westerdijk Fungal Biodiversity Institute, and Westerdijk Fungal Biodiversity Institute - Medical Mycology
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0301 basic medicine ,medicine.medical_specialty ,Antifungal Agents ,Virulence Factors ,030106 microbiology ,Drug resistance ,Opportunistic Infections ,Scedosporium ,Immunocompromised Host ,03 medical and health sciences ,Local infection ,Ascomycota ,[SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases ,Drug Resistance, Multiple, Fungal ,Epidemiology ,medicine ,Humans ,Intensive care medicine ,emergent ,[SDV.MP.MYC]Life Sciences [q-bio]/Microbiology and Parasitology/Mycology ,Lomentospora ,High rate ,Ecology ,business.industry ,Mortality rate ,Fungal genetics ,General Medicine ,Combined Modality Therapy ,infection ,3. Good health ,Molecular Typing ,Infectious Diseases ,Mycoses ,Surgical Procedures, Operative ,Host-Pathogen Interactions ,fungi ,business ,pathogen - Abstract
International audience; Species of Scedosporium and Lomentospora are considered as emerging opportunists, affecting immunosuppressed and otherwise debilitated patients, although classically they are known from causing trauma-associated infections in healthy individuals. Clinical manifestations range from local infection to pulmonary colonization and severe invasive disease, in which mortality rates may be over 80%. These unacceptably high rates are due to the clinical status of patients, diagnostic difficulties, and to intrinsic antifungal resistance of these fungi. In consequence, several consortia have been founded to increase research efforts on these orphan fungi. The current review presents recent findings and summarizes the most relevant points, including the Scedosporium/Lomentospora taxonomy, environmental distribution, epidemiology, pathology, virulence factors, immunology, diagnostic methods, and therapeutic strategies.
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- 2018
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29. Noninvasive Combined Diagnosis and Monitoring of Aspergillus and Pseudomonas Infections: Proof of Concept
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David A. Stevens, Miloš Petřík, Tomáš Pluháček, Eva Krejčí, Petr Hubacek, Eva Čubová, Marcela Káňová, Radim Dobiáš, Vladimír Havlíček, Anton Škríba, Goran Mitulović, Jiří Houšť, Jiří Novák, and Andrea Palyzová
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quorum-sensing molecules ,Microbiology (medical) ,QH301-705.5 ,Case Report ,Plant Science ,Urine ,medicine.disease_cause ,virulence factor ,Aspergillus fumigatus ,Microbiology ,Intensive care ,medicine ,Biology (General) ,Ecology, Evolution, Behavior and Systematics ,Aspergillus ,biology ,Pseudomonas aeruginosa ,business.industry ,siderophores ,Pseudomonas ,biology.organism_classification ,medicine.disease ,coinfection ,Coinfection ,invasive infection ,noninvasive diagnosis ,business ,Urine sample - Abstract
In acutely ill patients, particularly in intensive care units or in mixed infections, time to a microbe-specific diagnosis is critical to a successful outcome of therapy. We report the application of evolving technologies involving mass spectrometry to diagnose and monitor a patient’s course. As proof of this concept, we studied five patients and used two rat models of mono-infection and coinfection. We report the noninvasive combined monitoring of Aspergillus fumigatus and Pseudomonas aeruginosa infection. The invasive coinfection was detected by monitoring the fungal triacetylfusarinine C and ferricrocin siderophore levels and the bacterial metabolites pyoverdin E, pyochelin, and 2-heptyl-4-quinolone, studied in the urine, endotracheal aspirate, or breath condensate. The coinfection was monitored by mass spectrometry followed by isotopic data filtering. In the rat infection model, detection indicated 100-fold more siderophores in urine compared to sera, indicating the diagnostic potential of urine sampling. The tools utilized in our studies can now be examined in large clinical series, where we could expect the accuracy and speed of diagnosis to be competitive with conventional methods and provide advantages in unraveling the complexities of mixed infections.
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- 2021
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30. Ultra-high performance supercritical fluid chromatography-mass spectrometry procedure for analysis of monosaccharides from plant gum binders
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Karel Lemr, Vladimír Havlíček, Tomáš Pluháček, and Volodymyr Pauk
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Paper ,food.ingredient ,Formic acid ,01 natural sciences ,Biochemistry ,Analytical Chemistry ,chemistry.chemical_compound ,Hydrolysis ,food ,Polysaccharides ,Tandem Mass Spectrometry ,Paint ,Plant Gums ,Environmental Chemistry ,Monosaccharide ,Spectroscopy ,chemistry.chemical_classification ,Chromatography ,010405 organic chemistry ,Elution ,Tragacanth ,Monosaccharides ,010401 analytical chemistry ,Chromatography, Supercritical Fluid ,Supercritical fluid ,0104 chemical sciences ,chemistry ,Supercritical fluid chromatography ,Gum arabic - Abstract
The ultra-high performance supercritical fluid chromatography-mass spectrometry (UHPSFC/MS) procedure for analysis of native monosaccharides was developed. Chromatographic conditions were investigated to separate a mixture of four hexoses, three pentoses, two deoxyhexoses and two uronic acids. Increasing water content in methanol modifier to 5% and formic acid to 4% improved peak shapes of neutral monosaccharides and allowed complete elution of highly polar uronic acids in a single run. An Acquity HSS C18SB column outperformed other three tested stationary phases (BEH (silica), BEH 2-ethylpyridine, CSH Fluoro-Phenyl) in terms of separation of isomers and analysis time (4.5 min). Limits of detection were in the range 0.01–0.12 ng μL−1. Owing to separation of anomers, identification of critical pairs (arabinose-xylose and glucose-galactose) was possible. Feasibility of the new method was demonstrated on plant-derived polysaccharide binders. Samples of watercolor paints, painted paper and three plant gums widely encountered in painting media (Arabic, cherry and tragacanth) were decomposed prior the analysis by microwave-assisted hydrolysis at 40 bar initial pressure using 2 mol L−1 trifluoroacetic acid. Among tested temperatures, 120 °C ensured appropriate hydrolysis efficiency for different types of gum and avoided excessive degradation of labile monosaccharides. Procedure recovery tested on gum Arabic was 101% with an RSD below 8%. Aqueous hydrolysates containing monosaccharides in different ratios specific to each type of plant gum were diluted or analyzed directly. Filtration of samples before hydrolysis reduced interferences from a paper support and identification of gum Arabic in watercolor-painted paper samples was demonstrated. Successful identification of pure gum Arabic was confirmed for sample quantities as little as 1 μg. Two classification approaches were compared and principal component analysis was superior to analysis based on peak area ratios of monosaccharides. The proposed procedure using UHPSFC/MS represents an interesting alternative which can compete with other chromatographic methods in the field of saccharide analysis in terms of speed, sensitivity and simplicity of workflow.
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- 2017
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31. Resolution of isomeric new designer stimulants using gas chromatography – Vacuum ultraviolet spectroscopy and theoretical computations
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Ludovit Skultety, Ira S. Lurie, Cory Vaught, Vladimír Havlíček, Angelica Szewczak, Lindsey Shear-Laude, Changling Qiu, Jonathan Smuts, James X. Mao, Peter Kroll, Petr Fryčák, Kevin A. Schug, and Karel Lemr
- Subjects
Chromatography, Gas ,Vacuum ,Absorption spectroscopy ,Resolution (mass spectrometry) ,medicine.drug_class ,Analytical chemistry ,010402 general chemistry ,medicine.disease_cause ,01 natural sciences ,Biochemistry ,Designer Drugs ,Analytical Chemistry ,Isomerism ,medicine ,Environmental Chemistry ,Butylone ,Spectroscopy ,Chemistry ,Spectrum Analysis ,010401 analytical chemistry ,Time-dependent density functional theory ,0104 chemical sciences ,Designer drug ,Central Nervous System Stimulants ,Density functional theory ,Gas chromatography ,Ultraviolet ,medicine.drug - Abstract
Distinguishing isomeric representatives of “bath salts”, “plant food”, “spice”, or “legal high” remains a challenge for analytical chemistry. In this work, we used vacuum ultraviolet spectroscopy combined with gas chromatography to address this issue on a set of forty-three designer drugs. All compounds, including many isomers, returned differentiable vacuum ultraviolet/ultraviolet spectra. The pair of 3- and 4-fluoromethcathinones (m/z 181.0903), as well as the methoxetamine/meperidine/ethylphenidate (m/z 247.1572) triad, provided very distinctive vacuum ultraviolet spectral features. On the contrary, spectra of 4-methylethcathinone, 4-ethylmethcathinone, 3,4-dimethylmethcathinone triad (m/z 191.1310) displayed much higher similarities. Their resolution was possible only if pure standards were probed. A similar situation occurred with the ethylone and butylone pair (m/z 221.1052). On the other hand, majority of forty-three drugs was successfully separated by gas chromatography. The detection limits for all the drug standards were in the 2–4 ng range (on-column amount), which is sufficient for determinations of seized drugs during forensics analysis. Further, state-of-the-art time-dependent density functional theory was evaluated for computation of theoretical absorption spectra in the 125–240 nm range as a complementary tool.
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- 2017
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32. Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency
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Anton Škríba, Abdullah M. S. Al-Hatmi, Annelies Verbon, Vladimír Havlíček, Tokameh Mahmoudi, Elisa De Crignis, Mohammad Javad Najafzadeh, Raquel Crespo, Tsung Wai Kan, Renata Ptackova, Robert-Jan Palstra, Miroslav Sulc, Enrico Ne, Joyce Kang, Michael Roling, Casper Rokx, Elizabeth LeMasters, Peter D. Katsikis, Wilfred F. J. van IJcken, Joyce H.G. Lebbink, Sybren de Hoog, Pritha Biswas, Mateusz Stoszko, Yvonne M. Mueller, Alessia Bertoldi, Biochemistry, Immunology, Radiotherapy, Molecular Genetics, Internal Medicine, Medical Microbiology & Infectious Diseases, and Cell biology
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RNA polymerase II ,HIV Infections ,environment and public health ,03 medical and health sciences ,chemistry.chemical_compound ,All institutes and research themes of the Radboud University Medical Center ,Gliotoxin ,SDG 3 - Good Health and Well-being ,Transcription (biology) ,7SK RNA ,Virology ,Gene expression ,Humans ,snRNP ,Positive Transcriptional Elongation Factor B ,P-TEFb ,Molecular Biology ,Research Articles ,030304 developmental biology ,0303 health sciences ,Multidisciplinary ,biology ,030306 microbiology ,SciAdv r-articles ,RNA-Binding Proteins ,Ribonucleoproteins, Small Nuclear ,Molecular biology ,3. Good health ,lnfectious Diseases and Global Health Radboud Institute for Health Sciences [Radboudumc 4] ,chemistry ,Ribonucleoproteins ,biology.protein ,HIV-1 ,Small nuclear ribonucleoprotein ,Research Article ,HeLa Cells ,Transcription Factors - Abstract
Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb, and reverses HIV-1 latency., A leading pharmacological strategy toward HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with latency reversal agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs, we used fungal secondary metabolites as a source of bioactive molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the positive transcription elongation factor b (P-TEFb) inhibitory 7SK small nuclear ribonucleoprotein (snRNP) complex, to be significantly reduced upon GTX treatment of CD4+ T cells. GTX directly disrupted 7SK snRNP by targeting La-related protein 7 (LARP7), releasing active P-TEFb, which phosphorylated RNA polymerase II (Pol II) C-terminal domain (CTD), inducing HIV transcription.
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- 2019
33. Gliotoxin, identified from a screen of fungal metabolites, disrupts 7SK snRNP, releases P-TEFb and reverses HIV-1 latency
- Author
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Pritha Biswas, Mateusz Stoszko, Yvonne M. Mueller, Abdullah M. S. Al-Hatmi, Wilfred F. J. van IJcken, Alessia Bertoldi, Robert-Jan Palstra, Miroslav Sulc, Joyce H.G. Lebbink, Elisa De Crignis, Sybren de Hoog, Raquel Crespo, Enrico Ne, Joyce Kang, Renata Ptackova, Anton Škríba, Peter D. Katsikis, Mohammad Javad Najafzadeh, Tokameh Mahmoudi, Annelies Verbon, Tsung Wai Kan, Vladimír Havlíček, Casper Rokx, and Michael Roling
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chemistry.chemical_compound ,biology ,Gliotoxin ,Chemistry ,Transcription (biology) ,7SK RNA ,Gene expression ,biology.protein ,RNA ,RNA polymerase II ,snRNP ,P-TEFb ,Cell biology - Abstract
A leading pharmacological strategy towards HIV cure requires “shock” or activation of HIV gene expression in latently infected cells with Latency Reversal Agents (LRAs) followed by their subsequent clearance. In a screen for novel LRAs we used fungal secondary metabolites (extrolites) as a source of bio-active molecules. Using orthogonal mass spectrometry (MS) coupled to latency reversal bioassays, we identified gliotoxin (GTX) as a novel LRA. GTX significantly induced HIV-1 gene expression in latent ex vivo infected primary cells and in CD4+ T cells from all aviremic HIV-1+ participants. RNA sequencing identified 7SK RNA, the scaffold of the P-TEFb inhibitory 7SK snRNP complex to be significantly reduced upon GTX treatment of independent donor CD4+T cells. GTX disrupted 7SK snRNP, releasing active P-TEFb, which then phosphorylated RNA Pol II CTD, inducing HIV transcription. Our data highlight the power of combining a medium throughput bioassay, mycology and orthogonal mass spectrometry to identify novel potentially therapeutic compounds.
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- 2019
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34. Freeing Aspergillus fumigatus of Polymycovirus Infection Renders It More Resistant to Competition with Pseudomonas aeruginosa Due to Altered Iron-Acquiring Tactics
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Ioly Kotta-Loizou, Andrea Palyzová, Rutuja H. Patil, Vladimír Havlíček, Tomáš Pluháček, Robert H.A. Coutts, and David A. Stevens
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Microbiology (medical) ,Siderophore ,siderophore ,QH301-705.5 ,Plant Science ,medicine.disease_cause ,Article ,polymycovirus ,intermicrobial competition ,Aspergillus fumigatus ,Microbiology ,Exponential growth ,medicine ,Extracellular ,Biology (General) ,Ecology, Evolution, Behavior and Systematics ,Strain (chemistry) ,biology ,Pseudomonas aeruginosa ,Chemistry ,Pseudomonas ,biology.organism_classification ,Intracellular - Abstract
A virus-free (VF) A. fumigatus isolate has been shown to be resistant in competition with Pseudomonas as compared to the isogenic line infected with Aspergillus fumigatus polymycovirus 1 (AfuPmV-1), and this phenotype was apparently related to alterations in iron metabolism. Here we investigated further the mechanisms underpinning this phenotype. The extracellular siderophore profiles of five isogenic VF and virus-infected (VI) strains were sampled at 24, 31, 48, 54, and 72 h in submerged cultures and quantitatively examined by liquid chromatography and mass spectrometry. Intracellular profiles of conidia and cultures at the stationary growth phase were defined. VF A. fumigatus demonstrated the best fitness represented by the fastest onset of its exponential growth when grown on an iron-limited mineral medium. The exponential phase and transitional production phase of the extracellular triacetylfusarinine C (TafC) were achieved at 24 and 31 h, respectively, contrary to VI strains, which acted more slowly. As a result, the TafC reservoir was consumed sooner in the VF strain. Additionally, the VF strain had lower ferricrocin and higher hydroxyferricrocin content in the pellet during the stationary phase. All of these differences were significant (Kruskal–Wallis, p <, 0.01). In our study, the siderophore reservoir of a VF strain was consumed sooner, improving the fitness of the VF strain in competition with P. aeruginosa.
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- 2021
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35. Biodistribution of upconversion/magnetic silica-coated NaGdF4:Yb3+/Er3+ nanoparticles in mouse models
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Uliana Kostiv, Lenka Rajsiglová, Dominika Luptáková, Tomáš Pluháček, Luca Vannucci, Vladimír Havlíček, Hana Engstová, Daniel Jirák, Miroslav Šlouf, Peter Makovicky, Radislav Sedláček, and Daniel Horák
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General Chemical Engineering ,General Chemistry ,equipment and supplies - Abstract
Upconversion magnetic nanoparticles emit visible light after NIR irradiation. Gd renders them with MRI contrast. Localization of the particles is excellently visible in blood vasculature of tumor bearing mice after intravenous administration.
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- 2017
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36. Meet interesting abbreviations in clinical mass spectrometry: from compound classification by REIMS to multimodal and mass spectrometry imaging (MSI)
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Karel Lemr, Dominika Luptáková, Tomáš Pluháček, Vladimír Havlíček, Andrea Palyzová, Ivo Juránek, J Přichystal, and J Balog
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Spectrometry, Mass, Electrospray Ionization ,Chemistry ,010401 analytical chemistry ,Hyperspectral imaging ,Context (language use) ,02 engineering and technology ,General Medicine ,Computational biology ,021001 nanoscience & nanotechnology ,Mass spectrometry ,Bioinformatics ,01 natural sciences ,Mass spectrometry imaging ,0104 chemical sciences ,Infectious Diseases ,Molecular classification ,Virology ,Animals ,Humans ,Biomarker discovery ,0210 nano-technology ,Fiducial marker ,Pathogen inactivation ,Biomarkers - Abstract
This feature article discusses two modern mass spectrometry abbreviations in their clinical applications. Rapid evaporative ionization mass spectrometry (REIMS) is reported as a molecular classification tool useful for spectral features definition prior to mass spectrometry imaging (MSI). REIMS is appreciated not only as an ionization technique coupled with a surgical device but particularly as a biomarker discovery tool. For more complex understanding of pathological processes at cellular and molecular levels, the importance of multimodal approach in imaging applications is documented in the context of fiducial markers needed for hyperspectral data fusion collected by optical microscopy, elemental and molecular MSI. Finally, pathogen inactivation needed prior to the sectioning of the infected tissue is reported, and the impact of formaldehyde crosslinking to signal reduction is discussed.
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- 2017
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37. Aspergillus infection monitored by multimodal imaging in a rat model
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Oldrich Benada, Milos Petrik, Andrea Palyzová, Karel Lemr, Vladimír Havlíček, Dominika Luptáková, and Tomáš Pluháček
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0301 basic medicine ,Materials science ,Analytical chemistry ,Grocott's methenamine silver stain ,Multimodal Imaging ,Biochemistry ,Mass spectrometry imaging ,03 medical and health sciences ,chemistry.chemical_compound ,Microscopy ,Animals ,Aspergillosis ,Humans ,Lung ,Molecular Biology ,Detection limit ,Laser ablation ,Eosin ,Radiochemistry ,Rats ,Staining ,Aspergillus ,030104 developmental biology ,chemistry ,Positron-Emission Tomography ,Microscopy, Electron, Scanning ,Inductively coupled plasma - Abstract
Although myriads of experimental approaches have been published in the field of fungal infection diagnostics, interestingly, in 21st century there is no satisfactory early noninvasive tool for Aspergillus diagnostics with good sensitivity and specificity. In this work, we for the first time described the fungal burden in rat lungs by multimodal imaging approach. The Aspergillus infection was monitored by positron emission tomography and light microscopy employing modified Grocott's methenamine silver staining and eosin counterstaining. Laser ablation inductively coupled plasma mass spectrometry imaging has revealed a dramatic iron increase in fungi-affected areas, which can be presumably attributed to microbial siderophores. Quantitative elemental data were inferred from matrix-matched standards prepared from rat lungs. The iron, silver, and gold MS images collected with variable laser foci revealed that particularly silver or gold can be used as excellent elements useful for sensitively tracking the Aspergillus infection. The limit of detection was determined for both (107) Ag and (197) Au as 0.03 μg/g (5 μm laser focus). The selective incorporation of (107) Ag and (197) Au into fungal cell bodies and low background noise from both elements were confirmed by energy dispersive X-ray scattering utilizing the submicron lateral resolving power of scanning electron microscopy. The low limits of detection and quantitation of both gold and silver make ICP-MS imaging monitoring a viable alternative to standard optical evaluation used in current clinical settings.
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- 2016
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38. Analysis of hyaluronan and its derivatives using chromatographic and mass spectrometric techniques
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Martina Hermannová, Matěj Šimek, Karel Lemr, and Vladimír Havlíček
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Spectrometry, Mass, Electrospray Ionization ,Polymers and Plastics ,Electrospray ionization ,Substituent ,02 engineering and technology ,010402 general chemistry ,01 natural sciences ,Reductive amination ,Glycosaminoglycan ,chemistry.chemical_compound ,Hyaluronic acid ,Materials Chemistry ,Hyaluronic Acid ,Derivatization ,Amination ,Chromatography ,integumentary system ,Chemistry ,Depolymerization ,Organic Chemistry ,021001 nanoscience & nanotechnology ,0104 chemical sciences ,carbohydrates (lipids) ,Matrix-assisted laser desorption/ionization ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,0210 nano-technology - Abstract
The aim of this paper is to review chromatographic and mass-spectrometric methods and underline the best analytical approaches for successful analysis of various hyaluronic acid species in different types of samples. Hyaluronan-degrading enzymes and chemical depolymerization produce di- or oligosaccharides suitable for hyaluronan quantification or structural characterization of hyaluronan derivatives. Efficient purification and pre-column derivatization of hyaluronan disaccharides by reductive amination allow subnanogram quantification in biological samples. The chromatographic separation is capable to distinguish all glycosaminoglycans disaccharides and to resolve hyaluronan fragments with 2-40 monomers. Using electrospray ionization or matrix assisted laser desorption ionization, hyaluronan fragments up to 8 kDa or 41 kDa, respectively, can be observed. One- or two-dimensional chromatographic separation with higly sensitive mass-spectrometric detection is an indispensable tool for revealing substituent position, extent of modification and substitution patterns of chemically modified hyaluronan derivatives. It is essential for studying structure-biological function relationships of hyaluronan and its derivatives.
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- 2020
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39. Bringing SEM and MSI Closer Than Ever Before: Visualizing Aspergillus and Pseudomonas Infection in the Rat Lungs
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Jiří Novák, Anton Škríba, Miloš Petřík, Andrea Palyzová, Dominika Luptáková, Helena Marešová, Olga Kofroňová, Vladimír Havlíček, Oldřich Benada, and Tereza Juříková
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Microbiology (medical) ,Scanning electron microscope ,Plant Science ,01 natural sciences ,03 medical and health sciences ,Pseudomonas infection ,medicine ,rat lung tissue ,bacteria ,lcsh:QH301-705.5 ,Ecology, Evolution, Behavior and Systematics ,Fixative ,030304 developmental biology ,0303 health sciences ,Aspergillus ,Frozen section procedure ,Chromatography ,fixation ,biology ,Chemistry ,Communication ,010401 analytical chemistry ,biology.organism_classification ,medicine.disease ,Molecular biomarkers ,0104 chemical sciences ,matrix-assisted laser desorption/ionization mass spectrometry imaging ,lcsh:Biology (General) ,fungi ,Lung tissue ,scanning electron microscopy ,Fungal hyphae - Abstract
A procedure for processing frozen rat lung tissue sections for scanning electron microscopy (SEM) from deeply frozen samples initially collected and stored for matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) was developed. The procedure employed slow thawing of the frozen sections while floating on the surface and melting in a fixative solution. After the float-washing step, the sections were dehydrated in a graded ethanol series and dried in a critical point dryer. The SEM generated images with well-preserved structures, allowing for monitoring of bacterial cells and fungal hyphae in the infected tissue. Importantly, the consecutive nonfixed frozen sections were fully compatible with MALDI-MSI, providing molecular biomarker maps of Pseudomonas aeruginosa. The protocol enables bimodal image fusion in the in-house software CycloBranch, as demonstrated by SEM and MALDI-MSI.
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- 2020
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40. Analysis of Microbial Siderophores by Mass Spectrometry
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Dominika Luptáková, Vladimír Havlíček, Anton Škríba, Jirí K. Novák, and Tomáš Pluháček
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0301 basic medicine ,Siderophore ,Chromatography ,Molecular mass ,Chemistry ,010401 analytical chemistry ,Mass spectrometry ,01 natural sciences ,Mass spectrometry imaging ,0104 chemical sciences ,Characterization (materials science) ,03 medical and health sciences ,030104 developmental biology ,Mass spectrum ,Sample collection ,Inductively coupled plasma mass spectrometry - Abstract
Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.
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- 2019
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41. Imaging of Pseudomonas aeruginosa infection with Ga-68 labelled pyoverdine for positron emission tomography
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Marian Hajduch, Eva Umlaufova, Vladimír Havlíček, Milos Petrik, Clemens Decristoforo, Andrea Palyzová, Zbynek Novy, Hubertus Haas, Dalibor Dolezal, and Vladislav Raclavsky
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0301 basic medicine ,Siderophore ,Microbiological culture ,Low protein ,Iron ,lcsh:Medicine ,Siderophores ,Gallium Radioisotopes ,medicine.disease_cause ,Article ,Microbiology ,03 medical and health sciences ,chemistry.chemical_compound ,Mice ,Positron Emission Tomography Computed Tomography ,medicine ,Animals ,Pseudomonas Infections ,Tissue Distribution ,lcsh:Science ,Mice, Inbred BALB C ,Multidisciplinary ,Pyoverdine ,medicine.diagnostic_test ,biology ,Molecular Structure ,Chemistry ,Pseudomonas aeruginosa ,lcsh:R ,Pseudomonas ,Biological Transport ,biology.organism_classification ,In vitro ,Culture Media ,Rats ,030104 developmental biology ,Positron emission tomography ,Positron-Emission Tomography ,lcsh:Q ,Radiopharmaceuticals ,Oligopeptides - Abstract
Pseudomonas aeruginosa is an increasingly prevalent opportunistic pathogen that causes a variety of life-threatening nosocomial infections. Novel strategies for the development of new antibacterial treatments as well as diagnostic tools are needed. One of the novel diagnostic strategies for the detection of infection could be the utilization of siderophores. Siderophores are low-molecular-weight chelators produced by microbes to scavenge essential iron. Replacing iron in siderophores by suitable radiometals, such as Ga-68 for positron emission tomography (PET) imaging, opens approaches for targeted imaging of infection. Here we report on pyoverdine PAO1 (PVD-PAO1), a siderophore produced by P. aeruginosa, labelled with Ga-68 for specific imaging of Pseudomonas infections. PVD-PAO1 was labelled with Ga-68 with high radiochemical purity. The resulting complex showed hydrophilic properties, low protein binding and high stability in human serum. In vitro uptake of 68Ga-PVD-PAO1 was highly dependent on the type of microbial culture. In normal mice 68Ga-PVD-PAO1 showed rapid pharmacokinetics with urinary excretion. PET imaging in infected animals displayed specific accumulation of 68Ga-PVD-PAO1 in infected tissues and better distribution than clinically used 18F-fluorodeoxyglucose (18F-FDG) and 68Ga-citrate. Ga-68 labelled pyoverdine PAO1 seems to be a promising agent for imaging of P. aeruginosa infections by means of PET.
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- 2018
42. Membrane depolarization and aberrant lipid distributions in the neonatal rat brain following hypoxic-ischaemic insult
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Ivo Juránek, Dominika Luptáková, Vladimír Havlíček, Tomáš Pluháček, Anton Škríba, Ladislav Baciak, and Blanka Šedivá
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Male ,0301 basic medicine ,medicine.medical_specialty ,Pathology ,Nape ,Science ,Encephalopathy ,Article ,Membrane Potentials ,03 medical and health sciences ,0302 clinical medicine ,In vivo ,medicine ,Animals ,Neonatology ,Rats, Wistar ,Multidisciplinary ,medicine.diagnostic_test ,Chemistry ,Cell Membrane ,Brain ,Magnetic resonance imaging ,Depolarization ,medicine.disease ,Lipids ,Rats ,030104 developmental biology ,medicine.anatomical_structure ,Animals, Newborn ,Acute Disease ,Hypoxia-Ischemia, Brain ,Medicine ,Female ,030217 neurology & neurosurgery ,Ex vivo ,Intracellular - Abstract
Neonatal hypoxic-ischaemic (HI) encephalopathy is among the most serious complications in neonatology. In the present study, we studied the immediate (0 hour), subacute (36 hours) and late (144 hours) responses of the neonatal brain to experimental HI insult in laboratory rats. At the striatal level, the mass spectrometry imaging revealed an aberrant plasma membrane distribution of Na+/K+ ions in the oedema-affected areas. The failure of the Na+/K+ gradients was also apparent in the magnetic resonance imaging measurements, demonstrating intracellular water accumulation during the acute phase of the HI insult. During the subacute phase, compared with the control brains, an incipient accumulation of an array of N-acylphosphatidylethanolamine (NAPE) molecules was detected in the HI-affected brains, and both the cytotoxic and vasogenic types of oedema were detected. In the severely affected brain areas, abnormal distributions of the monosialogangliosides GM2 and GM3 were observed in two-thirds of the animals exposed to the insult. During the late stage, a partial restoration of the brain tissue was observed in most rats in both the in vivo and ex vivo studies. These specific molecular changes may be further utilized in neonatology practice in proposing and testing novel therapeutic strategies for the treatment of neonatal HI encephalopathy.
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- 2018
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43. Laser ablation inductively coupled plasma mass spectrometry imaging: A personal identification based on a gunshot residue analysis on latent fingerprints
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Vladimír Havlíček, Vítězslav Maier, Martin Švidrnoch, Karel Lemr, and Tomáš Pluháček
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Laser ablation ,Gunshot residue ,Chemistry ,Laser ablation inductively coupled plasma mass spectrometry ,010401 analytical chemistry ,Analytical chemistry ,02 engineering and technology ,021001 nanoscience & nanotechnology ,01 natural sciences ,Biochemistry ,Latent fingerprint ,Mass Spectrometry ,0104 chemical sciences ,Analytical Chemistry ,Identification (information) ,Fingerprint ,Metals ,Environmental Chemistry ,Metallic impurities ,Laser Therapy ,Dermatoglyphics ,0210 nano-technology ,Inductively coupled plasma mass spectrometry ,Spectroscopy - Abstract
A new laser ablation inductively coupled plasma mass spectrometry imaging (LA-ICP-MSI) method has been developed to visualize latent fingerprint of suspect persons possibly related to a criminal offence committed with a gun. Metallic impurities and metallic gunshot residues (GSRs) adhered on a latent fingerprint can link biometric information and shooting. The identification and spatial distribution of characteristic gunshot-related metals (Cu, Zn, Sb, Ba, Hg, Pb) was studied in detail. Observed limits of detection for the corresponding metals were below 0.3 ng cm−2. The distributions of the selected metals were simply transferred to images of fingerprints that can reveal a person somehow manipulating with a gun while the presence of characteristic and consistent GSR particles provided the evidence of the shooting. LA-ICP-MSI has been proved to be a unique tool in dactyloscopic identification of a suspect person from latent and commonly visualized fingerprints.
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- 2018
44. Lateral resolution of desorption nanoelectrospray: a nanospray tip without nebulizing gas as a source of primary charged droplets
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Petr Fryčák, T. Ingr, Michael Volný, Hana Chmelickova, Lucie Hartmanova, Iveta Lorencová, Karel Lemr, and Vladimír Havlíček
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Electrospray ,Analyte ,Chromatography ,Resolution (mass spectrometry) ,Chemistry ,010401 analytical chemistry ,Analytical chemistry ,010402 general chemistry ,01 natural sciences ,Biochemistry ,Mass spectrometry imaging ,0104 chemical sciences ,Analytical Chemistry ,Volumetric flow rate ,chemistry.chemical_compound ,Desorption ,Electrochemistry ,Rhodamine B ,Mass spectrum ,Environmental Chemistry ,Spectroscopy - Abstract
Desorption nanoelectrospray (nanoDESI) was described in 2007 and it represents a miniaturized version of desorption electrospray without the assistance of the nebulizing gas. Compared to DESI, a nanoelectrospray tip (2 ± 1 μm I.D.) generates primary charged droplets of smaller sizes and lower spray liquid flow rates. This is the first report on utilization of nanoDESI for mass spectrometry imaging (MSI). Its new coupling with a Q-TOF instrument allowed faster mass spectra acquisition (4 Hz) essential for MSI of fine surface details. To evaluate nanoDESI potential for mass spectrometry imaging, etched glass substrates with Rhodamine B patterns of different dimensions were prepared. The Rhodamine B lines were analysed in 1D scanning mode and their width was determined experimentally by nanoDESI measurement. The experimental data revealed that the lateral resolution of nanoDESI is close to 30 μm along the x-axis (orthogonal to the inlet). 2D scanning mode confirmed good resolution along both axes as dye squares with dimensions about 60 μm × 60 μm were easily distinguished. The low flow rate of the spray liquid reduced undesirable analyte washing effects, which allowed repeated scanning analysis of the surface. The presented results demonstrate the applicability of nanoDESI for high surface resolution mass spectrometry imaging.
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- 2016
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45. Reversed Phase Liquid Chromatography Hyphenated to Continuous Flow—Extractive Desorption Electrospray Ionization—Mass Spectrometry for Analysis and Charge State Manipulation of Undigested Proteins
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Vladimír Havlíček, Elisa M. Rice, Li Li, Samuel H. Yang, Veronika Vidová, Kevin A. Schug, and Aruna B. Wijeratne
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Chromatography, Reverse-Phase ,Spectrometry, Mass, Electrospray Ionization ,Desorption electrospray ionization ,Chromatography ,Chemistry ,Electrospray ionization ,Static Electricity ,Analytical chemistry ,Extractive electrospray ionization ,Proteins ,Atmospheric-pressure chemical ionization ,Equipment Design ,General Medicine ,Mass spectrometry ,Peptide Mapping ,Atomic and Molecular Physics, and Optics ,Sample preparation in mass spectrometry ,Specimen Handling ,Equipment Failure Analysis ,Direct electron ionization liquid chromatography–mass spectrometry interface ,Chromatography, High Pressure Liquid ,Spectroscopy ,Ambient ionization - Abstract
The application of continuous flow–extractive desorption electrospray ionization (CF-EDESI), an ambient ionization source demonstrated previously for use with intact protein analysis, is expanded here for the coupling of reversed phase protein separations to mass spectrometry. This configuration allows the introduction of charging additives to enhance detection without affecting the chromatographic separation mechanism. Two demonstrations of the advantages of CF-EDESI are presented in this work. First, a proof-of-principle is presented to demonstrate the applicability of hyphenation of liquid chromatography (LC) to CF-EDESI. LC-CF-EDESI-MS has good sensitivity compared to LC–electrospray ionization (ESI)–mass spectrometry. Second, the supercharging mechanism investigated in CF-EDESI provides an insight into a highly debated supercharging process in ESI. The results indicate that the mechanism of protein charging seen in HPLC-CF-EDESI is different from supercharging phenomena in conventional ESI. The surface tension mechanism and binding mechanism may both contribute to protein supercharging in ESI.
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- 2015
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46. Arrival time distributions of product ions reveal isomeric ratio of deprotonated molecules in ion mobility-mass spectrometry of hyaluronan-derived oligosaccharides
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Karel Lemr, Andreea-Maria Iordache, Vladimír Havlíček, Helena Pelantová, Kristína Slováková, and Martina Hermannová
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Fragmentation (mass spectrometry) ,Chemistry ,Computational chemistry ,Ion-mobility spectrometry ,Analytical chemistry ,Mass spectrum ,Molecule ,Nuclear magnetic resonance spectroscopy ,Spectroscopy ,Mass spectrometry ,Ion - Abstract
Hyaluronic acid is a naturally occurring linear polysaccharide with substantial medical potential. In this work, discrimination of tyramine-based hyaluronan derivatives was accessed by ion mobility–mass spectrometry of deprotonated molecules and nuclear magnetic resonance spectroscopy. As the product ion mass spectra did not allow for direct isomer discrimination in mixture, the reductivelabeling ofoligosaccharidesaswell asstable isotope labelingwasperformed. The ion mobility separation ofparent ions together with the characteristic fragmentation for reduced isomers providing unique product ions allowed us to identify isomers present in a mixture and determine their mutual isomeric ratio. The determination used simple recalculation of arrival time distribution areas ofuniqueions toareasofdeprotonatedmolecules. Massspectrometrydatawere confirmedbynuclear magneticresonance spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd. Additional supporting information may be found in the online version of this article at the publisher’s web site.
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- 2015
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47. Characterization of microbial siderophores by mass spectrometry
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Vladimír Havlíček, Karel Lemr, David Milde, Tomáš Pluháček, Jiří Novák, and Dipankar Ghosh
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0301 basic medicine ,Siderophore ,Chromatography ,Molecular mass ,Chemistry ,High resolution ,Condensed Matter Physics ,Mass spectrometry ,Tandem mass spectrometry ,General Biochemistry, Genetics and Molecular Biology ,Analytical Chemistry ,03 medical and health sciences ,Data filtering ,030104 developmental biology ,Secondary metabolism ,Spectroscopy - Abstract
Siderophores play important roles in microbial iron piracy, and are applied as infectious disease biomarkers and novel pharmaceutical drugs. Inductively coupled plasma and molecular mass spectrometry (ICP-MS) combined with high resolution separations allow characterization of siderophores in complex samples taking advantages of mass defect data filtering, tandem mass spectrometry, and iron-containing compound quantitation. The enrichment approaches used in siderophore analysis and current ICP-MS technologies are reviewed. The recent tools for fast dereplication of secondary metabolites and their databases are reported. This review on siderophores is concluded with their recent medical, biochemical, geochemical, and agricultural applications in mass spectrometry context.
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- 2015
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48. High-throughput workflow for identification of phosphorylated peptides by LC-MALDI-TOF/TOF-MS coupled toin situenrichment on MALDI plates functionalized by ion landing
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Marcela Strnadová, Michael Volný, Radovan Hynek, Lukáš Krásný, Vladimír Havlíček, Petr Pompach, Petr Novák, and Karel Valis
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Cell lysates ,In situ ,Workflow ,Chromatography ,Chemistry ,Phosphorylation ,Database search engine ,Jurkat cells ,Spectroscopy ,Ion ,Trypsinization - Abstract
We report an MS-based workflow for identification of phosphorylated peptides from trypsinized protein mixtures and cell lysates that is suitable for high-throughput sample analysis. The workflow is based on an in situ enrichment on matrix-assisted laser desorption/ionization (MALDI) plates that were functionalized by TiO2 using automated ion landing apparatus that can operate unsupervised. The MALDI plate can be functionalized by TiO2 into any array of predefined geometry (here, 96 positions for samples and 24 for mass calibration standards) made compatible with a standard MALDI spotter and coupled with high-performance liquid chromatography. The in situ MALDI plate enrichment was compared with a standard precolumn-based separation and achieved comparable or better results than the standard method. The performance of this new workflow was demonstrated on a model mixture of proteins as well as on Jurkat cells lysates. The method showed improved signal-to-noise ratio in a single MS spectrum, which resulted in better identification by MS/MS and a subsequent database search. Using the workflow, we also found specific phosphorylations in Jurkat cells that were nonspecifically activated by phorbol 12-myristate 13-acetate. These phosphorylations concerned the mitogen-activated protein kinase/extracellular signal-regulated kinase signaling pathway and its targets and were in agreement with the current knowledge of this signaling cascade. Control sample of non-activated cells was devoid of these phosphorylations. Overall, the presented analytical workflow is able to detect dynamic phosphorylation events in minimally processed mammalian cells while using only a short high-performance liquid chromatography gradient. Copyright © 2015 John Wiley & Sons, Ltd.
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- 2015
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49. Protein composition of the phase I Coxiella burnetii soluble antigen prepared by extraction with trichloroacetic acid
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Vladimír Havlíček, Maksym Danchenko, M. Kmeťová, Ľudovít Škultéty, Gabriela Flores-Ramirez, and Eva Špitalská
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Slovakia ,Q fever ,Biology ,Microbiology ,Disease Outbreaks ,chemistry.chemical_compound ,Bacterial Proteins ,Virology ,medicine ,Animals ,Humans ,Trichloroacetic acid ,Trichloroacetic Acid ,Gel electrophoresis ,Antigens, Bacterial ,Zoonosis ,Vaccination ,Outbreak ,General Medicine ,medicine.disease ,Coxiella burnetii ,biology.organism_classification ,Infectious Diseases ,chemistry ,Q Fever ,Bacteria - Abstract
Q fever is a highly infectious, widespread airborne zoonosis caused by Coxiella burnetii bacterium. Humans usually acquire the disease by inhalation of contaminated aerosol produced by infected livestock. Vaccination is the most practical way for prevention and control of the disease in the exposed population. In this work, we reviewed the most important Q-fever outbreaks in Slovakia as well as the progress in vaccine development. One of them represents a soluble antigen complex produced by extraction with trichloroacetic acid from a highly purified C. burnetii phase I strain Nine Mile. It was developed at the Institute of Virology in Bratislava. The protein content of this vaccine was separated by gel electrophoresis and analyzed by mass spectrometry. The study has resulted in the identification of 39 bacterial proteins from which 12 were recognized as immunoreactive. Most of the proteins were involved in bacterium pathogenicity (41.6%) and cell wall maintenance (25%). Four of the immunoreactive proteins may possess the moonlighting activity. Definition of the vaccine components represents a prerequisite for vaccine standardization and approval by governmental authorities.
- Published
- 2017
50. Specific storage of glycoconjugates with terminal α-galactosyl moieties in the exocrine pancreas of Fabry disease patients with blood group B
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Ludovit Skultety, Helena Hulkova, Vladimír Havlíček, Robert Dobrovolný, Ladislav Kuchar, Jitka Rybová, Befekadu Asfaw, Jana Ledvinová, and Jakub Sikora
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0301 basic medicine ,Male ,Pathology ,medicine.medical_specialty ,Cell type ,Glycoconjugate ,Acinar Cells ,Biochemistry ,Glycosphingolipids ,Lipofuscin ,ABO Blood-Group System ,03 medical and health sciences ,chemistry.chemical_compound ,Biosynthesis ,ABO blood group system ,Insulin-Secreting Cells ,medicine ,Humans ,Pancreas ,chemistry.chemical_classification ,geography ,geography.geographical_feature_category ,Galactose ,Middle Aged ,Islet ,medicine.disease ,Fabry disease ,030104 developmental biology ,medicine.anatomical_structure ,chemistry ,Case-Control Studies ,Fabry Disease ,lipids (amino acids, peptides, and proteins) - Abstract
Blood group B glycosphingolipids (B-GSLs) are substrates of the lysosomal alpha-galactosidase A (AGAL). Similar to its major substrate-globotriaosylceramide (Gb3Cer)-B-GSLs are not degraded and accumulate in the cells of patients affected by an inherited defect of AGAL activity (Fabry disease-FD).The pancreas is a secretory organ known to have high biosynthesis of blood group GSLs. Herein, we provide a comprehensive overview of the biochemical and structural abnormalities in pancreatic tissue from two male FD patients with blood group B. In both patients, we found major accumulation of a variety of complex B-GSLs carrying predominantly hexa- and hepta-saccharide structures. The subcellular pathology was dominated by deposits containing B-glycoconjugates and autofluorescent ceroid. The contribution of Gb3Cer to the storage was minor. This abnormal storage pattern was specific for the pancreatic acinar epithelial cells. Other pancreatic cell types including those of islets of Langerhans were affected much less or not at all.Altogether, we provide evidence for a key role of B-antigens in the biochemical and morphological pathology of the exocrine pancreas in FD patients with blood group B. We believe that our findings will trigger further studies aimed at assessing the potential pancreatic dysfunction in this disease.
- Published
- 2017
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