The coronavirus pandemic caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) directly or indirectly affects every individual worldwide. The fight against SARS-CoV-2 is based on the rapid and accurate diagnosis and subsequent isolation of infected individuals. Therefore, the demands for the scientific development of diagnostic methods for the confirmation of SARS-CoV-2 are enormous. Currently, reverse-transcription quantitative polymerase chain reaction (RT-qPCR) is the main method used for detecting viruses, including SARS-CoV-2, and is considered the gold standard for coronavirus disease 2019 (COVID-19) identification. However, various alternatives have been investigated due to the time and cost demands of this method or to shortages of reagents. In this review, we focus on matrix-assisted laser desorption and ionisation with time-of-flight analyser mass spectrometry (MALDI-TOF MS) techniques as potential tools for the diagnosis of viruses with an emphasis on SARS-CoV-2. MALDI-TOF is commonly used in clinical laboratories for bacterial characterization and identification, but in the field of clinical virology, MALDI-TOF remains only a promising technology for routine diagnosis. This review provides an overview of the development of clinical virology from the point of view of using MALDI-TOF for virus identification and as a possible diagnostic tool for SARS-CoV-2 detection. In addition, this review summarizes the current state of standard methods for virus diagnostics including the preparation of clinical samples.
- MeSH
- COVID-19 * diagnóza MeSH
- lidé MeSH
- pandemie MeSH
- SARS-CoV-2 MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- viry * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- přehledy MeSH
Identification of metal-binding sites in proteins and understanding metal-coupled protein folding mechanisms are aspects of high importance for the structure-to-function relationship. Mass spectrometry (MS) has brought a powerful adjunct perspective to structural biology, obtaining from metal-to-protein stoichiometry to quaternary structure information. Currently, the different experimental and/or instrumental setups usually require the use of multiple data analysis software, and in some cases, they lack some of the main data analysis steps (MS processing, scoring, identification). Here, we present a comprehensive data analysis pipeline that addresses charge-state deconvolution, statistical scoring, and mass assignment for native MS, bottom-up, and native top-down with emphasis on metal-protein complexes. We have evaluated all of the approaches using assemblies of increasing complexity, including free and chemically labeled proteins, from low- to high-resolution MS. In all cases, the results have been compared with common software and proved how MetaOdysseus outperformed them.
- Publikační typ
- abstrakt z konference MeSH
Respiratory infections are a real threat for humans, and therefore the pig model is of interest for studies. As one of a case for studies, Actinobacillus pleuropneumoniae (APP) caused infections and still worries many pig breeders around the world. To better understand the influence of pathogenic effect of APP on a respiratory system-lungs and tracheobronchial lymph nodes (TBLN), we aimed to employ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry imaging (MALDI-TOF MSI). In this study, six pigs were intranasally infected by APP and two were used as non-infected control, and 48 cryosections have been obtained. MALDI-TOF MSI and immunohistochemistry (IHC) were used to study spatial distribution of infectious markers, especially interleukins, in cryosections of porcine tissues of lungs (necrotic area, marginal zone) and tracheobronchial lymph nodes (TBLN) from pigs infected by APP. CD163, interleukin 1β (IL‑1β) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL‑1β were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL‑1β and protegrin‑4 precursor were all significantly (p < 0.001) more expressed in necrotic areas of lungs infected by APP than in marginal zone, TBLN and in control lungs.
- MeSH
- Actinobacillus pleuropneumoniae patogenita MeSH
- antigeny diferenciační myelomonocytární metabolismus MeSH
- biologické markery metabolismus MeSH
- bronchy metabolismus MeSH
- CD antigeny metabolismus MeSH
- infekce bakteriemi rodu Actinobacillus metabolismus mikrobiologie MeSH
- infekce dýchací soustavy metabolismus mikrobiologie MeSH
- interleukin-1beta metabolismus MeSH
- kationické antimikrobiální peptidy metabolismus MeSH
- lymfatické uzliny metabolismus MeSH
- plíce metabolismus MeSH
- prasata MeSH
- receptory buněčného povrchu metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Dietary supplementation with polyunsaturated fatty acids (PUFA) n-3 can affect cutaneous wound healing; however, recent findings demonstrate the variable extent of their influence on the quality of healing. Here, we compare the effect of several dietary oils, containing different levels of PUFA n-3 and PUFA n-6, on wound healing in the rat model. Rats were fed the feed mixture with 8% palm oil (P), safflower oil (S), fish oil (F) or Schizochytrium microalga extract (Sch) and compared to the animals fed by control feed mixture (C). Dorsal full-thickness cutaneous excisions were performed after 52 days of feeding and skin was left to heal for an additional 12 days. Histopathological analysis of skin wounds was performed, including immune cells immunolabeling and the determination of hydroxyproline amount as well as gene expression analyses of molecules contributing to different steps of the healing. Matrix-assisted-laser-desorption-ionization mass-spectrometry-imaging (MALDI-MSI) was used to determine the amount of collagen α-1(III) chain fragment in healing samples. Treatment by Schizochytrium extract resulted in decrease in the total wound area, in contrast to the safflower oil group where the size of the wound was larger when comparing to control animals. Diet with Schizochytrium extract and safflower oils displayed a tendency to increase the number of new vessels. The number of MPO-positive cells was diminished following any of oil treatment in comparison to the control, but their highest amount was found in animals with a fish oil diet. On the other hand, the number of CD68-positive macrophages was increased, with the most significant enhancement in the fish oil and safflower oil group. Hydroxyproline concentration was the highest in the safflower oil group but it was also enhanced in all other analyzed treatments in comparison to the control. MALDI-MSI signal intensity of a collagen III fragment decreased in the sequence C > S > Sch > P > F treatment. In conclusion, we observed differences in tissue response during healing between dietary oils, with the activation of inflammation observed following the treatment with oil containing high eicosapentaenoic acid (EPA) level (fish oil) and enhanced healing features were induced by the diet with high content of docosahexaenoic acid (DHA, Schizochytrium extract).
- MeSH
- antigeny CD8 metabolismus MeSH
- dietní tuky nenasycené aplikace a dávkování farmakologie MeSH
- hojení ran účinky léků MeSH
- indoly chemie MeSH
- kolagen typ III metabolismus MeSH
- krysa rodu rattus MeSH
- kůže účinky léků zranění metabolismus MeSH
- kyseliny mastné omega-6 analýza MeSH
- makrofágy imunologie MeSH
- modely nemocí na zvířatech MeSH
- omega-3 mastné kyseliny analýza MeSH
- palmový olej aplikace a dávkování chemie farmakologie MeSH
- rybí oleje aplikace a dávkování chemie farmakologie MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- světlicový olej aplikace a dávkování chemie farmakologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
An inexorable switch from antibiotics has become a major desideratum to overcome antibiotic resistance. Bacteriocin from Lactobacillus casei, a cardinal probiotic was used to design novel antibacterial peptides named as Probiotic Bacteriocin Derived and Modified (PBDM) peptides (PBDM1: YKWFAHLIKGLC and PBDM2: YKWFRHLIKKLC). The loop-shaped 3D structure of peptides was characterized in silico via molecular dynamics simulation as well as biophysically via spectroscopic methods. Thereafter, in vitro results against multidrug resistant bacterial strains and hospital samples demonstrated the strong antimicrobial activity of PBDM peptides. Further, in vivo studies with PBDM peptides showed downright recovery of balb/c mice from Vancomycin Resistant Staphylococcus aureus (VRSA) infection to its healthy condition. Thereafter, in vitro study with human epithelial cells showed no significant cytotoxic effects with high biocompatibility and good hemocompatibility. In conclusion, PBDM peptides displayed significant antibacterial activity against certain drug resistant bacteria which cause infections in human beings. Future analysis are required to unveil its mechanism of action in order to execute it as an alternative to antibiotics.
- Publikační typ
- časopisecké články MeSH
Looking insight pathological processes, metallothioneins (MTs) are considered to be potential biomarkers for monitoring of a development of various types of diseases, such as cancer. The early identification of the MTs in biological tissues could be important tool for the estimation of appropriate clinical therapy. Therefore, here we investigated the application of matrix assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI) together with immunohistochemical analyses (IHC) using MT-1/2 antibody for MT detection in formalin-fixed paraffin-embedded (FFPE) biopsy specimens of human skin. Principal component analyses revealed differences in the peptide/protein profiles separating healthy skin from the carcinoma specimens. Statistically significant ion peaks at m/z 6038, 6300, 6676, and 7026 were more frequently detected in squamous cell carcinoma (SCC), basal cell carcinoma (BCC) and melanoma. Using IHC, we found that MT-1/2 was significantly higher in SCC and melanoma compared to healthy skin. Surprisingly, significantly low levels of MT-1/2 were found in BCC. On one side, the results indicate important role of MTs in melanoma occurrence and progression, as on the second side, there are hidden processes associated with MTs based on differences of the occurrence of the MS peaks, which could be associated with cycling of MTs isoforms.
- MeSH
- imunohistochemie * MeSH
- kůže metabolismus MeSH
- lidé MeSH
- proteiny chemie metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Nanoparticular form of titanium dioxide (TiO2 NPs) belongs to important industrial material. Despite being widely used, serious contradictions regarding biosafety of TiO2 NPs remain. We anticipate that such discrepancies could be due to a lack of understanding of a linkage between TiO2 NPs phase composition and cytotoxicity. Therefore, we synthesized two types of biphasic TiO2 NPs differing in an anatase-brookite phase composition. The study presents an array of in vitro data suggesting that TiO2 NPs with a prevailing anatase phase composition possess higher cytotoxicity compared to TiO2 NPs with an equal anatase-brookite crystallinity. This phenomenon was evidenced by significantly higher inhibition of metabolic activity and growth of epithelial and neuroblast-like cells. Moreover, anatase-prevailing TiO2 NPs tend to produce higher amount of reactive oxygen species resulting in DNA fragmentation. Further insights into the molecular aspects of cytotoxicity of anatase-prevailing TiO2 NPs were obtained by comparative proteomics delineating that TiO2 NPs deregulate expression of a variety of proteins and associated pathways. This inevitably results in a decreased cellular ability to detoxify reactive oxygen species and respond to various stress conditions. The study provides novel data that add another piece to the jigsaw of the relation between structural features of NPs and biosafety.
- MeSH
- buněčné linie MeSH
- epitelové buňky účinky léků metabolismus MeSH
- kovové nanočástice chemie toxicita ultrastruktura MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- nádorové buněčné linie MeSH
- oxidační stres * MeSH
- reaktivní formy kyslíku metabolismus MeSH
- titan chemie toxicita MeSH
- transmisní elektronová mikroskopie MeSH
- viabilita buněk účinky léků MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
The objective of the present study was to compare the effects of dietary oils containing polyunsaturated fatty acids (PUFA) n-3 and n-6, respectively, on cutaneous wound healing in rats, and to demonstrate the usefulness of the matrix-assisted-laser-desorption-ionization mass spectroscopy-imaging (MALDI MSI) method in this type of experiment. Superiority of PUFA n-3 in this context was the tested hypothesis. Four groups of male Wistar rats by twelve animals each were fed a diet with added 5% of palm oil (P; control), fish oil (F), Schizochytrium microalga oil (Sch) and safflower oil (S), respectively, for eight weeks. Consequently, dorsal full-thickness cutaneous excisions were performed, and selected markers of wound healing were evaluated 18 days post excision. The median of signal intensity corresponding to an amount of collagen α-1 (III) fragment, quantified using MALDI MSI, decreased in a sequence P > F > Sch > S (P < 0.001). Using haematoxylin-eosin staining of the histological preparations, semi-quantitatively assessed epithelium height tended to decrease in the order of P > S > Sch > F; the wound extent in the sequence of P > Sch > S > F; and the sequence of the progress of neo-angiogenesis was assessed as S > P > F ≈ Sch. It was concluded that the tested hypothesis was confirmed only partially: PUFA n-3 showed better results regarding the wound extent, but were inferior in terms of epithelium height and progress of neo-angiogenesis. This was the first time MALDI MSI was successfully employed for evaluating skin wound healing in a rat model.
- MeSH
- dietní tuky nenasycené * terapeutické užití MeSH
- dietní tuky terapeutické užití MeSH
- hojení ran * MeSH
- kolagen typu I, řetězec alfa 1 analýza MeSH
- nenasycené mastné kyseliny terapeutické užití MeSH
- potkani Wistar MeSH
- rány a poranění dietoterapie veterinární MeSH
- reepitalizace MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice statistika a číselné údaje veterinární MeSH
- výsledek terapie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- klinická studie MeSH
DNA hypermethylation is one of the most common epigenetic modifications in prostate cancer (PCa). Several studies have delineated sarcosine as a PCa oncometabolite that increases the migration of malignant prostate cells while decreasing their doubling time. Here, we show that incubation of prostate cells with sarcosine elicited the upregulation of sarcosine N-demethylation enzymes, sarcosine dehydrogenase and pipecolic acid oxidase. This process was accompanied by a considerable increase in the production of the major methyl-donor S-adenosylmethionine (SAMe), together with an elevation of cellular methylation potential. Global DNA methylation analyses revealed increases in methylated CpG islands in distinct prostate cell lines incubated with sarcosine, but not in cells of nonprostate origin. This phenomenon was further associated with marked upregulation of DNA methyltransferases (Dnmts). Epigenetic changes were recapitulated through blunting of Dnmts using the hypomethylating agent 5-azacytidine, which was able to inhibit sarcosine-induced migration of prostate cells. Moreover, spatial mapping revealed concomitant increases in sarcosine, SAMe and Dnmt1 in histologically confirmed malignant prostate tissue, but not in adjacent or nonmalignant tissue, which is in line with the obtained in vitro data. In summary, we show here for the first time that sarcosine acts as an epigenetic modifier of prostate cells and that this may contribute to its oncometabolic role.
- MeSH
- buněčné linie MeSH
- CpG ostrůvky * MeSH
- epigeneze genetická účinky léků MeSH
- lidé MeSH
- nádory prostaty metabolismus patologie MeSH
- prostata metabolismus patologie MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- sarkosin farmakologie MeSH
- upregulace účinky léků MeSH
- Check Tag
- lidé MeSH
- mužské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
