ABSTRACT
Increased awareness of the impact of human activities on the environment has emerged in recent decades. One significant global environmental and human health issue is the development of materials that could potentially have negative effects. These materials can accumulate in the environment, infiltrate organisms, and move up the food chain, causing toxic effects at various levels. Therefore, it is crucial to assess materials comprising nano-scale particles due to the rapid expansion of nanotechnology. The aquatic environment, particularly vulnerable to waste pollution, demands attention. This review provides an overview of the behavior and fate of metallic nanoparticles (NPs) in the aquatic environment. It focuses on recent studies investigating the toxicity of different metallic NPs on aquatic organisms, with a specific emphasis on thiol-biomarkers of oxidative stress such as glutathione, thiol- and related-enzymes, and metallothionein. Additionally, the selection of suitable measurement methods for monitoring thiol-biomarkers in NPs' ecotoxicity assessments is discussed. The review also describes the analytical techniques employed for determining levels of oxidative stress biomarkers.
ABSTRACT
Herby, the interaction of metallothioneins with commonly used Pt-based anticancer drugs - cisplatin, carboplatin, and oxaliplatin - was investigated using the combined power of elemental (i.e. LA-ICP-MS, CE-ICP-MS) and molecular (i.e. MALDI-TOF-MS) analytical techniques providing not only required information about the interaction, but also the benefit of low sample consumption. The amount of Cd and Pt incorporated within the protein was determined for protein monomers and dimer/oligomers formed by non-oxidative dimerization. Moreover, fluorescence spectrometry using Zn2+-selective fluorescent indicator - FluoZin3 - was employed to monitor the ability of Pt drugs to release natively occurring Zn from the protein molecule. The investigation was carried out using two protein isoforms (i.e. MT2, MT3), and significant differences in behaviour of these two isoforms were observed. The main attention was paid to elucidating whether the protein dimerization/oligomerization may be the reason for the potential failure of the anticancer therapy based on these drugs. Based on the results, it was demonstrated that the interaction of MT2 (both monomers and dimers) interacted with Pt drugs significantly less compared to MT3 (both monomers and dimers). Also, a significant difference between monomeric and dimeric forms (both MT2 and MT3) was not observed. This may suggest that dimer formation is not the key factor leading to the inactivation of Pt drugs.
Subject(s)
Metallothionein , Spectrometry, Fluorescence , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Metallothionein/metabolism , Metallothionein/chemistry , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Spectrometry, Fluorescence/methods , Carboplatin/pharmacology , Oxaliplatin/pharmacology , Cisplatin/pharmacology , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Organoplatinum Compounds/pharmacology , Organoplatinum Compounds/chemistry , Platinum/chemistry , Metallothionein 3 , Cytostatic Agents/pharmacology , Cytostatic Agents/chemistry , Mass Spectrometry/methods , HumansABSTRACT
BACKGROUND & AIMS: Metallothionein-3 (hMT3) is a structurally unique member of the metallothioneins family of low-mass cysteine-rich proteins. hMT3 has poorly characterized functions, and its importance for hepatocellular carcinoma (HCC) cells has not yet been elucidated. Therefore, we investigated the molecular mechanisms driven by hMT3 with a special emphasis on susceptibility to sorafenib. METHODS: Intrinsically sorafenib-resistant (BCLC-3) and sensitive (Huh7) cells with or without up-regulated hMT3 were examined using cDNA microarray and methods aimed at mitochondrial flux, oxidative status, cell death, and cell cycle. In addition, in ovo/ex ovo chick chorioallantoic membrane (CAM) assays were conducted to determine a role of hMT3 in resistance to sorafenib and associated cancer hallmarks, such as angiogenesis and metastastic spread. Molecular aspects of hMT3-mediated induction of sorafenib-resistant phenotype were delineated using mass-spectrometry-based proteomics. RESULTS: The phenotype of sensitive HCC cells can be remodeled into sorafenib-resistant one via up-regulation of hMT3. hMT3 has a profound effect on mitochondrial respiration, glycolysis, and redox homeostasis. Proteomic analyses revealed a number of hMT3-affected biological pathways, including exocytosis, glycolysis, apoptosis, angiogenesis, and cellular stress, which drive resistance to sorafenib. CONCLUSIONS: hMT3 acts as a multifunctional driver capable of inducing sorafenib-resistant phenotype of HCC cells. Our data suggest that hMT3 and related pathways could serve as possible druggable targets to improve therapeutic outcomes in patients with sorafenib-resistant HCC.
ABSTRACT
Distinct monocyte subpopulations have been previously described in healthy pigs and pigs experimentally infected with Actinobacillus pleuropneumoniae (APP). The CD163+ subpopulation of bone marrow (BM), peripheral blood (PB) and lung monocytes was found to play an important role in the inflammatory process. The inflammation is accompanied by elevation of inflammatory cytokines. The aim of the study was to evaluate the contribution of CD163+ monocytes and macrophages to cytokine production during APP-induced lung inflammation. Cytokine production was assessed by flow cytometry (FC) and quantitative PCR (qPCR) in CD163+ monocytes and by qPCR, immunohistochemistry/fluorescence in lungs and tracheobronchial lymph nodes (TBLN). Despite the systemic inflammatory response after APP infection, BM and PB CD163+ monocytes did not express elevated levels of a wide range of cytokines compared to control pigs. In contrast, significant amounts of IL-1ß, IL-6, IL-8 and TNF-α were produced in lung lesions and IL-1ß in the TBLN. At the protein level, TNF-α was expressed by both CD163+ monocytes and macrophages in lung lesions, whereas IL-1ß, IL-6 and IL-8 expression was found only in CD163+ monocytes; no CD163+ macrophages were found to produce these cytokines. Furthermore, the quantification of CD163+ monocytes expressing the two cytokines IL-1ß and IL-8 that were most elevated was performed. In lung lesions, 36.5% IL-1ß positive CD163+ monocytes but only 18.3% IL-8 positive CD163+ monocytes were found. In conclusion, PB and BM CD163+ monocytes do not appear to contribute to the elevated cytokine levels in plasma. On the other hand, CD163+ monocytes contribute to inflammatory cytokine expression, especially IL-1ß at the site of inflammation during the inflammatory process.
Subject(s)
Actinobacillus Infections , Actinobacillus pleuropneumoniae , Swine , Animals , Actinobacillus pleuropneumoniae/physiology , Monocytes/metabolism , Cytokines , Tumor Necrosis Factor-alpha/metabolism , Interleukin-8/metabolism , Interleukin-6/metabolism , Actinobacillus Infections/veterinary , Inflammation/metabolism , Inflammation/veterinaryABSTRACT
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.
Subject(s)
COVID-19 , Viruses , COVID-19/diagnosis , Humans , Pandemics , SARS-CoV-2 , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methodsABSTRACT
Here we developed a powerful tool for comprehensive data collection and mapping of molecular and elemental signatures in the Melanoma-bearing Libechov Minipig (MeLiM) model. The combination of different mass spectrometric methods allowed for detail investigation of specific melanoma markers and elements and their spatial distribution in tissue sections. MALDI-MSI combined with HPLC-MS/MS analyses resulted in identification of seven specific proteins, S100A12, CD163, MMP-2, galectin-1, tenascin, resistin and PCNA that were presented in the melanoma signatures. Furthermore, the ICP-MS method allowed for spatial detection of zinc, calcium, copper, and iron elements linked with the allocation of the specific binding proteins.
Subject(s)
Melanoma , Tandem Mass Spectrometry , Animals , Melanoma/metabolism , Proteins , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , Swine , Swine, MiniatureABSTRACT
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 1ß (IL1ß) and a protegrin-4 precursor were successfully detected based on their tryptic fragments. CD163 and IL1ß were confirmed also by IHC. The protegrin-4 precursor was identified by MALDI-TOF/TOF directly on the tissue cryosections. CD163, IL1ß and protegrin4 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.
Subject(s)
Biomarkers/metabolism , Bronchi/metabolism , Lung/metabolism , Lymph Nodes/metabolism , Respiratory Tract Infections/metabolism , Actinobacillus Infections/metabolism , Actinobacillus Infections/microbiology , Actinobacillus pleuropneumoniae/pathogenicity , Animals , Antigens, CD/metabolism , Antigens, Differentiation, Myelomonocytic/metabolism , Antimicrobial Cationic Peptides/metabolism , Interleukin-1beta/metabolism , Receptors, Cell Surface/metabolism , Respiratory Tract Infections/microbiology , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization/methods , SwineABSTRACT
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).
Subject(s)
Dietary Fats, Unsaturated/administration & dosage , Fatty Acids, Omega-3/analysis , Fatty Acids, Omega-6/analysis , Skin/injuries , Wound Healing/drug effects , Animals , CD8 Antigens/metabolism , Collagen Type III/metabolism , Dietary Fats, Unsaturated/pharmacology , Disease Models, Animal , Fish Oils/administration & dosage , Fish Oils/chemistry , Fish Oils/pharmacology , Indoles/chemistry , Macrophages/immunology , Male , Palm Oil/administration & dosage , Palm Oil/chemistry , Palm Oil/pharmacology , Rats , Safflower Oil/administration & dosage , Safflower Oil/chemistry , Safflower Oil/pharmacology , Skin/drug effects , Skin/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
BACKGROUND: The high doses of zinc oxide (ZnO) administered orally to piglets for the prevention of diarrhea and increase of growth rate can contaminate pig farms and the surrounding environment. Therefore, there is a need to find a replacement of high doses of dietary ZnO with an equally effective alternative. In the present study, the effect of two formulations of zinc phosphate-based nanoparticles (ZnA and ZnC NPs) on growth performance, intestinal microbiota, antioxidant status, and intestinal and liver morphology was evaluated. A total of 100 weaned piglets were randomly divided into 10 equal groups with the base diet (control) or the base diet supplemented with ZnA, ZnC, or ZnO at concentrations 500, 1000, and 2000 mg Zn per kilogram of diet. Supplements were given to animals for 10 days. Fecal samples were collected on day 0, 5, 10 and 20. At the end of the treatment (day 10), three piglets from each group were sacrificed and analyzed. RESULTS: Comparing to that of control, the significantly higher piglet weight gain was observed in all piglet groups fed with ZnA (P < 0.05). Differences in the total aerobic bacteria and coliform counts in piglet feces after NPs supplementation compared to that of control and ZnO groups were also found (P < 0.05). The majority of aerobic culturable bacteria from the feces represented Escherichia (28.57-47.62%), Enterococcus (3.85-35.71%), and Streptococcus (3.70-42.31%) spp. A total of 542 Escherichia coli isolates were screened for the virulence genes STa, STb, Stx2, F4, and F18. The substantial occurrence of E. coli virulence factors was found on day 5, mainly in fimbrillary antigen and thermostable toxins, except for piglets fed by ZnC. Zn treatment decreased Zn blood levels in piglets fed with ZnO and ZnA (500 mg/kg) and increased in ZnC (2000 mg/kg) compared to that of control (P < 0.05). The antioxidant status of piglets was affected only by ZnA. While some changes in the liver and the intestinal morphology of piglets with NPs were observed, none were serious as reflected by the normal health status and increased weigh gain performance. CONCLUSIONS: Our results indicate that ZnA NPs have a positive effect on the piglet growth performance even at the lowest concentration. The prevalence of E. coli virulence factors was lowest in pigs supplemented with ZnC. Zinc phosphate-based nanoparticles may be an effective alternative to ZnO.
ABSTRACT
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.