RESUMO
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.
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COVID-19 , Vírus , COVID-19/diagnóstico , Humanos , Pandemias , SARS-CoV-2 , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodosRESUMO
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.
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Cisteína , Proteínas , Sítios de Ligação , Espectrometria de Massas , SoftwareRESUMO
Mammalian metallothioneins (MTs) are a group of cysteine-rich proteins that bind metal ions in two α- and ß-domains and represent a major cellular Zn(II)/Cu(I) buffering system in the cell. At cellular free Zn(II) concentrations (10-11-10-9 M), MTs do not exist in fully loaded forms with seven Zn(II)-bound ions (Zn7MTs). Instead, MTs exist as partially metal-depleted species (Zn4-6MT) because their Zn(II) binding affinities are on the nano- to picomolar range comparable to the concentrations of cellular Zn(II). The mode of action of MTs remains poorly understood, and thus, the aim of this study is to characterize the mechanism of Zn(II) (un)binding to MTs, the thermodynamic properties of the Zn1-6MT2 species, and their mechanostability properties. To this end, native mass spectrometry (MS) and label-free quantitative bottom-up and top-down MS in combination with steered molecular dynamics simulations, well-tempered metadynamics (WT-MetaD), and parallel-bias WT-MetaD (amounting to 3.5 µs) were integrated to unravel the chemical coordination of Zn(II) in all Zn1-6MT2 species and to explain the differences in binding affinities of Zn(II) ions to MTs. Differences are found to be the result of the degree of water participation in MT (un)folding and the hyper-reactive character of Cys21 and Cys29 residues. The thermodynamics properties of Zn(II) (un)binding to MT2 are found to differ from those of Cd(II), justifying their distinctive roles. The potential of this integrated strategy in the investigation of numerous unexplored metalloproteins is attested by the results highlighted in the present study.
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MetalotioneínaRESUMO
Here, using human metallothionein (MT2) as an example, we describe an improved strategy based on differential alkylation coupled to MS, assisted by zinc probe monitoring, for identification of cysteine-rich binding sites with nanomolar and picomolar metal affinity utilizing iodoacetamide (IAM) and N-ethylmaleimide reagents. We concluded that an SN2 reaction provided by IAM is more suitable to label free Cys residues, avoiding nonspecific metal dissociation. Afterward, metal-bound Cys can be easily labeled in a nucleophilic addition reaction after separation by reverse-phase C18 at acidic pH. Finally, we evaluated the efficiency of the method by mapping metal-binding sites of Zn7-xMT species using a bottom-up MS approach with respect to metal-to-protein affinity and element(al) resolution. The methodology presented might be applied not only for MT2 but to identify metal-binding sites in other Cys-containing proteins.
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Metalotioneína/química , Zinco/análise , Sítios de Ligação , Humanos , Concentração de Íons de Hidrogênio , Metalotioneína/genéticaRESUMO
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).
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Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Ômega-3/análise , Ácidos Graxos Ômega-6/análise , Pele/lesões , Cicatrização/efeitos dos fármacos , Animais , Antígenos CD8/metabolismo , Colágeno Tipo III/metabolismo , Gorduras Insaturadas na Dieta/farmacologia , Modelos Animais de Doenças , Óleos de Peixe/administração & dosagem , Óleos de Peixe/química , Óleos de Peixe/farmacologia , Indóis/química , Macrófagos/imunologia , Masculino , Óleo de Palmeira/administração & dosagem , Óleo de Palmeira/química , Óleo de Palmeira/farmacologia , Ratos , Óleo de Cártamo/administração & dosagem , Óleo de Cártamo/química , Óleo de Cártamo/farmacologia , Pele/efeitos dos fármacos , Pele/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
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.
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Biomarcadores/metabolismo , Brônquios/metabolismo , Pulmão/metabolismo , Linfonodos/metabolismo , Infecções Respiratórias/metabolismo , Infecções por Actinobacillus/metabolismo , Infecções por Actinobacillus/microbiologia , Actinobacillus pleuropneumoniae/patogenicidade , Animais , Antígenos CD/metabolismo , Antígenos de Diferenciação Mielomonocítica/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Interleucina-1beta/metabolismo , Receptores de Superfície Celular/metabolismo , Infecções Respiratórias/microbiologia , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , SuínosRESUMO
Novicidin (NVC), is a membrane-penetrating peptide, which forms a stable complex with Zn-Schiff base with interesting antitumor selectivity. We studied NVC derivatives to determine functional roles of key amino acids in toxicity, helicity, and binding of the Zn-Schiff base complex. Trimmed derivatives highlighted the role of peptide length and helicity in toxicity and membrane penetration. The removal of Lys from position 1 and 2 strongly increases the ability to disrupt the membranes. The trimming of the N-terminal residues significantly increases the stability of peptide helicity enhancing penetrating properties. Gly residue derivatives undermined a role of peptide bending in membrane penetration and toxicity. After the substitution of the central Gly derivatives with Ile or Lys, the peptides retained toxicity. These results illustrate the minor role of central helix bending in NVC toxicity. Binding-site-peptide derivatives identified His residue as the sole Zn-Schiff base binding site and eliminated the role of other aromatic residues.
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Peptídeos Catiônicos Antimicrobianos/administração & dosagem , Sistemas de Liberação de Medicamentos , Bases de Schiff/química , Zinco/administração & dosagem , Sequência de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/toxicidade , Sítios de Ligação , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Glicina/química , Humanos , Ligantes , Conformação Proteica , Espectroscopia de Prótons por Ressonância Magnética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectroscopia de Infravermelho com Transformada de Fourier , Zinco/químicaRESUMO
A novel microfluidic label-free bead-based metallothionein immunosensors was designed. To the surface of superparamagnetic agarose beads coated with protein A, polyclonal chicken IgY specifically recognizing metallothionein (MT) were immobilized via rabbit IgG. The Brdicka reaction was used for metallothionein detection in a microfluidic printed 3D chip. The assembled chip consisted of a single copper wire coated with a thin layer of amalgam as working electrode. Optimization of MT detection using designed microfluidic chip was performed in stationary system as well as in the flow arrangement at various flow rates (0-1800 µL/min). In stationary arrangement it is possible to detect MT concentrations up to 30 ng/mL level, flow arrangement allows reliable detection of even lower concentration (12.5 ng/mL). The assembled miniature flow chip was subsequently tested for the detection of MT elevated levels (at approx. level 100 µg/mL) in samples of patients with cancer. The stability of constructed device for metallothionein detection in flow arrangement was found to be several days without any maintenance needed.
Assuntos
Técnicas Eletroquímicas/instrumentação , Separação Imunomagnética/instrumentação , Metalotioneína/sangue , Animais , Anticorpos Imobilizados/química , Anticorpos Imobilizados/metabolismo , Galinhas , Técnicas Eletroquímicas/métodos , Eletrodos , Desenho de Equipamento , Neoplasias de Cabeça e Pescoço/sangue , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Imunoglobulinas/química , Imunoglobulinas/metabolismo , Separação Imunomagnética/métodos , Masculino , Pessoa de Meia-Idade , CoelhosRESUMO
Methicillin-resistant Staphylococcus aureus (MRSA) is a dangerous pathogen resistant to ß-lactam antibiotics. Due to its resistance, it is difficult to manage the infections caused by this strain. We examined this issue in terms of observation of the growth properties and ability to form biofilms in sensitive S. aureus and MRSA after the application of antibiotics (ATBs)-ampicillin, oxacillin and penicillin-and complexes of selenium nanoparticles (SeNPs) with these ATBs. The results suggest the strong inhibition effect of SeNPs in complexes with conventional ATBs. Using the impedance method, a higher disruption of biofilms was observed after the application of ATB complexes with SeNPs compared to the group exposed to ATBs without SeNPs. The biofilm formation was intensely inhibited (up to 99%±7% for S. aureus and up to 94%±4% for MRSA) after application of SeNPs in comparison with bacteria without antibacterial compounds whereas ATBs without SeNPs inhibited S. aureus up to 79%±5% and MRSA up to 16%±2% only. The obtained results provide a basis for the use of SeNPs as a tool for the treatment of bacterial infections, which can be complicated because of increasing resistance of bacteria to conventional ATB drugs.
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Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Nanopartículas/química , Selênio/farmacologia , Staphylococcus aureus/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Selênio/químicaRESUMO
In this work, we focused on the differences between bacterial cultures of E. coli obtained from swabs of infectious wounds of patients compared to laboratory E. coli. In addition, blocking of the protein responsible for the synthesis of glutathione (γ-glutamylcysteine synthase-GCL) using 10 mM buthionine sulfoximine was investigated. Each E. coli showed significant differences in resistance to antibiotics. According to the determined resistance, E. coli were divided into experimental groups based on a statistical evaluation of their properties as more resistant and more sensitive. These groups were also used for finding the differences in a dependence of the glutathione pathway on resistance to antibiotics. More sensitive E. coli showed the same kinetics of glutathione synthesis while blocking GCL (Km 0.1 µM), as compared to non-blocking. In addition, the most frequent mutations in genes of glutathione synthetase, glutathione peroxidase and glutathione reductase were observed in this group compared to laboratory E.coli. The group of "more resistant" E. coli exhibited differences in Km between 0.3 and 0.8 µM. The number of mutations compared to the laboratory E. coli was substantially lower compared to the other group.
Assuntos
Farmacorresistência Bacteriana/genética , Escherichia coli/genética , Glutationa/genética , Transdução de Sinais/genética , Butionina Sulfoximina/farmacologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Glutationa Peroxidase/genética , Glutationa Redutase/genética , Glutationa Sintase/genética , Humanos , Cinética , Mutação/efeitos dos fármacos , Mutação/genética , Transdução de Sinais/efeitos dos fármacosRESUMO
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.
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The Kékfrankos is the most frequently cultivated wine grape in Hungary, with a significant national and regional impact, resulting in considerable amounts of byproducts (e.g. pomace, seeds). To the best of our knowledge no research has been conducted on the antioxidant and antibacterial properties of its seed extracts (GSE). A novel apporach of applying direct microwave treatment on grape seeds was implemented for the first time to enhance antioxidant and antimicrobial properties of GSE. Antioxidant properties were assayed using the DPPH (2,2-diphenyl-1-picrylhydrazyl), FRAP (Ferric Reducing Antioxidant Power) and TPC (Folin-Ciocâlteu's Total Polyphenol Content) methods. Profile and content of polyphenols was studied using high-performance liquid chromatography/tandem mass spectrometry and matrix-assisted laser desorption/ionization mass spectrometry. Antibacterial properties were evaluated using Gram-positive Staphylococcus aureus (SA), methicillin-resistant Staphylococcus aureus (ST239) (MRSA) and Gram-negative Escherichia coli (EC) bacteria strains. Results proved that the mild direct microwave treatment of grape seeds significantly increased total polyphenol, (+)-catechin, (-)-epicatechin as well as antioxidant capacity levels by 20-30 % compared to untreated samples and resulted the best antibacterial properties based on bacterial growth curves (SA and MRSA: 0.015625 mg/mL, EC: 0.25 mg/mL). Results justify the importance of further pharmacological investigations on Kékfrankos grape seed extracts and that the direct microwave treatment of grape seeds is an innovative approach for the fast and cost efficient improvement of the antibacterial properties of grape seed extracts.
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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.
Assuntos
Infecções por Actinobacillus , Actinobacillus pleuropneumoniae , Suínos , Animais , Actinobacillus pleuropneumoniae/fisiologia , Monócitos/metabolismo , Citocinas , Fator de Necrose Tumoral alfa/metabolismo , Interleucina-8/metabolismo , Interleucina-6/metabolismo , Infecções por Actinobacillus/veterinária , Inflamação/metabolismo , Inflamação/veterináriaRESUMO
Bees originally developed their stinging apparatus and venom against members of their own species from other hives or against predatory insects. Nevertheless, the biological and biochemical response of arthropods to bee venom is not well studied. Thus, in this study, the physiological responses of a model insect species (American cockroach, Periplaneta americana) to honeybee venom were investigated. Bee venom toxins elicited severe stress (LD50 = 1.063 uL venom) resulting in a significant increase in adipokinetic hormones (AKHs) in the cockroach central nervous system and haemolymph. Venom treatment induced a large destruction of muscle cell ultrastructure, especially myofibrils and sarcomeres. Interestingly, co-application of venom with cockroach Peram-CAH-II AKH eliminated this effect. Envenomation modulated the levels of carbohydrates, lipids, and proteins in the haemolymph and the activity of digestive amylases, lipases, and proteases in the midgut. Bee venom significantly reduced vitellogenin levels in females. Dopamine and glutathione (GSH and GSSG) insignificantly increased after venom treatment. However, dopamine levels significantly increased after Peram-CAH-II application and after co-application with bee venom, while GSH and GSSG levels immediately increased after co-application. The results suggest a general reaction of the cockroach body to bee venom and at least a partial involvement of AKHs.
Assuntos
Venenos de Abelha/efeitos adversos , Hemolinfa/efeitos dos fármacos , Imunidade Inata , Hormônios de Inseto/farmacologia , Oligopeptídeos/farmacologia , Periplaneta/imunologia , Ácido Pirrolidonocarboxílico/análogos & derivados , Animais , Sistema Nervoso Central/química , Sistema Nervoso Central/efeitos dos fármacos , Hemolinfa/química , Periplaneta/química , Periplaneta/efeitos dos fármacos , Ácido Pirrolidonocarboxílico/farmacologiaRESUMO
Metallothionein-3 has poorly characterized functions in neuroblastoma. Cisplatin-based chemotherapy is a major regimen to treat neuroblastoma, but its clinical efficacy is limited by chemoresistance. We investigated the impact of human metallothionein-3 (hMT3) up-regulation in neuroblastoma cells and the mechanisms underlying the cisplatin-resistance. We confirmed the cisplatin-metallothionein complex formation using mass spectrometry. Overexpression of hMT3 decreased the sensitivity of neuroblastoma UKF-NB-4 cells to cisplatin. We report, for the first time, cisplatin-sensitive human UKF-NB-4 cells remodelled into cisplatin-resistant cells via high and constitutive hMT3 expression in an in vivo model using chick chorioallantoic membrane assay. Comparative proteomic analysis demonstrated that several biological pathways related to apoptosis, transport, proteasome, and cellular stress were involved in cisplatin-resistance in hMT3 overexpressing UKF-NB-4 cells. Overall, our data confirmed that up-regulation of hMT3 positively correlated with increased cisplatin-chemoresistance in neuroblastoma, and a high level of hMT3 could be one of the causes of frequent tumour relapses.
Assuntos
Cisplatino/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metalotioneína 3/biossíntese , Proteínas de Neoplasias/biossíntese , Animais , Linhagem Celular Tumoral , Embrião de Galinha , Resistencia a Medicamentos Antineoplásicos/genética , Humanos , Metalotioneína 3/genética , Proteínas de Neoplasias/genéticaRESUMO
Antibiotic-resistant bacterial infections have become global issues for public health, which increases the utter need to develop alternatives to antibiotics. Here, the HSER (Homo sapiens retinoic acid receptor) peptide was designed from retinoic acid receptor responder protein 2 of Homo sapiens, and was conjugated with synthesized CQDs (carbon quantum dots) for enhanced antibacterial activity in combination, as individually they are not highly effective. The HSER-CQDs were characterized using spectrophotometer, HPLC coupled with electrospray-ionization quadrupole time-of-flight mass spectrometer (ESI-qTOF) mass spectrometer, zeta potential, zeta size, and FTIR. Thereafter, the antibacterial activity against Vancomycin-Resistant Staphylococcus aureus (VRSA) and Escherichia coli (carbapenem resistant) was studied using growth curve analysis, further supported by microscopic images showing the presence of cell debris and dead bacterial cells. The antibacterial mechanism of HSER-CQDs was observed to be via cell wall disruption and also interaction with gDNA (genomic DNA). Finally, toxicity test against normal human epithelial cells showed no toxicity, confirmed by microscopic analysis. Thus, the HSER-CQDs conjugate, having high stability and low toxicity with prominent antibacterial activity, can be used as a potential antibacterial agent.
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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.
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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.
Assuntos
Imuno-Histoquímica , Proteínas/química , Proteínas/metabolismo , Pele/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , HumanosRESUMO
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.
Assuntos
Nanopartículas Metálicas/química , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Titânio/química , Linhagem Celular , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Cristalografia por Raios X , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Humanos , Nanopartículas Metálicas/toxicidade , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Titânio/toxicidadeRESUMO
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.