RESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of malignancy with one of the worst prognoses amongst any type of cancer. Surgery is applicable only to the limited number of patients with locally resectable tumors and currently represents the only curative treatment option. Treatment with chemotherapy and radiotherapy can only extend patient survival. Despite advances in conventional therapies, the five-year survival of PDAC remained largely unchanged. New in vitro and in vivo models are therefore urgently needed to investigate this type of cancer. Here, we present the establishment and characterization of a novel pancreatic cancer cell line, isolated from a patient with PDAC. Cell line abbreviated as PANDA (PANncreatic Ductal Adenocarcinoma) was established with an optimized 3D culture protocol published previously by our group. The new cancer cell line "PANDA" represents a novel in vitro approach for PDAC cancer research and new therapy testing.
Assuntos
Adenocarcinoma , Neoplasias Pancreáticas , Técnicas de Cultura de Células em Três Dimensões , Linhagem Celular , Humanos , TecnologiaRESUMO
no Abstract Keywords.
Assuntos
Anti-Infecciosos , Antibacterianos/uso terapêutico , Anti-Infecciosos/uso terapêuticoRESUMO
Cold atmospheric plasma has great potential for use in modern medicine. It has been used in the clinical treatment of skin diseases and chronic wounds, and in laboratory settings it has shown effects on selective decrease in tumour-cell viability, reduced tumour mass in animal models and stem-cell proliferation. Many researchers are currently focusing on its application to internal structures and the use of plasma-activated liquids in tolerated and effective human treatment. There has also been analysis of plasma's beneficial synergy with standard pharmaceuticals to enhance their effect. Cold atmospheric plasma triggers various responses in tumour cells, and this can result in epigenetic changes in both DNA methylation levels and histone modification. The expression and activity of non-coding RNAs with their many important cell regulatory functions can also be altered by cold atmospheric plasma action. Finally, there is ongoing debate whether plasma-produced radicals can directly affect DNA damage in the nucleus or only initiate apoptosis or other forms of cell death. This article therefore summarises accepted knowledge of cold atmospheric plasma's influence on epigenetic changes, the expression and activity of non-coding RNAs, and DNA damage and its effect in synergistic treatment with routinely used pharmaceuticals.
Assuntos
Antineoplásicos/uso terapêutico , Dano ao DNA , Epigênese Genética/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Gases em Plasma/uso terapêutico , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias/genética , Neoplasias/patologiaRESUMO
Population aging has been a global trend for the last decades, which increases the pressure to develop new cell-based or drug-based therapies, including those that may cure bone diseases. To understand molecular processes that underlie bone development and turnover, we followed osteogenic differentiation of human dental pulp stem cells (DPSCs) using a specific induction medium. The differentiation process imitating in vivo osteogenesis is triggered by various signaling pathways and is associated with massive proteome and metabolome changes. Proteome was profiled by ultrahigh-performance liquid chromatography and comprehensively quantified by ion mobility-enhanced mass spectrometry. From 2667 reproducibly quantified and identified proteins, 432 were differentially abundant by strict statistic criteria. Metabolome profiling was carried out by nuclear magnetic resonance. From 27 detected metabolites, 8 were differentially accumulated. KEGG and MetaboAnalyst hinted metabolic pathways that may be involved in the osteogenic process. Enrichment analysis of differentially abundant proteins highlighted PPAR, FoxO, JAK-STAT, IL-17 signaling pathways, biosynthesis of thyroid hormones and steroids, mineral absorption, and fatty acid metabolism as processes with prominent impact on osteoinduction. In parallel, metabolomic data showed that aminoacyl-tRNA biosynthesis, as well as specific amino acids, likely promote osteodifferentiation. Targeted immunoassays validated and complemented omic results. Our data underlined the complexity of the osteogenic mechanism. Finally, we proposed promising targets for future validation in patient samples, a step toward the treatment of bone defects.
Assuntos
Osteoblastos/metabolismo , Osteogênese , Transdução de Sinais , Células-Tronco/fisiologia , Diferenciação Celular , Linhagem Celular , Polpa Dentária/citologia , Humanos , Redes e Vias Metabólicas , Metabolômica , ProteômicaRESUMO
Alveolar epithelial type II (ATII) cells and their proper function are essential for maintaining lung integrity and homeostasis. However, they can be damaged by lipopolysaccharide (LPS) during Gram-negative bacterial infection. Thus, this study evaluated and compared the effects of LPS on short and long-term cultures of A549 cells by determining the cell viability, levels of oxidative stress and antimicrobial peptide cathelicidin LL-37 and changes in the expression of surfactant proteins (SPs). Moreover, we compared A549 cell response to LPS in the presence of different serum concentrations. Additionally, the effect of N-acetylcysteine (NAC) on LPS-induced oxidative stress as a possible treatment was determined. Our results indicate that A549 cells are relatively resistant to LPS and able to maintain integrity even at high LPS concentrations. Their response to endotoxin is partially dependent on serum concentration. NAC failed to lower LPS-induced oxidative stress in A549 cells. Finally, LPS modulates SP gene expression in A549 cells in a time dependent manner and differences between short and long-term cultures were present. Our results support the idea that long-term cultivation of A549 cells could promote a more ATII-like phenotype and thus could be a more suitable model for ATII cells, especially for in vitro studies dealing with surfactant production.
Assuntos
Células Epiteliais Alveolares/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Lipopolissacarídeos/metabolismo , Estresse Oxidativo , Proteínas Associadas a Surfactantes Pulmonares/metabolismo , Células A549 , Células Epiteliais Alveolares/citologia , Técnicas de Cultura de Células , Sobrevivência Celular , Humanos , CatelicidinasRESUMO
Cold atmospheric plasma use in clinical studies is mainly limited to the treatment of chronic wounds, but its application in a wide range of medical fields is now the goal of many analyses. It is therefore likely that its application spectrum will be expanded in the future. Cold atmospheric plasma has been shown to reduce microbial load without any known significant negative effects on healthy tissues, and this should enhance its possible application to any microbial infection site. It has also been shown to have anti-tumour effects. In addition, it acts proliferatively on stem cells and other cultivated cells, and the highly increased nitric oxide levels have a very important effect on this proliferation. Cold atmospheric plasma use may also have a beneficial effect on immunotherapy in cancer patients. Finally, it is possible that the use of plasma devices will not remain limited to surface structures, because current endeavours to develop sufficiently miniature microplasma devices could very likely lead to its application in subcutaneous and internal structures. This study summarises the available literature on cold plasma action mechanisms and analyses of its current in vivo and in vitro use, primarily in the fields of regenerative and dental medicine and oncology.
Assuntos
Gases em Plasma/uso terapêutico , Proliferação de Células/efeitos da radiação , Assistência Odontológica/métodos , Humanos , Regeneração/efeitos dos fármacos , Medicina Regenerativa/métodos , Células-Tronco/efeitos da radiação , Cicatrização/efeitos da radiaçãoRESUMO
One of the greatest breakthroughs of regenerative medicine in this century was the discovery of induced pluripotent stem cell (iPSC) technology in 2006 by Shinya Yamanaka. iPSCs originate from terminally differentiated somatic cells that have newly acquired the developmental capacity of self-renewal and differentiation into any cells of three germ layers. Before iPSCs can be used routinely in clinical practice, their efficacy and safety need to be rigorously tested; however, iPSCs have already become effective and fully-fledged tools for application under in vitro conditions. They are currently routinely used for disease modeling, preparation of difficult-to-access cell lines, monitoring of cellular mechanisms in micro- or macroscopic scales, drug testing and screening, genetic engineering, and many other applications. This review is a brief summary of the reprogramming process and subsequent differentiation and culture of reprogrammed cells into neural precursor cells (NPCs) in two-dimensional (2D) and three-dimensional (3D) conditions. NPCs can be used as biomedical models for neurodegenerative diseases (NDs), which are currently considered to be one of the major health problems in the human population.
Assuntos
Diferenciação Celular/genética , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Neurais/citologia , Doenças Neurodegenerativas/tratamento farmacológico , Linhagem da Célula/genética , Autorrenovação Celular/efeitos dos fármacos , Autorrenovação Celular/genética , Reprogramação Celular/genética , Descoberta de Drogas , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Neurais/efeitos dos fármacos , Medicina RegenerativaRESUMO
Dental pulp stem cells (DPSCs) have excellent proliferative properties, mineralization potential and can be easily obtained from third molar teeth. Recently, many studies have focused on isolation and differentiation of DPSCs. In our study, we focused on biological properties of non-differentiated DPSCs in comparison with osteogenic differentiated cells from DPSCs. We analyzed morphology as well as mineralization potential using three varied osteogenic differentiation media. After fifteen days of differentiation, calcium deposit production was observed in all three osteogenic differentiation media. However, only one osteogenic medium, without animal serum supplement, showed rapid and strong calcification-OsteoMAX-XF™ Differentiation Medium. Therefore, we examined specific surface markers, and gene and protein expression of cells differentiated in this osteogenic medium, and compared them to non-differentiated DPSCs. We proved a decrease in expression of CD9 and CD90 mesenchymal stem cell surface markers, as well as downregulation in the expression of pluripotency genes (NANOG and OCT-4) and increased levels of expression in osteogenic genes (ALP, BSP, OCN and RUNX2). Moreover, osteogenic proteins, such as BSP and OCN, were only produced in differentiated cells. Our findings confirm that carefully selected differentiation conditions for stem cells are essential for their translation into future clinical applications.
Assuntos
Diferenciação Celular , Técnicas de Reprogramação Celular/métodos , Polpa Dentária/citologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Adulto , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Cálcio/metabolismo , Células Cultivadas , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Meios de Cultura Livres de Soro/química , Meios de Cultura Livres de Soro/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Proteína Homeobox Nanog/genética , Proteína Homeobox Nanog/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Osteoblastos/metabolismo , Osteocalcina/genética , Osteocalcina/metabolismoRESUMO
The study aimed to prove the hypothesis that exogenous surfactant and an antibiotic polymyxin B (PxB) can more effectively reduce lipopolysaccharide (LPS)-induced acute lung injury (ALI) than surfactant treatment alone, and to evaluate the effect of this treatment on the gene expression of surfactant proteins (SPs). Anesthetized rats were intratracheally instilled with different doses of LPS to induce ALI. Animals with LPS 500 µg/kg have been treated with exogenous surfactant (poractant alfa, Curosurf®, 50 mg PL/kg b.w.) or surfactant with PxB 1% w.w. (PSUR + PxB) and mechanically ventilated for 5 hrs. LPS at 500 µg/kg increased lung edema, oxidative stress, and the levels of proinflammatory mediators in lung tissue and bronchoalveolar lavage fluid (BALF). PSUR reduced lung edema and oxidative stress in the lungs and IL-6 in BALF. This effect was further potentiated by PxB added to PSUR. Exogenous surfactant enhanced the gene expression of SP-A, SP-B, and SP-C, however, gene expression for all SPs was reduced after treatment with PSUR + PxB. In mechanically ventilated rats with LPS-induced ALI, the positive effect of exogenous surfactant on inflammation and oxidative stress was potentiated with PxB. Due to the tendency for reduced SPs gene expression after surfactant/PxB treatment topical use of PxB should be considered with caution.
Assuntos
Homeostase/efeitos dos fármacos , Lipopolissacarídeos/efeitos adversos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Polimixina B/farmacologia , Respiração Artificial , Tensoativos/farmacologia , Animais , Antibacterianos/farmacologia , Biomarcadores/metabolismo , Citocinas/metabolismo , Interações Medicamentosas , Regulação da Expressão Gênica/efeitos dos fármacos , Contagem de Leucócitos , Pulmão/citologia , Pulmão/imunologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , SuínosRESUMO
The main function of the lungs is oxygen transport from the atmosphere into the blood circulation, while it is necessary to keep the pulmonary tissue relatively free of pathogens. This is a difficult task because the respiratory system is constantly exposed to harmful substances entering the lungs by inhalation or via the blood stream. Individual types of lung cells are equipped with the mechanisms that maintain pulmonary homeostasis. Because of the clinical significance of acute respiratory distress syndrome (ARDS) the article refers to the physiological role of alveolar epithelial cells type I and II, endothelial cells, alveolar macrophages, and fibroblasts. However, all these cells can be damaged by lipopolysaccharide (LPS) which can reach the airspaces as the major component of the outer membrane of Gram-negative bacteria, and lead to local and systemic inflammation and toxicity. We also highlight a negative effect of LPS on lung cells related to alveolar-capillary barrier and their response to LPS exposure. Additionally, we describe the molecular mechanism of LPS signal transduction pathway in lung cells.
Assuntos
Lesão Pulmonar Aguda/metabolismo , Células Epiteliais Alveolares/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , Síndrome do Desconforto Respiratório/metabolismo , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Capilares/efeitos dos fármacos , Capilares/patologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Pulmão/patologia , Oxigênio/metabolismo , Alvéolos Pulmonares/efeitos dos fármacos , Alvéolos Pulmonares/patologia , Síndrome do Desconforto Respiratório/induzido quimicamente , Síndrome do Desconforto Respiratório/patologia , Transdução de Sinais/genéticaRESUMO
Utilization of liquid biopsy in the management of cancerous diseases is becoming more attractive. This method can overcome typical limitations of tissue biopsies, especially invasiveness, no repeatability, and the inability to monitor responses to medication during treatment as well as condition during follow-up. Liquid biopsy also provides greater possibility of early prediction of cancer presence. Corpus uteri mesenchymal tumors are comprised of benign variants, which are mostly leiomyomas, but also a heterogenous group of malignant sarcomas. Pre-surgical differentiation between these tumors is very difficult and the final description of tumor characteristics usually requires excision and histological examination. The leiomyomas and malignant leiomyosarcomas are especially difficult to distinguish and can, therefore, be easily misdiagnosed. Because of the very aggressive character of sarcomas, liquid biopsy based on early diagnosis and differentiation of these tumors would be extremely helpful. Moreover, after excision of the tumor, liquid biopsy can contribute to an increased knowledge of sarcoma behavior at the molecular level, especially on the formation of metastases which is still not well understood. In this review, we summarize the most important knowledge of mesenchymal uterine tumors, the possibilities and benefits of liquid biopsy utilization, the types of molecules and cells that can be analyzed with this approach, and the possibility of their isolation and capture. Finally, we review the typical abnormalities of leiomyomas and sarcomas that can be searched and analyzed in liquid biopsy samples with the final aim to pre-surgically differentiate between benign and malignant mesenchymal tumors.
Assuntos
Biomarcadores Tumorais , Leiomioma/diagnóstico , Sarcoma/diagnóstico , Neoplasias Uterinas/diagnóstico , Micropartículas Derivadas de Células , Aberrações Cromossômicas , DNA Tumoral Circulante , Metilação de DNA , Diagnóstico Diferencial , Feminino , Humanos , Leiomioma/genética , Leiomioma/metabolismo , Biópsia Líquida , MicroRNAs/sangue , Mutação , Estadiamento de Neoplasias , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Sarcoma/genética , Sarcoma/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
Statins are the inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This enzyme catalyzes conversion of HMG-CoA to mevalonate, which are the intermediates in cholesterol biosynthetic pathway. Statins also play an important role in carcinogenesis, because they are able to affect the cancer cell metabolism. Their effect has been observed in several cellular processes, such as angiogenesis, metastasis, apoptosis and cell proliferation. However, these effects are highly dependent on type of cancer and individual statins vary in their antitumor potential. This review summarizes the recent epidemiological evidence and preclinical studies that showed effects of all clinically used statins in vitro and in vivo. We also consider the results of different observational and retrospective studies focused on association among statins and cancer risk which are still under open discussion.
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinogênese/metabolismo , Colesterol/metabolismo , Inibidores de Hidroximetilglutaril-CoA Redutases/administração & dosagem , Ácido Mevalônico/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Carcinogênese/patologia , Relação Dose-Resposta a Droga , Medicina Baseada em Evidências , Feminino , Humanos , Masculino , Modelos Biológicos , Neoplasias/patologia , Resultado do TratamentoRESUMO
Dihydropyrimidine dehydrogenase (DPD) acts as the first-step enzyme catabolizing pyrimidines in vivo. DPYD gene mutations interfere with the breakdown of uracil and thymine. Genetic variations of DPYD can cause an enzyme deficiency state, which results in severe toxicity or other adverse side effects such as DNA damage or RNA damage caused by imbalance of the nucleotide pool. Our case-control study investigates the possible association between seven DPYD gene polymophisms (rs1801267, rs72547602, rs1801160, rs3918290, rs1801159, rs1801158, rs1801265) and risk of colorectal cancer (CRC). The association analysis for DPD was performed on 273 CRC patients and 187 healthy controls. There is significant allele association of SNP rs1801160 with colorectal cancer (p = 0.003, OR = 3.264, 95% CI = 1.425-7.475) in present analysis. Haplotype analysis of four DPYD polymorphisms showed significant difference in the distribution "IISt" haplotype between cases and controls. In comparison to the most common haplotype (VISt), the "IISt" haplotype was associated with increased risk for CRC (p = 0.038, OR = 2.733, 95% CI = 1.019-7.326). The present study suggests that the SNP rs1801160 and the "IISt" haplotype in the DPYD gene may also have a role in colorectal cancer risk.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/epidemiologia , Neoplasias Colorretais/genética , Di-Hidrouracila Desidrogenase (NADP)/genética , Predisposição Genética para Doença/epidemiologia , Estudos de Casos e Controles , Neoplasias Colorretais/enzimologia , Feminino , Marcadores Genéticos/genética , Genótipo , Haplótipos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Polimorfismo de Nucleotídeo Único/genética , Prevalência , Prognóstico , Reprodutibilidade dos Testes , Medição de Risco , Sensibilidade e Especificidade , Eslováquia/epidemiologiaRESUMO
Homocysteine (Hcy) is a sulfur-containing non-proteinogenic amino acid derived in methionine metabolism. The increased level of Hcy in plasma, hyperhomocysteinemia, is considered to be an independent risk factor for cardio and cerebrovascular diseases. However, it is still not clear if Hcy is a marker or a causative agent of diseases. More and more research data suggest that Hcy is an important indicator for overall health status. This review represents the current understanding of molecular mechanism of Hcy metabolism and its link to hyperhomocysteinemia-related pathologies in humans. The aberrant Hcy metabolism could lead to the redox imbalance and oxidative stress resulting in elevated protein, nucleic acid and carbohydrate oxidation and lipoperoxidation, products known to be involved in cytotoxicity. Additionally, we examine the role of Hcy in thiolation of proteins, which results in their molecular and functional modifications. We also highlight the relationship between the imbalance in Hcy metabolism and pathogenesis of diseases, such as cardiovascular diseases, neurological and psychiatric disorders, chronic kidney disease, bone tissue damages, gastrointestinal disorders, cancer, and congenital defects.
Assuntos
Nível de Saúde , Homocisteína/metabolismo , Hiper-Homocisteinemia/metabolismo , Estresse Oxidativo/fisiologia , Doenças Ósseas/patologia , Doenças Cardiovasculares/patologia , Transtornos Cerebrovasculares/patologia , Gastroenteropatias/patologia , Homocisteína/sangue , Humanos , Nefropatias/patologia , Neoplasias/patologia , Oxirredução , Fatores de RiscoRESUMO
Mycobacterium tuberculosis lipoarabinomannan (LAM) and biosynthetically related lipoglycans and glycans play an important role in host-pathogen interactions. Therefore, the elucidation of the complete biosynthetic pathways of these important molecules is expected to afford novel therapeutic targets. The characterization of biosynthetic enzymes and transporters involved in the formation and localization of these complex macromolecules in the bacterial cell envelope largely relies on genetic manipulation of mycobacteria and subsequent analyses of lipoglycan structural alterations. However, lipoglycans are present in relatively low amounts. Their purification to homogeneity remains tedious and time-consuming. To overcome these issues and to reduce the biomass and time required for lipoglycan purification, we report here the development of a methodology to efficiently purify lipoglycans by sodium deoxycholate-polyacrylamide gel electrophoresis. This faster purification method can be applied on a small amount of mycobacterial cells biomass (10-50 mg), resulting in tens of micrograms of purified lipoglycans. This amount of purified products was found to be sufficient to undertake structural analyses of lipoglycans and glycans carbohydrate domains by a combination of highly sensitive analytical procedures, involving cryoprobe NMR analysis of intact macromolecules and chemical degradations monitored by gas chromatography and capillary electrophoresis. This glycomic approach was successfully applied to the purification and structural characterization of a newly identified polysaccharide, structurally related to LAM, in the model fast-growing species Mycobacterium smegmatis.
Assuntos
Lipopolissacarídeos/química , Mycobacterium tuberculosis/química , Polissacarídeos Bacterianos/química , Glicômica/métodos , Lipopolissacarídeos/metabolismo , Mycobacterium tuberculosis/metabolismo , Polissacarídeos Bacterianos/metabolismoRESUMO
Metabolomics has proven to be a sensitive tool for monitoring biochemical processes in cell culture. It enables multi-analysis, clarifying the correlation between numerous metabolic pathways. Together with other analysis, it thus provides a global view of a cell's physiological state. A comprehensive analysis of molecular changes is also required in the case of mesenchymal stem cells (MSCs), which currently represent an essential portion of cells used in regenerative medicine. Reproducibility and correct measurement are closely connected to careful metabolite extraction, and sample preparation is always a critical point. Our study aimed to compare the efficiencies of four harvesting and six extraction methods. Several organic reagents (methanol, ethanol, acetonitrile, methanol-chloroform, MTBE) and harvesting approaches (trypsinization vs. scraping) were tested. We used untargeted nuclear magnetic resonance spectroscopy (NMR) to determine the most efficient method for the extraction of metabolites from human adherent cells, specifically human dermal fibroblasts adult (HDFa) and dental pulp stem cells (DPSCs). A comprehensive dataset of 29 identified and quantified metabolites were determined to possess statistically significant differences in the abundances of several metabolites when the cells were detached mechanically to organic solvent compared to when applying enzymes mainly in the classes of amino acids and peptides for both types of cells. Direct scraping to organic solvent is a method that yields higher abundances of determined metabolites. Extraction with the use of different polar reagents, 50% and 80% methanol, or acetonitrile, mostly showed the same quality. For both HDFa and DPSC cells, the MTBE method, methanol-chloroform, and 80% ethanol extractions showed higher extraction efficiency for the most identified and quantified metabolites Thus, preparation procedures provided a cell sample processing protocol that focuses on maximizing extraction yield. Our approach may be useful for large-scale comparative metabolomic studies of human mesenchymal stem cell samples.
RESUMO
The analysis of volatile organic compounds (VOCs) in exhaled air has attracted the interest of the scientific community because it provides the possibility of monitoring physiological and metabolic processes and non-invasive diagnostics of various diseases. However, this method remains underused in clinical practice as well as in research because of the lack of standardized procedures for the collection, storage and transport of breath samples, which would guarantee good reproducibility and comparability of results. The method of sampling, as well as the storage time of the breath samples in the polymer bags used for sample storage and transport, affect the composition and concentration of VOCs present in the breath samples. The aim of our study was to compare breath samples obtained using two methods with fully disposable equipment: a Haldane sampling tube intended for direct breath collection and breath samples exhaled into a transparent Tedlar bag. The second task was to monitor the stability of selected compounds of real breath samples stored in a Tedlar bag for 6 h. Gas chromatography coupled with ion mobility spectrometry (GC-IMS) implemented in the BreathSpec®device was used to analyse exhaled breath. Our results showed a significant difference in the signal intensity of some volatiles when taking a breath sample with a Haldane tube and a Tedlar bag. Due to its endogenous origin, acetone levels were significantly higher when the Haldane tube sampler was used while elevated levels of 2-propanol and unidentified VOC (designated as VOC 3) in the Tedlar bag samples likely originated from contamination of the Tedlar bags. The VOC stability study revealed compound-specific signal intensity changes of the selected VOCs with storage time in the Tedlar bags, with some volatiles showing increasing signal intensity during storage in Tedlar bags. This limits the use of Tedlar bags only for very limited time and carefully selected purpose. Our results highlight the importance of careful design and implementation of experiments and clinical protocols to obtain relevant and reliable results.
Assuntos
Testes Respiratórios , Manejo de Espécimes , Compostos Orgânicos Voláteis , Humanos , Testes Respiratórios/instrumentação , Testes Respiratórios/métodos , Compostos Orgânicos Voláteis/análise , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodos , Espectrometria de Mobilidade Iônica/métodos , Espectrometria de Mobilidade Iônica/instrumentação , Masculino , Feminino , Reprodutibilidade dos Testes , Adulto , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cromatografia Gasosa-Espectrometria de Massas/instrumentação , Expiração , Pessoa de Meia-Idade , Fatores de TempoRESUMO
In recent decades, we have seen significant technical progress in the modern world, leading to the widespread use of telecommunications systems, electrical appliances, and wireless technologies. These devices generate electromagnetic radiation (EMR) and electromagnetic fields (EMF) most often in the extremely low frequency or radio-frequency range. Therefore, they were included in the group of environmental risk factors that affect the human body and health on a daily basis. In this study, we tested the effect of exposure EMF generated by a new prototype wireless charging system on four human cell lines (normal cell lines-HDFa, NHA; tumor cell lines-SH-SY5Y, T98G). We tested different operating parameters of the wireless power transfer (WPT) device (87-207 kHz, 1.01-1.05 kW, 1.3-1.7 mT) at different exposure times (pulsed 6 × 10 min; continuous 1 × 60 min). We observed the effect of EMF on cell morphology and cytoskeletal changes, cell viability and mitotic activity, cytotoxicity, genotoxicity, and oxidative stress. The results of our study did not show any negative effect of the generated EMF on either normal cells or tumor cell lines. However, in order to be able to estimate the risk, further population and epidemiological studies are needed, which would reveal the clinical consequences of EMF impact.
Assuntos
Campos Eletromagnéticos , Neuroblastoma , Humanos , Campos Eletromagnéticos/efeitos adversos , Neurônios , Linhagem Celular Tumoral , Sobrevivência CelularRESUMO
The biosynthesis of the major cell envelope glycoconjugates of Mycobacterium tuberculosis is topologically split across the plasma membrane, yet nothing is known of the transporters required for the translocation of lipid-linked sugar donors and oligosaccharide intermediates from the cytoplasmic to the periplasmic side of the membrane in mycobacteria. One of the mechanisms used by prokaryotes to translocate lipid-linked phosphate sugars across the plasma membrane relies on translocases that share resemblance with small multidrug resistance transporters. The presence of an small multidrug resistance-like gene, Rv3789, located immediately upstream from dprE1/dprE2 responsible for the formation of decaprenyl-monophosphoryl-ß-D-arabinose (DPA) in the genome of M. tuberculosis led us to investigate its potential involvement in the formation of the major arabinosylated glycopolymers, lipoarabinomannan (LAM) and arabinogalactan (AG). Disruption of the ortholog of Rv3789 in Mycobacterium smegmatis resulted in a reduction of the arabinose content of both AG and LAM that accompanied the accumulation of DPA in the mutant cells. Interestingly, AG and LAM synthesis was restored in the mutant not only upon expression of Rv3789 but also upon that of the undecaprenyl phosphate aminoarabinose flippase arnE/F genes from Escherichia coli. A bacterial two-hybrid system further indicated that Rv3789 interacts in vivo with the galactosyltransferase that initiates the elongation of the galactan domain of AG. Biochemical and genetic evidence is thus consistent with Rv3789 belonging to an AG biosynthetic complex, where its role is to reorient DPA to the periplasm, allowing this arabinose donor to then be used in the buildup of the arabinan domains of AG and LAM.
Assuntos
Proteínas de Bactérias/metabolismo , Farmacorresistência Bacteriana Múltipla/fisiologia , Galactanos/metabolismo , Lipopolissacarídeos/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mycobacterium tuberculosis/metabolismo , Arabinose/genética , Arabinose/metabolismo , Proteínas de Bactérias/genética , Galactanos/genética , Teste de Complementação Genética , Glicosilação , Lipopolissacarídeos/genética , Proteínas de Membrana Transportadoras/genética , Mutação , Mycobacterium smegmatis/genética , Mycobacterium smegmatis/metabolismo , Mycobacterium tuberculosis/genéticaRESUMO
Here, we present newly derived in vitro model for modeling Duchenne muscular dystrophy. Our new cell line was derived by reprogramming of peripheral blood mononuclear cells (isolated from blood from pediatric patient) with Sendai virus encoding Yamanaka factors. Derived iPS cells are capable to differentiate in vitro into three germ layers as verified by immunocytochemistry. When differentiated in special medium, our iPSc formed spontaneously beating cardiomyocytes. As cardiomyopathy is the main clinical complication in patients with Duchenne muscular dystrophy, the cell line bearing the dystrophin gene mutation might be of interest to the research community.