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Background: Neoadjuvant radiotherapy (neo-RT) is widely used in locally advanced rectal cancer (LARC) as a component of radical treatment. Despite the advantages of neo-RT, which typically improves outcomes in LARC patients, the lack of reliable biomarkers that predict response and monitor the efficacy of therapy, can result in the application of unnecessary aggressive therapy affecting patients' quality of life. Hence, the search for molecular biomarkers for assessing the radio responsiveness of this cancer represents a relevant issue. Methods: Here, we combined proteomic and metabolomic approaches to identify molecular signatures, which could discriminate LARC tumors with good and poor responses to neo-RT. Results: The integration of data on differentially accumulated proteins and metabolites made it possible to identify disrupted metabolic pathways and signaling processes connected with response to irradiation, including ketone bodies synthesis and degradation, purine metabolism, energy metabolism, degradation of fatty acid, amino acid metabolism, and focal adhesion. Moreover, we proposed multi-component panels of proteins and metabolites which could serve as a solid base to develop biomarkers for monitoring and predicting the efficacy of preoperative RT in rectal cancer patients. Conclusion: We proved that an integrated multi-omic approach presents a valid look at the analysis of the global response to cancer treatment from the perspective of metabolomic reprogramming.
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Wild strawberry (Fragaria vesca L.) seed oil (WSO) recovered by two methods-cold pressing (CP) and extraction with supercritical carbon dioxide (SCO2E)-taking into account the different extraction times, was characterized for its composition and quality. The cytotoxicity assessment of WSOs was also carried out using the normal human dermal fibroblast (NHDF) cell line. Tocopherol and total polyphenol contents were significantly higher in WSO recovered by SCO2E, up to 1901.0 and 58.5 mg/kg, respectively, in comparison with CP oil. In CP oil, the highest content of carotenoids and squalene was determined (123.8 and 31.4 mg/kg, respectively). Phytosterol summed up to 5396 mg/kg in WSO collected in 30 min of SCO2E. Moreover, the highest oxidative stability was found for this oil. All studied WSOs were non-cytotoxic in lactate dehydrogenase (LDH) leaching and sulforhodamine B (SRB) assays; however, oils collected by SCO2E in 15 and 30 min were found to be cytotoxic in the tetrazolium salt (MTT) test, with the CC50 at a concentration of 3.4 and 5.5%, respectively. In conclusion, the composition of WSO indicates that, depending on the method of its recovery, seeds can have different bio-potencies and various applications.
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Background: Sleep bruxism (SB) is a common sleep-related movement behavior with a complex etiology. A recent hypothesis suggests psychoactive substance usage, tobacco smoking, and alcohol intake are risk factors for SB. This study aimed to evaluate SB intensity in tobacco smokers and alcohol drinkers. Methods: A total of 133 adults underwent full-night audio- and video-polysomnography, and the polysomnograms were evaluated using the American Academy of Sleep Medicine guidelines. The study group was divided into smoker and nonsmoker groups as well as drinker and non-drinker groups. Results: The results of the polysomnographic analysis confirmed that tobacco smoking has a significant effects on SB. Tobacco smokers showed increased bruxism intensity (5.50 ± 4.71 vs. 3.83 ± 3.26, p < 0.05), especially the mixed phenotype (0.93 ± 1.00 vs. 0.59 ± 0.59, p < 0.05), in the N1 sleep stage (22.84 ± 20.45 vs. 15.66 ± 13.60, p < 0.05) and the nonsupine position (4.93 ± 5.56 vs. 2.50 ± 2.31, p < 0.05). They also showed a higher number of bruxism episodes with arousal compared with nonsmokers (2.91 ± 2.83 vs. 1.61 ± 1.49, p < 0.05), indicating increased sleep fragmentation. However, no significant effect of alcohol on SB intensity was observed, and the bruxism episode index was similar in alcohol drinkers and nondrinkers. In addition, electrolyte disturbances and lipid disorders were evaluated. Compared with nonsmokers, tobacco smokers showed a higher concentration of plasma triglycerides (177.67 ± 106.9 vs. 129.18 ± 65.61) and lower levels of iron and magnesium (96.68 ± 43.58 vs. 123.83 ± 52.36 and 1.85 ± 0.22 vs. 1.96 ± 0.21, respectively). Conclusions: In summary, this study showed that tobacco smoking, but not alcohol consumption, is related to bruxism intensity and lipid and electrolyte disturbances in individuals with sleep disorders.
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(1) Background: Sleep bruxism (SB) is a common sleep behavior. Obstructive sleep apnea (OSA) is a prevalent sleep-related breathing disorder with potential long-term major neurocognitive and cardiovascular sequelae. Although the co-occurrence of SB and OSA has been described previously, the exact relationship remains unclear. The present study aimed to evaluate the incidence of SB in different phenotypes of OSA. (2) Methods: The participants of this study were adult patients referred to the Department and Clinic of Internal Medicine, Occupational Diseases, Hypertension and Clinical Oncology at the Wroclaw Medical University. They underwent a single-night video polysomnography in a sleep laboratory. The data related to common OSA phenotypes were analyzed in two separate groups of patients: body position related (n = 94) and rapid eye movement (REM) related (n = 85). (3) Results: The obtained results showed that the incidence of SB and severe SB was higher for body position-related OSA phenotype (p < 0.05 for all comparisons). No statistically significant differences were observed for REM-related OSA phenotype (p > 0.05 for all comparisons). (4) Conclusions: Body position-related OSA phenotype seems to be associated with higher SB and severe SB incidence, but the relationship is not independent. However, in the light of the unclear relationship between SB and sleep-disordered breathing, the topic needs further study.
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The purpose of this study was to determine the relationship between cadmium exposure and the likelihood of developing or exacerbating symptoms of inflammatory arthritis (IA). The study included 51 IA patients and 46 control subjects. Demographic and lifestyle data were collected. Haematological and biochemical parameters and blood cadmium levels (Cd-B) were determined. Cd-B correlated positively with age, smoking, living in a high-traffic area, and serum levels of inflammatory markers and negatively with mean corpuscular haemoglobin concentration (MCHC). The binary logistic regression model implied that high Cd-B (≥0.65 µg/L) is linked with an increased risk of IA in the studied population (odds ratio: 4.4). High levels of DNA oxidative damage marker (8-hydroxy-2'-deoxyguanosine) (≥7.66 ng/mL) and cyclooxygenase-2 (≥22.9 ng/mL) and frequent consumption of offal was also associated with increased risk of IA. High Cd-B was related to increased risk of disease symptoms onset in the group of IA patients, decreased the level of interleukin 10, and positively correlated with the disease activity. Increased Cd-B is associated with intensified inflammatory processes and decreased haemoglobin levels; in IA patients with decreased anti-inflammatory interleukin 10. These changes partly explain why cadmium exposure and a high cadmium body burden may raise the risk of IA and of disease symptoms exacerbation.
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Artrite , Cádmio , Artrite/epidemiologia , Biomarcadores , Carga Corporal (Radioterapia) , Humanos , Interleucina-10 , Projetos Piloto , Polônia/epidemiologiaRESUMO
Identification of biomarkers that could be used for the prediction of the response to neoadjuvant radiotherapy (neo-RT) in locally advanced rectal cancer remains a challenge addressed by different experimental approaches. Exosomes and other classes of extracellular vesicles circulating in patients' blood represent a novel type of liquid biopsy and a source of cancer biomarkers. Here, we used a combined proteomic and metabolomic approach based on mass spectrometry techniques for studying the molecular components of exosomes isolated from the serum of rectal cancer patients with different responses to neo-RT. This allowed revealing several proteins and metabolites associated with common pathways relevant for the response of rectal cancer patients to neo-RT, including immune system response, complement activation cascade, platelet functions, metabolism of lipids, metabolism of glucose, and cancer-related signaling pathways. Moreover, the composition of serum-derived exosomes and a whole serum was analyzed in parallel to compare the biomarker potential of both specimens. Among proteins that the most properly discriminated good and poor responders were GPLD1 (AUC = 0.85, accuracy of 74%) identified in plasma as well as C8G (AUC = 0.91, accuracy 81%), SERPINF2 (AUC = 0.91, accuracy 79%) and CFHR3 (AUC = 0.90, accuracy 81%) identified in exosomes. We found that the proteome component of serum-derived exosomes has the highest capacity to discriminate samples of patients with different responses to neo-RT when compared to the whole plasma proteome and metabolome. We concluded that the molecular components of exosomes are associated with the response of rectal cancer patients to neo-RT and could be used for the prediction of such response.
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The energy-dense western diet significantly increases the risk of obesity, type 2 diabetes, cardiovascular episodes, stroke, and cancer. Recently more attention has been paid to the contribution of an unhealthy lifestyle on the development of central nervous system disorders. Exposure to long-lasting stress is one of the key lifestyle modifications associated with the increased prevalence of obesity and metabolic diseases. The main goal of the present study was to verify the hypothesis that exposure to chronic stress modifies alterations in the brain proteome induced by the western diet. Female adult rats were fed with the prepared chow reproducing the human western diet and/or subjected to chronic stress induced by social instability for 6 weeks. A control group of lean rats were fed with a standard diet. Being fed with the western diet resulted in an obese phenotype and induced changes in the serum metabolic parameters. The combination of the western diet and chronic stress exposure induced more profound changes in the rat cerebrocortical proteome profile than each of these factors individually. The down-regulation of proteins involved in neurotransmitter secretion (Rph3a, Snap25, Syn1) as well as in learning and memory processes (Map1a, Snap25, Tnr) were identified, while increased expression was detected for 14-3-3 protein gamma (Ywhag) engaged in the modulation of the insulin-signaling cascade in the brain. An analysis of the rat brain proteome reveals important changes that indicate that a combination of the western diet and stress exposure may lead to impairments of neuronal function and signaling.
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Diabetes Mellitus Tipo 2 , Dieta Ocidental , Animais , Dieta Hiperlipídica , Dieta Ocidental/efeitos adversos , Feminino , Insulina , Obesidade/etiologia , Obesidade/metabolismo , Proteoma/metabolismo , RatosRESUMO
BACKGROUND: Sleep bruxism (SB) is a complex behaviour that seems to be associated with the serotoninergic pathway. OBJECTIVES: This exploratory research aimed to evaluate the levels of serotonin in individuals with sleep bruxism diagnosed by video polysomnography. The study also evaluated whether the levels of serotonin were associated with body mass index, heart rate, and sleep parameters. METHODS: The study participants were adults hospitalised in the Department and Clinic of Internal Medicine, Occupational Diseases, Hypertension and Clinical Oncology at the Wroclaw Medical University. They underwent a single-night video polysomnography during which sleep and SB parameters and heart rate were evaluated. Additionally, body mass index and blood serotonin levels were evaluated for each patient. RESULTS: A total of 105 patients were included in this study (80 women and 25 men). All the patients were Caucasians aged 18-63 years, with a mean age ± (standard deviation) of 33.43± 10.8 years. Seventy-five patients (71.43%) presented sleep bruxism (bruxism episodes index ≥2) and 30 (28.57%) did not. Fifty patients (47.62%) presented severe sleep bruxism (bruxism episodes index >4). The results showed that lower blood serotonin levels were associated with severe sleep bruxism; increased bruxism episodes index, rapid eye movement sleep, and body mass index; and decreased maximal pulse. CONCLUSION: Severe sleep bruxism and the associated phenomena seem to co-occur with lower blood serotonin levels. The study supports the hypothesis on the relationship between the serotoninergic pathway and sleep bruxism.
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Serotonina , Bruxismo do Sono , Adolescente , Adulto , Índice de Massa Corporal , Feminino , Frequência Cardíaca , Humanos , Masculino , Pessoa de Meia-Idade , Sono , Adulto JovemRESUMO
OBJECTIVES: This exploratory research aimed to evaluate the levels of tryptophan hydroxylase 1 (TPH1) and aromatic l-amino acid decarboxylase (DDC), which play an important role in the serotonin synthesis pathway, in individuals with sleep bruxism (SB) diagnosed using audio-video polysomnography (vPSG) and compare them with that of individuals not presenting with SB. MATERIALS AND METHODS: The study included adult patients hospitalized in the Department and Clinic of Internal Medicine, Occupational Diseases, Hypertension and Clinical Oncology at the Wroclaw Medical University. The participants underwent a single-night vPSG for the evaluation of the SB parameters. Peripheral blood samples were also collected from them for estimating the serum levels of TPH1 and DDC. RESULTS: A total of 105 patients (80 women and 25 men) were included in the study. All the patients were Caucasians and aged 18-63 years (mean age: 33.43 ± 10.8 years). Seventy-five patients (71.43%) presented with SB, of which 50 (47.62%) had severe SB, while the remaining 30 patients (28.57%) did not. No statistically significant differences in TPH1 and DDC levels were observed between the individuals with SB and without SB. A significant negative correlation was found between tonic SB episodes and DDC levels (p = 0.0012). Other correlations between the SB parameters and the levels of the studied enzymes were statistically insignificant (p > 0.05 for all comparisons). CONCLUSIONS: The levels of the enzymes that are crucial for serotonin synthesis (TPH1 and DDC) did not seem to influence SB. CLINICAL RELEVANCE: This study provides important insights for further research on the relationship between the serotonin pathway and SB, which should take into account not only the process of serotonin synthesis but also the effect of serotonin-dependent neurotransmission on SB.
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Serotonina , Bruxismo do Sono , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Polissonografia , Adulto JovemRESUMO
Small extracellular vesicles (sEV), which are released to body fluids (e.g., serum, urine) by all types of human cells, may stimulate or inhibit the innate and adaptive immune response through multiple mechanisms. Exosomes or sEV have on their surface many key receptors of immune response, including major histocompatibility complex (MHC) components, identical to their cellular origin. They also exhibit an ability to carry antigen and target leukocytes either via interaction with cell surface receptors or intracellular delivery of inflammatory mediators, receptors, enzymes, mRNAs, and noncoding RNAs. By the transfer of donor MHC antigens to recipient antigen presenting cells sEV may also contribute to T cell allorecognition and alloresponse. Here, we review the influence of sEV on the development of rejection or tolerance in the setting of solid organ and tissue allotransplantation. We also summarize and discuss potential applications of plasma and urinary sEV as biomarkers in the context of transplantation. We focus on the attempts to use sEV as a noninvasive approach to detecting allograft rejection. Preliminary studies show that both sEV total levels and a set of specific molecules included in their cargo may be an evidence of ongoing allograft rejection.
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Vesículas Extracelulares/metabolismo , Rejeição de Enxerto/metabolismo , Animais , Anticorpos/metabolismo , Rejeição de Enxerto/imunologia , Humanos , Tolerância Imunológica , Biópsia Líquida , Transplante de ÓrgãosRESUMO
Many of the metabolic effects evoked by the ketogenic diet mimic the actions of fasting and the benefits of the ketogenic diet are often attributed to these similarities. Since fasting is a potent autophagy inductor in vivo and in vitro it has been hypothesized that the ketogenic diet may upregulate autophagy. The aim of the present study was to provide a comprehensive evaluation of the influence of the ketogenic diet on the hepatic autophagy. C57BL/6N male mice were fed with two different ketogenic chows composed of fat of either animal or plant origin for 4 weeks. To gain some insight into the time frame for the induction of autophagy on the ketogenic diet, we performed a short-term experiment in which animals were fed with ketogenic diets for only 24 or 48 h. The results showed that autophagy is upregulated in the livers of animals fed with the ketogenic diet. Moreover, the size of the observed effect was likely dependent on the diet composition. Subsequently, the markers of regulatory pathways that may link ketogenic diet action to autophagy were measured, i.e., the activity of mTORC1, activation of AMPK, and the levels of SIRT1, p53, and FOXO3. Overall, observed treatment-specific effects including the upregulation of SIRT1 and downregulation of FOXO3 and p53. Finally, a GC/MS analysis of the fatty acid composition of animals' livers and the chows was performed in order to obtain an idea about the presence of specific compounds that may shape the effects of ketogenic diets on autophagy.
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Autofagia/fisiologia , Dieta Cetogênica , Gorduras na Dieta/farmacologia , Cetose/metabolismo , Fígado/fisiologia , Regulação para Cima/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Gorduras na Dieta/análise , Proteína Forkhead Box O3/genética , Proteína Forkhead Box O3/metabolismo , Regulação da Expressão Gênica/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plantas , Transdução de Sinais , Sirtuína 1/genética , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismoRESUMO
INTRODUCTION: Environmental arsenic contamination is a major toxicological problem worldwide due to its carcinogenic and nephrotoxic potential. AIM: The purpose of this observational study was to determine the suspected association between urinary arsenic (uAs) and urinary leucine (or leucyl) aminopeptidase 3 (uLAP3) to evaluate uLAP3 as a candidate biomarker of exposure to airborne arsenic. MATERIALS AND METHODS: A total of 918 adults occupationally and/or environmentally exposed to airborne arsenic were enrolled in the study. Baseline information (age; sex; history of smoking; alcohol, fish and seafood consumption) was gathered. Total uAs concentrations [µg/L] of 918 subjects, as well as the sum of arsenic species (ΣiAs) in 259 subjects, were obtained. Urinary LAP3 was measured by an immune-enzymatic assay using an ELISA kit. Urinary creatinine concentration was assessed with the IB/lAB/1289 research protocol (version II, 2015-09-17). The values of uAs and uLAP3 were recalculated per unit of creatinine. The association between uAs and uLAP3 was assessed using a logistic regression model adjusted for confounders. RESULTS: The study identified a positive correlation between the logarithm of uAs and the logarithm of uLAP3 in the study population (r = 0.1737, p < 0.0000) and between urinary creatinine and uLAP3 concentration not adjusted for creatinine level (r = 0.1871, p < 0.001). In the logistic regression model, there was also an association between increased (≥15 µg/L) uAs and decreased (below the 25th quartile) uLAP3 [OR uLAP3 = 1.22 (95% CI 1.03 to 1.44, p < 0.02)]. CONCLUSIONS: These data suggest that urinary LAP3 may be a potential biomarker of arsenic exposure, which warrants further study.
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Poluentes Atmosféricos/urina , Arsênio/urina , Creatinina/urina , Exposição Ambiental , Leucil Aminopeptidase/urina , Adulto , Idoso , Idoso de 80 Anos ou mais , Cobre , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mineração , Fumar/urinaRESUMO
BACKGROUND: In general, the serum metabolome reflects the patient's body response to both disease state and implemented treatment. Though serum-derived exosomes are an emerging type of liquid biopsy, the metabolite content of these vesicles remains under researched. The aim of this pilot study was to compare the metabolite profiles of the whole serum and serum-derived exosomes in the context of differences between cancer patients and healthy controls as well as patients' response to radiotherapy (RT). METHODS: Serum samples were collected from 10 healthy volunteers and 10 patients with head and neck cancer before and after RT. Metabolites extracted from serum and exosomes were analyzed by the gas chromatography-mass spectrometry (GC-MS). RESULTS: An untargeted GC-MS-based approach identified 182 and 46 metabolites in serum and exosomes, respectively. Metabolites that differentiated cancer and control samples, either serum or exosomes, were associated with energy metabolism. Serum metabolites affected by RT were associated with the metabolism of amino acids, sugars, lipids, and nucleotides. CONCLUSIONS: cancer-related features of energy metabolism could be detected in both types of specimens. On the other hand, in contrast to RT-induced changes observed in serum metabolome, this pilot study did not reveal a specific radiation-related pattern of exosome metabolites.
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PURPOSE: Sleep bruxism (SB) is characterized by repetitive phasic, tonic, or mixed masticatory muscle activity during sleep with multifactorial etiology. Previous studies have shown that the complex origin of SB can be related to the psychological features of the affected individual, consumption of caffeine and alcohol, smoking, obstructive sleep apnea, diabetes, increased body mass index, hypertension, thyroid diseases, and probable genetic vulnerability. This study aimed to investigate the inflammatory markers, hormonal disturbances, and sleepiness associated with SB, which have a potential effect on the total cardiovascular (CV) risk among relatively young and healthy patients. PATIENTS AND METHODS: A total of 74 individuals with probable SB were subjected to single-night polysomnography, followed by blood panel and 24-h urinary excretion tests. The level of daytime sleepiness was assessed in the participants using the Epworth Sleepiness Scale. RESULTS: SB was found in 78.4% of participants. The bruxism episode index (BEI) positively correlated with the concentrations of 17-hydroxycorticosteroids, C-reactive protein, and fibrinogen in the collected urine samples. A positive correlation was also found between phasic BEI and glucose concentration 2 h after the consumption of glucose solution. Sleep bruxers showed significantly increased sleepiness compared to nonbruxers (p = 0.02). The scores on sleepiness were positively correlated with mixed BEI, minimal oxygen saturation, and mean heart rate. CONCLUSION: The results of this study revealed that participants with SB had metabolic and hormonal disturbances, probably due to stress and sympathetic activity. Moreover, it was found that young sleep bruxers potentially have a high CV risk due to the increased level of inflammatory and stress markers.
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Exosomes and other classes of extracellular vesicles (EVs) have gained interest due to their role in cell-to-cell communication. Knowledge of the molecular content of EVs may provide important information on features of parental cells and mechanisms of cross-talk between cells. To study functions of EVs it is essential to know their composition, that includes proteins, nucleic acids, and other classes biomolecules. The metabolome, set of molecules the most directly related to the cell phenotype, is the least researched component of EVs. However, the metabolome of EVs circulating in human blood and other bio-fluids is of particular interest because of its potential diagnostic value in cancer and other health conditions. On the other hand, the metabolome of EVs released to culture media in controlled conditions in vitro could shed light on important aspects of communication between cells in model systems. This paper summarizes the most common approaches implemented in EV metabolomics and integrates currently available data on the composition of the metabolome of EVs obtained in different models with particular focus on human body fluids and cancer cells.
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Exossomos/metabolismo , Metaboloma , Metabolômica , Líquidos Corporais/metabolismo , Comunicação Celular , Vesículas Extracelulares/metabolismo , Humanos , Metabolismo dos Lipídeos , Biópsia Líquida/métodos , Espectrometria de Massas/métodos , Metabolômica/métodos , Neoplasias/metabolismoRESUMO
Exosomes and other extracellular vesicles are key players in cell-to-cell communication, and it has been proposed that they are involved in different aspects of the response to ionizing radiation, including transmitting the radiation-induced bystander effect and mediating radioresistance. The functional role of exosomes depends on their molecular cargo, including proteome content. Here we aimed to establish the proteome profile of exosomes released in vitro by irradiated UM-SCC6 cells derived from human head-and-neck cancer and to identify processes associated with radiation-affected proteins. Exosomes and other small extracellular vesicles were purified by size-exclusion chromatography from cell culture media collected 24 h after irradiation of cells with a single 2, 4 or 8 Gy dose, and then proteins were identified using a shotgun LC-MS/MS approach. Exosome-specific proteins encoded by 1217 unique genes were identified. There were 472 proteins whose abundance in exosomes was significantly affected by radiation (at any dose), including 425 upregulated and 47 downregulated species. The largest group of proteins affected by radiation (369 species) included those with increased abundance at all radiation doses (≥2 Gy). Several gene ontology terms were associated with radiation-affected exosome proteins. Among overrepresented processes were those involved in the response to radiation, the metabolism of radical oxygen species, DNA repair, chromatin packaging, and protein folding. Hence, the protein content of exosomes released by irradiated cells indicates their actual role in mediating the response to ionizing radiation.
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Vesículas Extracelulares/metabolismo , Neoplasias de Cabeça e Pescoço/metabolismo , Proteoma/metabolismo , Radiação Ionizante , Linhagem Celular Tumoral , Regulação para Baixo/efeitos da radiação , Exossomos/metabolismo , Vesículas Extracelulares/efeitos da radiação , Vesículas Extracelulares/ultraestrutura , Ontologia Genética , Humanos , Proteínas de Neoplasias/metabolismo , Regulação para Cima/efeitos da radiaçãoRESUMO
Exosomes, the smallest subset of extracellular vesicles (EVs), have recently attracted much attention in the scientific community. Their involvement in intercellular communication and molecular reprogramming of different cell types created a demand for a stringent characterization of the proteome which exosomes carry and deliver to recipient cells. Mass spectrometry (MS) has been extensively used for exosome protein profiling. Unfortunately, no standards have been established for exosome isolation and their preparation for MS, leading to accumulation of artefactual data. These include the presence of high-abundance exosome-contaminating serum proteins in culture media which mask low-abundance exosome-specific components, isolation methods that fail to yield "pure" vesicles or variability in protein solubilization protocols. There is an unmet need for the development of standards for exosome generation, harvesting, and isolation from cellular supernatants and for optimization of protein extraction methods before proteomics analysis by MS. In this communication, we illustrate the existing problems in this field and provide a set of recommendations that are expected to harmonize exosome processing for MS and provide the faithful picture of the proteomes carried by exosomes. The recommended workflow for effective and specific identification of proteins in exosomes released by the low number of cells involves culturing cells in medium with a reduced concentration of exosome-depleted serum, purification of exosomes by size-exclusion chromatography, a combination of different protein extraction method and removal of serum-derived proteins from the final dataset using an appropriate sample of cell-unexposed medium as a control. Application of this method allowed detection of >250 vesicle-specific proteins in exosomes from 10 mL of culture medium.
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Técnicas de Cultura de Células , Meios de Cultura , Exossomos , Proteômica/métodos , Linhagem Celular Tumoral , Sobrevivência Celular , Cromatografia em Gel , Humanos , Espectrometria de Massas , Proteínas/isolamento & purificação , Fluxo de TrabalhoRESUMO
BACKGROUND: Linseed oil has cardio-protective effects. However, its antihypertensive action has not yet been well characterised. AIM: The primary purpose of the study was to evaluate the effect of short-term dietary supplementation with linseed oil on blood pressure (BP) and lipid metabolism in patients with mild hypercholesterolaemia. The secondary aim was to assess the effect of linseed oil on nitric oxide pathway and selected serum trace metals. METHODS: 150 volunteers: 43 men (49.9 ± 11.5 years) and 107 women (53.2 ± 10.3 years), diagnosed with mild hyper-cholesterolaemia, were assessed prospectively for BP and lipid levels, before and after lipid-lowering diet plus linseed oil supplementation at a dose of 15 mL daily for four weeks (study groups) or four-weekly lipid-lowering diet (control group). Multivariate logistic regression analysis was used to determine the effect of linseed oil on BP after adjustment for age, sex, height, body weight, body mass index, smoking status, and alcohol consumption. RESULTS: Supplementation with linseed oil significantly decreased low-density lipoprotein (LDL)- and non-high-density lipo-protein (HDL) cholesterol, and increased HDL- and HDL3- cholesterol levels. Additionally, linseed oil decreased diastolic BP in men (95% confidence interval [CI]: -6.0 to -1.1, p < 0.006), whereas in women linseed oil reduced (p < 0.001) systolic BP (-3.6 mmHg; 95% CI: -5.8 to -1.5) as well as diastolic BP (-4 mmHg; 95% CI: -5.8 to -2.1). Women with higher BP displayed an increase in serum L-arginine level (p < 0.01). In the logistic regression model oil consumption was associated with a decrease in mean BP (adjusted odds ratio 3.85; 95% CI 1.32-11.33). CONCLUSIONS: Our findings confirm the benefit of short-term linseed oil use in mild hypercholesterolaemia, particularly in patients with increased blood pressure.
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HDL-Colesterol/sangue , Hipercolesterolemia/tratamento farmacológico , Hipertensão/tratamento farmacológico , Óleo de Semente do Linho/farmacologia , Adulto , HDL-Colesterol/efeitos dos fármacos , LDL-Colesterol/sangue , LDL-Colesterol/efeitos dos fármacos , Feminino , Humanos , Hipercolesterolemia/sangue , Óleo de Semente do Linho/uso terapêutico , Masculino , Pessoa de Meia-IdadeRESUMO
INTRODUCTION: The distinction of papillary thyroid carcinomas from benign thyroid lesions has important implication for clinical man-agement. Classification based on histopathological features can be supported by molecular biomarkers, including lipidomic signatures, identified with the use of high-throughput mass spectrometry techniques. Formalin fixation is a standard procedure for stabilization and preservation of tissue samples, therefore this type of samples constitute highly valuable source of clinical material for retrospective molecular studies. In this study we used mass spectrometry imaging to detect lipids discriminating papillary cancer from not cancerous thyroid directly in formalin-fixed tissue sections. MATERIAL AND METHODS: For this purpose imaging and profiling of lipids present in non-malignant and cancerous thyroid tissue specimens were conducted. High resolution MALDI-Q-Ion Mobility-TOF-MS technique was used for lipidomic analysis of formalin fixed thyroid tissue samples. Lipids were identified by the comparison of the exact molecular masses and fragmentation pathways of the protonated molecule ions, recorded during the MS/MS experiments, with LIPID MAPS database. RESULTS: Several phosphatidylcholines (32:0, 32:1, 34:1 and 36:3), sphingomyelins (34:1 and 36:1) and phosphatidic acids (36:2 and 36:3) were detected and their abundances were significantly higher in cancerous tissue compared to non-cancerous tissue. The same lipid species were detected in formalin-fixed as in fresh-frozen tissue, but [M + Na]+ ions were the most abundant in formalin fixed whereas [M + K]+ ions were predominant in fresh tissue. CONCLUSIONS: Our results prove the viability of MALDI-MSI for analysis of lipid distribution directly in formalin-fixed tissue, and the potential for their use in the classification of thyroid diseases.
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Carcinoma Papilar/diagnóstico , Lipídeos/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Neoplasias da Glândula Tireoide/diagnóstico , Carcinoma Papilar/química , Carcinoma Papilar/diagnóstico por imagem , Formaldeído/química , Ensaios de Triagem em Larga Escala/métodos , Humanos , Imagem Molecular/métodos , Câncer Papilífero da Tireoide , Neoplasias da Glândula Tireoide/química , Neoplasias da Glândula Tireoide/diagnóstico por imagem , Fixação de TecidosRESUMO
Proteomics profiling of tissue specimens representative for major types of thyroid cancers: papillary (classical and follicular variant), follicular, anaplastic and medullary, as well as benign follicular adenoma, was performed using shotgun LC-MS/MS approaches. A combination of Orbitrap and MALDI-TOF approach allowed to identify protein products of 3700 unique genes and revealed large differences between medullary, anaplastic and epithelium-derived differentiated cancers (papillary and follicular). Proteins characteristic for medullary and anaplastic cancers included factors associated with neuroendocrine functions and factors typically associated with advanced malignancies, respectively. Proteomes of different types of epithelium-derived differentiated cancers and follicular adenoma were compared using multi-enzyme LC-MS/MS approach, which revealed products of 4800 unique genes. A comparable overall similarity of follicular cancers to both variants of papillary cancers was found. Moreover, follicular adenoma showed higher overall similarity to follicular cancer than to either variant of papillary cancer. Proteins discriminating differentiated thyroid neoplasms included factors associated with lipid and hormone metabolism, regulation of gene expression and maintenance of DNA structure. Importantly, proteome data matched several features of transcriptome and metabolome profiles of thyroid cancers contributing to systems biology of this malignancy.