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Host Defense Peptides (HDPs) have, in previous studies, been demonstrating antimicrobial, anti-inflammatory, and immunomodulatory capacity, important factors in the repair process. Knowing these characteristics, this article aims to evaluate the potential of HDPs IDR1018 and DJK-6 associated with MTA extract in the repair process of human pulp cells. Antibacterial activity of HDPs, MTA and HDPs combined with MTA in Streptococcus mutans planktonic bacteria and antibiofilm activity was evaluated. Cell toxicity was assayed with MTT and cell morphology was observed by scanning electron microscopy (SEM). Proliferation and migration of pulp cells were evaluated by trypan blue and wound healing assay. Inflammatory and mineralization related genes were evaluated by qPCR (IL-6, TNFRSF, DSPP, TGF-ß). Alkaline phosphatase, phosphate quantification and alizarin red staining were also verified. The assays were performed in technical and biological triplicate (n = 9). Results were submitted for the calculation of the mean and standard deviation. Then, normality verification by Kolmogorov Smirnov test, analyzing one-way ANOVA. Analyses were considered at a 95% significance level, with a p-value < 0.05. Our study demonstrated that HDPs combined with MTA were able to reduce biofilms performed in 24 h and biofilm performed over 7 days S. mutans biofilm (p < 0.05). IDR1018 and MTA, as well as their combination, down-regulated IL-6 expression (p < 0.05). Tested materials were not cytotoxic to pulp cells. IDR1018 induced high cell proliferation and combined with MTA induced high cellular migration rates in 48 h (p < 0.05). Furthermore, the combination of IDR1018 and MTA also induced high expression levels of DSPP, ALP activity, and the production of calcification nodules. So, IDR-1018 and its combination with MTA could assist in pulp-dentine complex repair process in vitro.
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Calcinosis , Pulpa Dental , Humanos , Interleucina-6 , Péptidos Catiónicos Antimicrobianos , Fosfatasa Alcalina , Análisis de VarianzaRESUMEN
Lower SIRT1 and insulin resistance are associated with accelerated telomere shortening. This study investigated whether the lifestyle of master athletes can attenuate these age-related changes and thereby slow aging. We compared insulin, SIRT1, and telomere length in highly trained male master athletes (n=52; aged 49.9±7.2 yrs) and age-matched non-athletes (n=19; aged 47.3±8.9 yrs). This is a cross-sectional study, in which all data were collected in one visit. Overnight fasted SIRT1 and insulin levels in whole blood were assessed using commercial kits. Relative telomere length was determined in leukocytes through qPCR analyses. Master athletes had higher SIRT1, lower insulin, and longer telomere length than age-matched non-athletes (p<0.05 for all). Insulin was inversely associated with SIRT1 (r=-0.38; p=0.001). Telomere length correlated positively with SIRT1 (r=0.65; p=0.001), whereas telomere length and insulin were not correlated (r=0.03; p=0.87). In conclusion, master athletes have higher SIRT1, lower insulin, and longer telomeres than age-matched non-athletes. Furthermore, SIRT1 was negatively associated with insulin and positively associated with telomere length. These findings suggest that in this sample of middle-aged participants reduced insulin, increased SIRT1 activity, and attenuation of biological aging are connected.
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Atletas , Insulina/sangre , Longevidad , Sirtuina 1 , Telómero/ultraestructura , Adulto , Envejecimiento , Estudios Transversales , Humanos , Leucocitos , Masculino , Persona de Mediana Edad , Sirtuina 1/genéticaRESUMEN
ABSTRACT: Aguiar, SS, Rosa, TS, Sousa, CV, Santos, PA, Barbosa, LP, Deus, LA, Rosa, EC, Andrade, RV, and Simões, HG. Influence of body fat on oxidative stress and telomere length of master athletes. J Strength Cond Res 35(6): 1693-1699, 2021-The present investigation analyzed the role of body fat and training history on biological aging of master athletes by comparing and verifying the relationships between markers of adiposity, oxidative balance, and telomere length (TL) in middle-aged runners and untrained individuals. Master athletes (sprinters and endurance runners, n = 21; 51.62 ± 8.19 years) and untrained age-matched controls (n = 11; 45.41 ± 10.34 years) had blood samples collected for biochemical and biomolecular analyzes. Pro-oxidant and antioxidant measures as well as DNA extraction were performed using commercial kits. Relative TL (T/S) was determined in leukocytes through quantitative polymerase chain reaction analyses. Master athletes had lower body fat and longer TL than untrained controls (body fat: 12.21 ± 4.14% vs. 26.03 ± 4.29%; TL: 1.10 ± 0.84 vs. 0.56 ± 0.56 T/S; p < 0.05). Furthermore, master athletes also showed a better oxidative balance than untrained controls (p < 0.05). A negative correlation was observed between TL and body fat (r = -0.471; p = 0.007), and conicity index (r = -0.407; p = 0.021), catalase activity (r = -0.569; p = 0.001), and CAT/TBARS ratio (r = -0.463; p = 0.008) for the whole sample. In conclusion, master athletes have longer TL, better oxidative profile, and lower body fat than untrained individuals. Moreover, for this middle-aged sample, body fat was inversely correlated with both TL and markers of oxidative balance, demonstrating the key role of adiposity in biological aging.
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Atletas , Telómero , Tejido Adiposo , Envejecimiento , Humanos , Persona de Mediana Edad , Estrés Oxidativo , Telómero/genéticaRESUMEN
This study aimed to verify the association between autonomic cardiac function (CAF) and the integration of caloric expenditure by physical activity (PA) intensity, sedentary behavior (SB), and sleep quality (PSQI) in active young men. Thirty-five subjects were included, and caloric expenditure in moderate-to-vigorous and light-intensity PA, SB, and PSQI were assessed using questionnaires. Heart rate variability (HRV) was recorded for short periods of time in the supine and orthostatic positions. Multiple linear regression was realized unadjusted and adjusted for covariables, such as age, body mass index, and fat mass. No adjusted analysis indicated that, in the supine position, there were negative associations between the SB and the TP, HF, and NorHF indices, and positive associations between SB and NorLF and LF/HF. In the orthostatic position, an interaction between SB and NorLF was found. Significance of proportion with the TP, HF, and LF/HF indices was confirmed. When adjusted, for the supine position, negative interactions were documented between SB and the TP as well as the HF indices, and between PSQI and the LF/HF index, with interference under the HF and LF/HF indices. Finally, our findings indicate that the proposed approach interacts with CAF, and SB is significantly related to CAF in young active men.
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Ejercicio Físico , Corazón/fisiología , Estilo de Vida , Conducta Sedentaria , Adolescente , Adulto , Sistema Nervioso Autónomo/fisiología , Frecuencia Cardíaca , Humanos , Modelos Lineales , Masculino , Adulto JovenAsunto(s)
Cryptosporidium/genética , Monitoreo del Ambiente/métodos , Ríos/microbiología , Brasil , Genotipo , Humanos , Población RuralRESUMEN
Leukocyte telomere length (LTL), a biological marker of aging that is associated with age-related diseases, is longer in master endurance runners (ER) than age-matched controls, but the underlying mechanisms are poorly investigated. The LTL, nitric oxide (NO), and redox balance of ER master runners were analyzed and compared to untrained middle-aged and young adults. We hypothesized that NO and redox balance at baseline would be related to longer LTL in ER athletes. Participants (nâ¯=â¯38) were long-term ER runners (nâ¯=â¯10; 51.6⯱â¯5.2â¯yrs.; 28.4⯱â¯9.4â¯yrs. of experience) and untrained age-matched (nâ¯=â¯17; 46.6⯱â¯7.1â¯yrs) and young controls (nâ¯=â¯11; 21.8⯱â¯4.0â¯yrs). Volunteers were assessed for anamnesis, anthropometrics, and blood sampling. Measurements of pro-and anti-oxidant status and DNA extraction were performed using commercial kits. Relative LTL was determined with qPCR analyses (T/S). While the middle-aged controls had shorter LTL than the young group, no difference was observed between ER athletes and young participants. A large effect size between the LTL of ER athletes and middle-aged controls (dâ¯=â¯0.85) was also observed. The ER athletes and untrained young group had better redox balance according to antioxidant/pro-oxidant ratios compared to middle-aged untrained participants, which also had lower values for redox parameters (TEAC/TBARS, SOD/TBARS, and CAT/TBARS; all pâ¯<â¯0.05). Furthermore, the NO level of ER athletes (175.2⯱â¯31.9⯵M) was higher (pâ¯<â¯0.05) than middle-aged controls (67.2⯱â¯23.3⯵M) and young participants (129.2⯱â¯17.3⯵M), with a significant correlation with LTL (râ¯=â¯0.766; pâ¯=â¯0.02). In conclusion, ER runners have longer LTL than age-matched controls, which in turn may be related to better NO bioavailability and redox balance status.
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Envejecimiento/fisiología , Óxido Nítrico/fisiología , Carrera/fisiología , Homeostasis del Telómero/fisiología , Adulto , Anciano , Envejecimiento/genética , Atletas , Composición Corporal/fisiología , Estudios de Casos y Controles , Humanos , Leucocitos/metabolismo , Peroxidación de Lípido/fisiología , Masculino , Persona de Mediana Edad , Oxidación-Reducción , Estrés Oxidativo/fisiología , Resistencia Física/fisiologíaRESUMEN
Accumulation of connective tissue, particularly extracellular matrix (ECM) proteins, has been observed in skeletal muscles with advancing age. Resistance training (RT) has been widely recommended to attenuate age-induced sarcopenia, even though its effects on the components that control ECM turnover in skeletal muscles remain to be elucidated. Thus, the aim of this study was to determine the effects of RT on connective tissue content and gene expression of key components of ECM in the skeletal muscles of aged rats. Young (3 mo.) and older (21 mo.) adult male Wistar rats were submitted to a RT protocol (ladder climbing with 65, 85, 95, and 100% load), 3 times a week for 12 weeks. Forty-eight hours post-training, the soleus (SOL) and gastrocnemius (GAS) muscles were dissected for histological and mRNA analysis. RT mitigated the age-associated increase of connective tissue content in both muscles, even though mRNA levels of COL-1 and-3 were elevated in older trained rats. Overall, RT significantly elevated the gene expression of key components of connective tissue deposition (TGFß and CTGF; MMP-2 and-9; TIMP-1 and-2) in the GAS and SOL muscles of older rats. In conclusion, RT blunted the age-induced accumulation of connective tissue concomitant to the upregulation of genes related to synthesis and degradation of the ECM network in the SOL and GAS muscles of older rats. Although our findings indicate that RT plays a crucial role reducing connective tissue accumulation in aged hindlimb muscles, key components of ECM turnover were paradoxically elevated. The phenotypic responses induced by RT were not accompanied by the gene expression of those components related to ECM turnover.
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Rare genetic diseases collectively impact a significant portion of the world's population. For many diseases there is limited information available, and clinicians can find difficulty in differentiating between clinically similar conditions. This leads to problems in genetic counseling and patient treatment. The biomedical market is affected because pharmaceutical and biotechnology industries do not see advantages in addressing rare disease treatments, or because the cost of the treatments is too high. By contrast, technological advances including DNA sequencing and analysis, together with computer-aided tools and online resources, are allowing a more thorough understanding of rare disorders. Here, we discuss how the collection of various types of information together with the use of new technologies is facilitating diagnosis and, consequently, treatment of rare diseases.
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Enfermedades Genéticas Congénitas , Enfermedades Raras , Animales , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/tratamiento farmacológico , Enfermedades Genéticas Congénitas/genética , Educación en Salud , Personal de Salud , Humanos , Participación del Paciente , Enfermedades Raras/diagnóstico , Enfermedades Raras/tratamiento farmacológico , Enfermedades Raras/genéticaRESUMEN
The fungus Sclerotinia sclerotiorum (Lib.) de Bary, one of the most important plant pathogens, causes white mold on a wide range of crops. Crop yield can be dramatically decreased due to this disease, depending on the plant cultivar and environmental conditions. In this study, a suppression subtractive hybridization cDNA library approach was used for the identification of pathogen and plant genes that were differentially expressed during infection of the susceptible cultivar BRS Pérola of Phaseolus vulgaris L. A total of 979 unigenes (430 contigs and 549 singletons) were obtained and classified according to their functional categories. The transcriptional profile of 11 fungal genes related to pathogenicity and virulence were evaluated by reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR). Additionally, the temporal expression profile obtained by RT-qPCR was evaluated for the following categories of plant defense-related genes: pathogenesis-related genes (PvPR1, PvPR2, and PvPR3), phenylpropanoid pathway genes (PvIsof, PvFPS1, and 4CL), and genes involved in defense and stress-related categories (PvLox, PvHiprp, PvGST, PvPod, and PvDox). Data obtained in this study provide a starting point for achieving a better understanding of the pathosystem S. sclerotiorum-P. vulgaris.
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Genetic disorders of the skeleton comprise a large group of more than 450 clinically distinct and genetically heterogeneous diseases associated with mutations in more than 300 genes. Achieving a definitive diagnosis is complicated due to the genetic heterogeneity of these disorders, their individual rarity and their diverse radiographic presentations. We used targeted exome sequencing and designed a 1.4 Mb panel for simultaneous testing of more than 4,800 exons in 309 genes involved in skeletal disorders. DNA from 69 individuals from 66 families with a known or suspected clinical diagnosis of a skeletal disorder was analyzed. Of 36 cases with a specific clinical hypothesis with a known genetic basis, mutations were identified for eight cases (22%). Of 20 cases with a suspected skeletal disorder but without a specific diagnosis, four causative mutations were identified. Also included were 11 cases with a specific skeletal disorder but for which there was at the time no known associated gene. For these cases, one mutation was identified in a known skeletal disease genes, and re-evaluation of the clinical phenotype in this case changed the diagnoses from osteodysplasia syndrome to Apert syndrome. These results suggest that the NGS panel provides a fast, accurate and cost-effective molecular diagnostic tool for identifying mutations in a highly genetically heterogeneous set of disorders such as genetic skeletal disorders. The data also stress the importance of a thorough clinical evaluation before DNA sequencing. The strategy should be applicable to other groups of disorders in which the molecular basis is largely known.
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Enfermedades del Desarrollo Óseo/genética , Exoma/genética , Técnicas de Diagnóstico Molecular/métodos , Análisis de Secuencia de ADN/métodos , Enfermedades del Desarrollo Óseo/diagnóstico , Estudios de Cohortes , Análisis Mutacional de ADN/métodos , Enfermedades Genéticas Congénitas/diagnóstico , Enfermedades Genéticas Congénitas/genética , Predisposición Genética a la Enfermedad , Pruebas Genéticas/métodos , Humanos , Polimorfismo de Nucleótido SimpleRESUMEN
BACKGROUND: Regular exercises are commonly described as an important factor in health improvement, being directly related to contractile force development in cardiac cells.In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG's), with low, moderate and high intensity of exercises.In order to evaluate the links between swimming exercise intensity and cardiac adaptation by using high molecular mass proteomics, isogenic Wistar rats were divided into four groups: one control (CG) and three training groups (TG's), with low, moderate and high intensity of exercises. RESULTS: Findings here reported demonstrated clear morphologic alterations, significant cellular injury and increased energy supplies at high exercise intensities. α-MyHC, as well proteins associated with mitochondrial oxidative metabolism were shown to be improved. α-MyHC expression increase 1.2 fold in high intensity training group when compared with control group. α-MyHC was also evaluated by real-time PCR showing a clear expression correlation with protein synthesis data increase in 8.48 fold in high intensity training group. Other myofibrillar protein, troponin , appear only in high intensity group, corroborating the cellular injury data. High molecular masses proteins such as MRS2 and NADH dehydrogenase, involved in metabolic pathways also demonstrate increase expression, respectily 1.5 and 1.3 fold, in response to high intensity exercise. CONCLUSIONS: High intensity exercise demonstrated an increase expression in some high molecular masses myofibrilar proteins, α-MyHC and troponin. Furthermore this intensity also lead a significant increase of other high molecular masses proteins such as MRS2 and NADH dehydrogenase in comparison to low and moderate intensities. However, high intensity exercise also represented a significant degree of cellular injury, when compared with the individuals submitted to low and moderate intensities.
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Corazón/fisiopatología , Miocardio/metabolismo , Condicionamiento Físico Animal/fisiología , Proteoma/metabolismo , Natación/fisiología , Animales , Proteínas de Transporte de Catión/metabolismo , Metabolismo Energético/fisiología , Ventrículos Cardíacos/metabolismo , Ventrículos Cardíacos/fisiopatología , Masculino , Fase I de la Desintoxicación Metabólica/fisiología , Mitocondrias/metabolismo , Mitocondrias/fisiología , Proteínas Mitocondriales/metabolismo , NADH Deshidrogenasa/metabolismo , Proteómica/métodos , Ratas , Ratas Wistar , Troponina/metabolismoRESUMEN
OBJECTIVE(S): To investigate the prevalence of microsatellite instability (MSI) in endometrial polyps and to evaluate whether there are clinical and histopathological parameters associated with this kind of instability. STUDY DESIGN: Between September 2008 and April 2009, endometrial polyps were collected from 109 patients. MSI was evaluated using the NCI recommended markers BAT25, BAT26, D2S123, D5S346 and D17S250. Histopathological analysis was performed, and clinical information was obtained from patients' records. RESULT(S): MSI low was detected in 6.4% of the validated samples (7/109). Of the seven MSI that were detected, six were positive for instability at D17S250 and one at D5S346. There were no significant differences between polyps with or without MSI with regard to age, BMI, menarche, parity, miscarriage or menopause; however, MSI was more frequent in polyps with simple hyperplasia without atypia (3/20; 15%). Furthermore, patients with multiple polyps had a marginally but statistically insignificant increase in the frequency of MSI (p<0.07). CONCLUSION(S): This is the first prospective study of MSI in endometrial polyps using hysteroscopically obtained samples. In a population of 109 patients, MSI was infrequent in endometrial polyps. Although MSI appears to be more frequent in multiple polyps and polyps with simple hyperplasia without atypia, this was not statistically significant.
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Neoplasias Endometriales/genética , Inestabilidad de Microsatélites , Pólipos/genética , Enfermedades Uterinas/genética , Neoplasias Endometriales/patología , Femenino , Humanos , Persona de Mediana Edad , Pólipos/patología , Enfermedades Uterinas/patologíaRESUMEN
BACKGROUND: Paracoccidioides brasiliensis is a dimorphic fungus that causes the most prevalent systemic mycosis in Latin America. The response to heat shock is involved in pathogenesis, as this pathogen switches from mycelium to yeast forms in a temperature dependent fashion that is essential to establish infection. HSP90 is a molecular chaperone that helps in the folding and stabilization of selected polypeptides. HSP90 family members have been shown to present important roles in fungi, especially in the pathogenic species, as an immunodominant antigen and also as a potential antifungal therapeutic target. RESULTS: In this work, we decided to further study the Pbhsp90 gene, its expression and role in cell viability because it plays important roles in fungal physiology and pathogenesis. Thus, we have sequenced a Pbhsp90 cDNA and shown that this gene is present on the genome as a single copy. We have also confirmed its preferential expression in the yeast phase and its overexpression during dimorphic transition and oxidative stress. Treatment of the yeast with the specific HSP90 inhibitors geldanamycin and radicicol inhibited growth at 2 and 10 microM, respectively. CONCLUSION: The data confirm that the Pbhsp90 gene encodes a morphologically regulated and stress-responsive protein whose function is essential to cell viability of this pathogen. This work also enforces the potential of HSP90 as a target for antifungal therapies, since the use of HSP90 inhibitors is lethal to the P. brasiliensis yeast cells in a dose-responsive manner.
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Regulación Fúngica de la Expresión Génica , Proteínas HSP90 de Choque Térmico/genética , Estrés Oxidativo/genética , Paracoccidioides/fisiología , Secuencia de Aminoácidos , Benzoquinonas/farmacología , Supervivencia Celular , Dosificación de Gen , Perfilación de la Expresión Génica , Proteínas HSP90 de Choque Térmico/química , Lactamas Macrocíclicas/farmacología , Macrólidos/farmacología , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Paracoccidioides/efectos de los fármacos , Paracoccidioides/genética , Paracoccidioides/metabolismo , Alineación de SecuenciaRESUMEN
Paracoccidioides brasiliensis is a dimorphic fungus that infects humans and establishes infection in the yeast form. We are interested in the mechanisms this fungus uses to evade the human immune system, and in its survival strategies within infected host cells. Reactive oxygen species play an important role in host defence, but are detoxified by pathogen-derived antioxidant enzymes to prevent oxidative damage. The transcriptional and post-transcriptional regulation of P. brasiliensis catalase and cytochrome-c peroxidase (CCP) antioxidant enzymes upon culture treatment with hydrogen peroxide (H(2)O(2)) is described. High H(2)O(2) concentrations (up to 100 mm) still permitted 70-100% survival of exponential and stationary phase yeast cells, though stationary phase cells were consistently more resistant. P. brasiliensis has both cytosolic and peroxisomal catalase isoenzymes and a single cytochrome-c peroxidase. High-dose treatments with H(2)O(2) led to an early increase in total catalase and CCP enzymatic activities, indicative of post-transcriptional regulation. The expression levels of the catalase genes increased three to fourfold when the cells were treated with 50 mm H(2)O(2) for 40 or 50 min. Lipid peroxidation, as assessed by the thiobarbituric acid method, was relatively low upon treatment with H(2)O(2), which was consistent with our results demonstrating that P. brasiliensis has a powerful antioxidant defence system enabling it to survive H(2)O(2)-mediated stress.
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Catalasa/metabolismo , Citocromo-c Peroxidasa/metabolismo , Estrés Oxidativo , Paracoccidioides/efectos de los fármacos , Paracoccidioides/enzimología , Paracoccidioidomicosis/metabolismo , Especies Reactivas de Oxígeno/farmacología , Antioxidantes/metabolismo , Catalasa/genética , Citocromo-c Peroxidasa/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Humanos , Paracoccidioides/crecimiento & desarrollo , Paracoccidioides/metabolismo , Paracoccidioidomicosis/microbiologíaRESUMEN
Paracoccidioides brasiliensis, a thermal dimorphic fungus, is the etiologic agent of the most common systemic mycosis in Latin America, paracoccidioidomycosis. The yeast form of P. brasiliensis acts as a facultative intracellular pathogen being able to survive and replicate within the phagosome of nonactivated murine and human macrophages. This ability has been proposed to be crucial to the development of disease. Thus, P. brasiliensis may have evolved mechanisms that counteract the constraints imposed by phagocytic cells. By using cDNA microarray technology we evaluated the early transcriptional response of this fungus to the environment of peritoneal murine macrophages in order to shed light on the mechanisms used by P. brasiliensis to survive within phagocytic cells. Of the 1152 genes analyzed, we identified 152 genes that were differentially transcribed. Intracellularly expressed genes were primarily associated with glucose and amino acid limitation, cell wall construction, and oxidative stress. For the first time, a comprehensive gene expression tool is used for the expression analysis of P. brasiliensis genes when interacting with macrophages. Overall, our data show a transcriptional plasticity of P. brasiliensis in response to the harsh environment of macrophages which may lead to adaptation and consequent survival of this pathogen.
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Perfilación de la Expresión Génica , Macrófagos/microbiología , Paracoccidioides/genética , Paracoccidioides/metabolismo , Transcripción Genética , Animales , ADN de Hongos/análisis , Regulación Fúngica de la Expresión Génica , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Análisis por MicromatricesRESUMEN
BACKGROUND: Mycelium-to-yeast transition in the human host is essential for pathogenicity by the fungus Paracoccidioides brasiliensis and both cell types are therefore critical to the establishment of paracoccidioidomycosis (PCM), a systemic mycosis endemic to Latin America. The infected population is of about 10 million individuals, 2% of whom will eventually develop the disease. Previously, transcriptome analysis of mycelium and yeast cells resulted in the assembly of 6,022 sequence groups. Gene expression analysis, using both in silico EST subtraction and cDNA microarray, revealed genes that were differential to yeast or mycelium, and we discussed those involved in sugar metabolism. To advance our understanding of molecular mechanisms of dimorphic transition, we performed an extended analysis of gene expression profiles using the methods mentioned above. RESULTS: In this work, continuous data mining revealed 66 new differentially expressed sequences that were MIPS(Munich Information Center for Protein Sequences)-categorised according to the cellular process in which they are presumably involved. Two well represented classes were chosen for further analysis: (i) control of cell organisation - cell wall, membrane and cytoskeleton, whose representatives were hex (encoding for a hexagonal peroxisome protein), bgl (encoding for a 1,3-beta-glucosidase) in mycelium cells; and ags (an alpha-1,3-glucan synthase), cda (a chitin deacetylase) and vrp (a verprolin) in yeast cells; (ii) ion metabolism and transport - two genes putatively implicated in ion transport were confirmed to be highly expressed in mycelium cells - isc and ktp, respectively an iron-sulphur cluster-like protein and a cation transporter; and a putative P-type cation pump (pct) in yeast. Also, several enzymes from the cysteine de novo biosynthesis pathway were shown to be up regulated in the yeast form, including ATP sulphurylase, APS kinase and also PAPS reductase. CONCLUSION: Taken together, these data show that several genes involved in cell organisation and ion metabolism/transport are expressed differentially along dimorphic transition. Hyper expression in yeast of the enzymes of sulphur metabolism reinforced that this metabolic pathway could be important for this process. Understanding these changes by functional analysis of such genes may lead to a better understanding of the infective process, thus providing new targets and strategies to control PCM.
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Regulación Fúngica de la Expresión Génica/genética , Micelio/genética , Paracoccidioides/genética , Levaduras/genética , Transporte Biológico/genética , Northern Blotting/métodos , Proteínas de Transporte de Catión/genética , Pared Celular/genética , Pared Celular/metabolismo , Cisteína/biosíntesis , Citoesqueleto/genética , Citoesqueleto/metabolismo , Etiquetas de Secuencia Expresada , Proteínas Fúngicas/genética , Perfilación de la Expresión Génica/métodos , Iones/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Levaduras/citología , beta-Glucosidasa/genéticaRESUMEN
Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America. Pathogenicity is assumed to be a consequence of the cellular differentiation process that this fungus undergoes from mycelium to yeast cells during human infection. In an effort to elucidate the molecular mechanisms involved in this process a network of Brazilian laboratories carried out a transcriptome project for both cell types. This review focuses on the data analysis yielding a comprehensive view of the fungal metabolism and the molecular adaptations during dimorphism in P. brasiliensis from analysis of 6022 groups, related to expressed genes, which were generated from both mycelium and yeast phases.
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Proteínas Fúngicas/metabolismo , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Paracoccidioides/crecimiento & desarrollo , Paracoccidioidomicosis/microbiología , Etiquetas de Secuencia Expresada , Proteínas Fúngicas/genética , Humanos , Paracoccidioides/genética , Paracoccidioides/metabolismo , Paracoccidioides/patogenicidad , Transcripción GenéticaRESUMEN
Paracoccidioides brasiliensis is the causative agent of paracoccidioidomycosis, a disease that affects 10 million individuals in Latin America. This report depicts the results of the analysis of 6,022 assembled groups from mycelium and yeast phase expressed sequence tags, covering about 80% of the estimated genome of this dimorphic, thermo-regulated fungus. The data provide a comprehensive view of the fungal metabolism, including overexpressed transcripts, stage-specific genes, and also those that are up- or down-regulated as assessed by in silico electronic subtraction and cDNA microarrays. Also, a significant differential expression pattern in mycelium and yeast cells was detected, which was confirmed by Northern blot analysis, providing insights into differential metabolic adaptations. The overall transcriptome analysis provided information about sequences related to the cell cycle, stress response, drug resistance, and signal transduction pathways of the pathogen. Novel P. brasiliensis genes have been identified, probably corresponding to proteins that should be addressed as virulence factor candidates and potential new drug targets.
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Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Micelio/metabolismo , Paracoccidioides/metabolismo , Transcripción Genética , Northern Blotting , ADN Complementario/metabolismo , Regulación hacia Abajo , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Internet , Modelos Biológicos , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Paracoccidioides/genética , ARN Mensajero/metabolismo , Análisis de Secuencia de ADN , Transducción de Señal , Regulación hacia ArribaRESUMEN
A gene encoding the entire highly expressed protein previously identified in the proteome of Paracoccidioides brasiliensis yeast cells as PbY20 has been isolated. The pby20 sequence reveals an open reading frame of 1364bp and a deduced amino acid sequence of 203 residues, which shows high identity to benzoquinone reductase from Phanerochaete chrysosporium (72.0%), Saccharomyces cerevisiae Ycp4 (65%), and Schizosaccharomyces pombe p25 (59%), and to allergens from Alternaria alternata Alt a7 (70%) and from Cladosporium herbarum, Cla h5 (68%). Low levels of the pby20 transcript in the mycelium and highly induced ones in infective yeast cells during the transition of this dimorphic fungus indicate transcriptional control of its expression. PbY20 was immunologically detected only in yeast cell extract, suggesting an important role in cell differentiation or even in the maintenance of the yeast form. Immunoelectron microscopy showed that PbY20 is found inside large granules and vacuoles, in the nucleus, and also in the cytoplasm. Through sequence comparisons analysis and fluorescence emission assay, PbY20 was recognized as a member of the flavin mononucleotide flavodoxin-like WrbA family, which are involved in heat shock and oxidative stress in biological systems. Assuming that PbY20 belongs to this family, a similar role could be attributed to this protein.
Asunto(s)
Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Paracoccidioides/genética , Alérgenos/genética , Alternaria/genética , Secuencia de Aminoácidos , Secuencia de Bases , Núcleo Celular/química , Cladosporium/genética , Gránulos Citoplasmáticos , Vesículas Citoplasmáticas/química , ADN de Hongos/química , ADN de Hongos/aislamiento & purificación , Flavodoxina/química , Flavodoxina/genética , Regulación Fúngica de la Expresión Génica , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Oxidorreductasas/genética , Phanerochaete/genética , ARN de Hongos/análisis , ARN Mensajero/análisis , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
Paracoccidioides brasiliensis is a dimorphic and thermo-regulated fungus which is the causative agent of paracoccidioidomycosis, an endemic disease widespread in Latin America that affects 10 million individuals. Pathogenicity is assumed to be a consequence of the dimorphic transition from mycelium to yeast cells during human infection. This review shows the results of the P. brasiliensis transcriptome project which generated 6,022 assembled groups from mycelium and yeast phases. Computer analysis using the tools of bioinformatics revealed several aspects from the transcriptome of this pathogen such as: general and differential metabolism in mycelium and yeast cells; cell cycle, DNA replication, repair and recombination; RNA biogenesis apparatus; translation and protein fate machineries; cell wall; hydrolytic enzymes; proteases; GPI-anchored proteins; molecular chaperones; insights into drug resistance and transporters; oxidative stress response and virulence. The present analysis has provided a more comprehensive view of some specific features considered relevant for the understanding of basic and applied knowledge of P. brasiliensis.