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OBJECTIVES: The Italian Society of Clinical Biochemistry and Clinical Molecular Biology (SIBioC) Big Data and Artificial Intelligence (BAI) Working Group promoted a survey to frame the knowledge, skills and technological predisposition in clinical laboratories. METHODS: A questionnaire, focussing on digitization, information technology (IT) infrastructures, data accessibility, and BAI projects underway was sent to 1,351 SIBioC participants. The responses were evaluated using SurveyMonkey software and Google Sheets. RESULTS: The 227 respondents (17%) from all over Italy (47% of 484 labs), mainly biologists, laboratory physicians and managers, mostly from laboratories of public hospitals, revealed lack of hardware, software and corporate Wi-Fi, and dearth of PCs. Only 25% work daily on clouds, while 65%-including Laboratory Directors-cannot acquire health data from sources other than laboratories. Only 50% of those with access can review a clinical patient's health record, while the other access only to laboratory information. The integration of laboratory data with other health data is mostly incomplete, which limits BAI-type analysis. Many are unaware of integration platforms. Over 90% report pulling data from the Laboratory Information System, with varying degrees of autonomy. Very few have already undertaken BAI projects, frequently relying on IT partnerships. The majority consider BAI as crucial in helping professional judgements, indicating a growing interest. CONCLUSIONS: The questionnaire received relevant feedback from SIBioC participants. It highlighted the level of expertise and interest in BAI applications. None of the obstacles stands out more than the others, emphasising the need to all-around work: IT infrastructures, data warehouses, BAI analysis software acquisition, data accessibility and training.
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Macrodatos , Servicios de Laboratorio Clínico , Humanos , Inteligencia Artificial , Laboratorios Clínicos , Encuestas y Cuestionarios , LaboratoriosRESUMEN
Background As defined by ISO 15189 competence is the "demonstrated ability to apply knowledge and skills" thus, its assessment is fundamental for ensuring the quality of the total testing process in order to reduce the risk for the patient. We have developed a functional software for the measurement of professional competences in order to standardize the procedure and to collect all the data in a single platform, avoiding redundancy and dispersion. Methods Our model objectively assesses the skills, as they become measurable and comparable with appropriate standards and involves both managers and operators, to increase their active engagement. The assessment concerns everyone, but the standards to be met (numerical values) can vary according to the responsibilities. Several subjective and objective criteria are evaluated: each parameter can contribute in a variable proportion to the total skills measured according to the needs of the organization. Results The data are automatically analyzed and can be easily monitored in real time in the form of indicators, thanks to dashboards. The comparison between the skills required and those measured allows highlighting the gap useful for planning personalized training paths. Conclusions Our tool is reliable and highly adaptable to laboratories about competences to track criteria, standards and monitored indicators. The computerized management is a strategic action as it fulfills the requirements of registration, traceability, communication, data analysis and indicators development, which are the tenets of continuous improvement, and allows planning to be made on the basis of the actual training needs.
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Simulación por Computador , Personal de Laboratorio/normas , Competencia Profesional/normas , Humanos , Capacitación en ServicioRESUMEN
OBJECTIVE: Our scope is to provide methodological elements on how to manage effectively the preanalytical phase in the laboratory testing process, by objectively measuring the risk connected to the phases handled by man with respect to those managed by machines. BACKGROUND: Preanalytical errors account for most of the mistakes related to laboratory testing and can affect patient care. Hence, it is necessary to manage the risk connected to the preanalytical phase, as required by certification and accreditation bodies. The risk assessment discloses the steps at greater risk and gives indications to make decisions. METHOD: We have reviewed the state of art in the automation of the preanalytical phase, addressing needs and problems. We have used the proactive risk assessment methodology FMECA (Failure Mode, Effects, and Criticality Analysis) to identify the most critical phases in our preanalytical process and have calculated the risk associated. RESULTS: The most critical phases were the human controlled ones. In particular, the highest risk indexes were associated to manual acceptance of test orders, identification of the patients, tube labeling, and sample collection. CONCLUSION: Automation in the preanalytical phase is fundamental to replace, support, or extend the human contribution. Nevertheless each organization is different about workloads and competencies, so the most suitable management must be tailor-made in each context. APPLICATION: We present a method by which each organization is able to find its best balance between automation and human contribution in the control of the preanalytical phase.
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Automatización , Errores Diagnósticos , Informática Médica , Personal de Laboratorio Clínico , Seguridad del Paciente , Fase Preanalítica , Evaluación de Procesos, Atención de Salud , Humanos , Medición de RiesgoRESUMEN
BACKGROUND: Mutations in the cyclin-dependent kinase-like 5 gene cause a clinical variant of Rett syndrome (CDKL5-RTT). A role for the acute-phase response (APR) is emerging in typical RTT caused by methyl-CpG-binding protein 2 gene mutations (MECP2-RTT). No information is, to date, available on the inflammatory protein response in CDKL5-RTT. We evaluated, for the first time, the APR protein response in CDKL5-RTT. METHODS: Protein patterns in albumin- and IgG-depleted plasma proteome from CDKL5-RTT patients were evaluated by two-dimensional gel electrophoresis/mass spectrometry. The resulting data were related to circulating cytokines and compared to healthy controls or MECP2-RTT patients. The effects of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) were evaluated. RESULTS: CDKL5-RTT mutations resulted in a subclinical attenuated inflammation, specifically characterized by an overexpression of the complement component C3 and CD5 antigen-like, both strictly related to the inflammatory response. Cytokine dysregulation featuring a bulk increase of anti-inflammatory cytokines, predominantly IL-10, could explain the unchanged erythrocyte sedimentation rate and atypical features of inflammation in CDKL5-RTT. Omega-3 PUFAs were able to counterbalance the pro-inflammatory status. CONCLUSION: For the first time, we revealed a subclinical smouldering inflammation pattern in CDKL5-RTT consisting in the coexistence of an atypical APR coupled with a dysregulated cytokine response.
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Reacción de Fase Aguda/inmunología , Citocinas/inmunología , Síndrome de Rett/inmunología , Espasmos Infantiles/inmunología , Reacción de Fase Aguda/genética , Reacción de Fase Aguda/metabolismo , Adolescente , Proteínas Sanguíneas/inmunología , Proteínas Sanguíneas/metabolismo , Niño , Preescolar , Citocinas/sangre , Suplementos Dietéticos , Síndromes Epilépticos , Ácidos Grasos Omega-3/farmacología , Femenino , Humanos , Lactante , Proteína 2 de Unión a Metil-CpG/genética , Proteínas Serina-Treonina Quinasas/genética , Síndrome de Rett/genética , Síndrome de Rett/metabolismo , Espasmos Infantiles/genética , Espasmos Infantiles/metabolismoRESUMEN
Rett syndrome (RTT) is a rare neurodevelopmental disorder usually caused by mutations in the X-linked gene methyl-CpG-binding protein 2 (MECP2). Several Mecp2 mutant mouse lines have been developed recapitulating part of the clinical features. In particular, Mecp2-308 female heterozygous mice, bearing a truncating mutation, are a validated model of the disease. While recent data suggest a role for inflammation in RTT, little information on the inflammatory status in murine models of the disease is available. Here, we investigated the inflammatory status by proteomic 2-DE/MALDI-ToF/ToF analyses in symptomatic Mecp2-308 female mice. Ten differentially expressed proteins were evidenced in the Mecp2-308 mutated plasma proteome. In particular, 5 positive acute-phase response (APR) proteins increased (i.e., kininogen-1, alpha-fetoprotein, mannose-binding protein C, alpha-1-antitrypsin, and alpha-2-macroglobulin), and 3 negative APR reactants were decreased (i.e., serotransferrin, albumin, and apolipoprotein A1). CD5 antigen-like and vitamin D-binding protein, two proteins strictly related to inflammation, were also changed. These results indicate for the first time a persistent unresolved inflammation of unknown origin in the Mecp2-308 mouse model.
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Inflamación/inmunología , Inflamación/metabolismo , Proteína 2 de Unión a Metil-CpG/metabolismo , Síndrome de Rett/inmunología , Síndrome de Rett/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Proteína 2 de Unión a Metil-CpG/genética , Ratones , ProteómicaRESUMEN
Inflammation has been advocated as a possible common central mechanism for developmental cognitive impairment. Rett syndrome (RTT) is a devastating neurodevelopmental disorder, mainly caused by de novo loss-of-function mutations in the gene encoding MeCP2. Here, we investigated plasma acute phase response (APR) in stage II (i.e., "pseudo-autistic") RTT patients by routine haematology/clinical chemistry and proteomic 2-DE/MALDI-TOF analyses as a function of four major MECP2 gene mutation types (R306C, T158M, R168X, and large deletions). Elevated erythrocyte sedimentation rate values (median 33.0 mm/h versus 8.0 mm/h, P < 0.0001) were detectable in RTT, whereas C-reactive protein levels were unchanged (P = 0.63). The 2-DE analysis identified significant changes for a total of 17 proteins, the majority of which were categorized as APR proteins, either positive (n = 6 spots) or negative (n = 9 spots), and to a lesser extent as proteins involved in the immune system (n = 2 spots), with some proteins having overlapping functions on metabolism (n = 7 spots). The number of protein changes was proportional to the severity of the mutation. Our findings reveal for the first time the presence of a subclinical chronic inflammatory status related to the "pseudo-autistic" phase of RTT, which is related to the severity carried by the MECP2 gene mutation.
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Proteína 2 de Unión a Metil-CpG/genética , Síndrome de Rett/genética , Síndrome de Rett/inmunología , Reacción de Fase Aguda , Sedimentación Sanguínea , Proteína C-Reactiva/metabolismo , Estudios de Casos y Controles , Niño , Preescolar , Electroforesis en Gel Bidimensional , Femenino , Humanos , Inflamación/fisiopatología , Mutación , Fenotipo , Mapeo de Interacción de ProteínasRESUMEN
Sepsis remains a significant global health challenge due to its high mortality and morbidity, compounded by the difficulty of early detection given its variable clinical manifestations. The integration of machine learning (ML) into laboratory medicine for timely sepsis identification and outcome forecasting is an emerging field of interest. This comprehensive review assesses the current body of research on ML applications for sepsis within the realm of laboratory diagnostics, detailing both their strengths and shortcomings. An extensive literature search was performed by two independent investigators across PubMed and Scopus databases, employing the keywords "Sepsis," "Machine Learning," and "Laboratory" without publication date limitations, culminating in January 2023. Each selected study was meticulously evaluated for various aspects, including its design, intent (diagnostic or prognostic), clinical environment, demographics, sepsis criteria, data gathering period, and the scope and nature of features, in addition to the ML methodologies and their validation procedures. Out of 135 articles reviewed, 39 fulfilled the criteria for inclusion. Among these, the majority (30 studies) were focused on devising ML algorithms for diagnosis, fewer (8 studies) on prognosis, and one study addressed both aspects. The dissemination of these studies across an array of journals reflects the interdisciplinary engagement in the development of ML algorithms for sepsis. This analysis highlights the promising role of ML in the early diagnosis of sepsis while drawing attention to the need for uniformity in validating models and defining features, crucial steps for ensuring the reliability and practicality of ML in clinical setting.
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Sepsis , Humanos , Reproducibilidad de los Resultados , Sepsis/diagnóstico , Algoritmos , Aprendizaje Automático , Proyectos de InvestigaciónRESUMEN
The mechanism of action of omega-3 polyunsaturated fatty acids (ω-3 PUFAs) is only partially known. Prior reports suggest a partial rescue of clinical symptoms and oxidative stress (OS) alterations following ω -3 PUFAs supplementation in patients with Rett syndrome (RTT), a devastating neurodevelopmental disorder with transient autistic features, affecting almost exclusively females and mainly caused by sporadic mutations in the gene encoding the methyl CpG binding protein 2 (MeCP2) protein. Here, we tested the hypothesis that ω-3 PUFAs may modify the plasma proteome profile in typical RTT patients with MECP2 mutations and classic phenotype. A total of 24 RTT girls at different clinical stages were supplemented with ω-3 PUFAs as fish oil for 12 months and compared to matched healthy controls. The expression of 16 proteins, mainly related to acute phase response (APR), was changed at the baseline in the untreated patients. Following ω-3 PUFAs supplementation, the detected APR was partially rescued, with the expression of 10 out of 16 (62%) proteins being normalized. ω-3 PUFAs have a major impact on the modulation of the APR in RTT, thus providing new insights into the role of inflammation in autistic disorders and paving the way for novel therapeutic strategies.
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Proteínas Sanguíneas/análisis , Ácidos Grasos Omega-3/farmacología , Proteoma , Síndrome de Rett/sangre , Proteínas de Fase Aguda/análisis , Adolescente , Adulto , Niño , Preescolar , Suplementos Dietéticos , Ácidos Grasos Omega-3/administración & dosificación , Femenino , Humanos , FN-kappa B/fisiologíaRESUMEN
Autism spectrum disorders (ASDs) are a complex group of neurodevelopment disorders steadily rising in frequency and treatment refractory, where the search for biological markers is of paramount importance. Although red blood cells (RBCs) membrane lipidomics and rheological variables have been reported to be altered, with some suggestions indicating an increased lipid peroxidation in the erythrocyte membrane, to date no information exists on how the oxidative membrane damage may affect cytoskeletal membrane proteins and, ultimately, RBCs shape in autism. Here, we investigated RBC morphology by scanning electron microscopy in patients with classical autism, that is, the predominant ASDs phenotype (age range: 6-26 years), nonautistic neurodevelopmental disorders (i.e., "positive controls"), and healthy controls (i.e., "negative controls"). A high percentage of altered RBCs shapes, predominantly elliptocytes, was observed in autistic patients, but not in both control groups. The RBCs altered morphology in autistic subjects was related to increased erythrocyte membrane F2-isoprostanes and 4-hydroxynonenal protein adducts. In addition, an oxidative damage of the erythrocyte membrane ß-actin protein was evidenced. Therefore, the combination of erythrocyte shape abnormalities, erythrocyte membrane oxidative damage, and ß-actin alterations constitutes a previously unrecognized triad in classical autism and provides new biological markers in the diagnostic workup of ASDs.
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Actinas/sangre , Trastorno Autístico/sangre , Membrana Eritrocítica/metabolismo , Eritrocitos/patología , Adolescente , Adulto , Aldehídos/metabolismo , Trastorno Autístico/psicología , Niño , Preescolar , Recuento de Eritrocitos , Membrana Eritrocítica/química , Femenino , Humanos , Inteligencia , Masculino , Proteínas de la Membrana/análisis , Estrés OxidativoRESUMEN
Rett syndrome (RTT) is a progressive neurodevelopmental disorder mainly caused by mutations in the gene encoding the methyl-CpG-binding protein 2 (MeCP2). Although over 200 mutations types have been identified so far, nine of which the most frequent ones. A wide phenotypical heterogeneity is a well-known feature of the disease, with different clinical presentations, including the classical form and the preserved speech variant (PSV). Aim of the study was to unveil possible relationships between plasma proteome and phenotypic expression in two cases of familial RTT represented by two pairs of sisters, harbor the same MECP2 gene mutation while being dramatically discrepant in phenotype, that is, classical RTT versus PSV. Plasma proteome was analysed by 2-DE/MALDI-TOF MS. A significant overexpression of six proteins in the classical sisters was detected as compared to the PSV siblings. A total of five out of six (i.e., 83.3%) of the overexpressed proteins were well-known acute phase response (APR) proteins, including alpha-1-microglobulin, haptoglobin, fibrinogen beta chain, alpha-1-antitrypsin, and complement C3. Therefore, the examined RTT siblings pairs proved to be an important benchmark model to test the molecular basis of phenotypical expression variability and to identify potential therapeutic targets of the disease.
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Proteínas Sanguíneas/análisis , Proteómica/métodos , Síndrome de Rett/sangre , Reacción de Fase Aguda , Adulto , Femenino , Humanos , Proteína 2 de Unión a Metil-CpG/genética , Fenotipo , Síndrome de Rett/clasificación , Síndrome de Rett/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Background: Deficiency of Vitamin B12 and folate may determine hematological, neurological, and metabolic alterations; therefore, an accurate quantification of their serum levels is required, especially in the presence of symptoms that might suggest a deficiency. CHORUS VIT B12 and CHORUS FOLATE are two automated immunoassays, developed to quantify vitamin B12 and folate, respectively, in human serum. Design and methods: This single-center, non-pharmacological, diagnostic study described the validation and characterization of CHORUS VIT B12 and CHORUS FOLATE, with a specific focus on performance, precision, and reliability. For each assay, 500 serum samples were analyzed. A comparison between CHORUS assays and commercially available kit was also performed. Results: For CHORUS VIT B12 the lower limit of quantification (LLoQ) was 165.0 pg/mL and the upper LoQ (ULoQ) was 1846.8 pg/mL. The assay was linear within the calibration range (150-2000 pg/mL) and the accuracy was described with the International Standard Vitamin B12, Serum Folate, HOLO TC (NIBSC code: 03/178), with a mean recovery on two lots of 111%. For CHORUS FOLATE (calibration range of 2.0-20.0 ng/mL), LLoQ was 2.0 ng/mL and ULoQ 19.6 ng/mL. The linearity was demonstrated from 2.4 to 20.0 ng/mL; the accuracy was described with the International Standard mentioned above, achieving a mean recovery on three lots of 92%. The lowest and highest values of both CHORUS and COBAS kits were similar and the median values did not significantly vary. Conclusion: CHORUS VIT B12 and CHORUS FOLATE performed well, accurately, and reliably in quantifying vitamin B12 and folate in human serum.
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The medicinal plant Mucuna pruriens, with reputed anti-snake venom properties has been reported to contain a kunitz-type trypsin inhibitor. This study was undertaken to further evaluate the protease inhibitory potential of gpMuc, a multiform glycoprotein, and other protein fractions from M. pruriens seeds against trypsin, chymotrypsin, Echis carinatus snake venom, ecarin and thrombin. The results showed that gpMuc inhibited both trypsin and chymotrypsin activities and was thermally stable, maintaining its trypsin inhibitory activity at temperatures of up to 50°C. Its structural conformation was also maintained at pH ranges of 4-7. Immunoreactivity study confirms that it contains protease-recognizing epitope on one of its isoforms. The whole protein extract of M. pruriens seeds inhibited prothrombin activation by ecarin and whole E. carinatus venom, and also thrombin-like activity using chromogenic assay.
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Glicoproteínas/química , Mucuna/química , Inhibidores de Proteasas/química , Venenos de Víboras/antagonistas & inhibidores , Animales , Quimotripsina/antagonistas & inhibidores , Endopeptidasas/metabolismo , Glicoproteínas/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Plantas Medicinales , Inhibidores de Proteasas/aislamiento & purificación , Isoformas de Proteínas/química , Trombina/antagonistas & inhibidores , Tripsina/metabolismo , ViperidaeRESUMEN
Westergren method is considered as the reference procedure to measure Erythrocyte Sedimentation Rate (ESR) by the International Council for Standardization in Haematology. However, a closed automated method, VES Matic Cube 80 (DIESSE S.p.A., Siena, Italy), has been introduced as a new ESR measurement instrument. In this article, we report two different studies: first, we compared the two methods (Westergren and VES Matic Cube 80) and second, we correlated the inflammatory state of 248 patients with their ESR values. Total protein, albumin, C-reactive protein, and other inflammatory proteins were detected in each sample. The results obtained using VES Matic Cube 80 demonstrated a good correlation with those obtained using the Westergren method (Ordinary linear regression: y=0.955x-0.205, r(2) =0.816, P<0.05; Passing-Bablock regression equation: y=0.9153x-0.5763; Bland-Altman analysis: bias 1.2; limits of agreement -17.4-19.9) and with the inflammatory protein levels (CRP: r=0.554 and r=0.498 and Fibrinogen: r=0.699 and r=0.663 for Ves Matic Cube 80 and Westergren, respectively), supporting the hypothesis that VES Matic Cube 80 offers a fast and safe ESR determination, ensuring precision and a very good correlation with the reference method.
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Proteínas Sanguíneas/metabolismo , Pruebas Hematológicas/instrumentación , Pruebas Hematológicas/métodos , Inflamación/sangre , Sedimentación Sanguínea , Femenino , Humanos , Modelos Lineales , Masculino , Persona de Mediana EdadRESUMEN
Mucuna pruriens has been used by native Nigerians as a prophylactic for snakebite. The protective effects of M. pruriens seed extract (MPE) were investigated against the pharmacological actions of N. sputatrix (Javan spitting cobra) venom in rats. The results showed that MPE-pretreatment protected against cardiorespiratory and, to a lesser extent, neuromuscular depressant effects of N. sputatrix venom. These may be explained at least in part by the neutralisation of the cobra venom toxins by anti-MPE antibodies elicited by the MPE pretreatment.
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Venenos Elapídicos/antagonistas & inhibidores , Mucuna , Animales , Sistema Cardiovascular/efectos de los fármacos , Depresión Química , Venenos Elapídicos/toxicidad , Masculino , Medicinas Tradicionales Africanas , Mucuna/química , Sistema Nervioso/efectos de los fármacos , Nigeria , Fitoterapia , Extractos Vegetales/farmacología , Plantas Medicinales/química , Ratas , Ratas Sprague-Dawley , Sistema Respiratorio/efectos de los fármacos , Semillas/químicaRESUMEN
OBJECTIVE: Sex-/age-differentiated cutoffs and the magnitude of serial changes in high-sensitivity cardiac troponins (hs-cTn) for acute coronary syndrome (ACS) diagnosis algorithms are still under discussion. This study presents a methodology to evaluate decision-making limits and to assess whether sex-specific cutoffs could improve diagnostic accuracy. METHODS: A high-sensitivity cardiac troponin T (hs-cTnT) 0-/3-hour protocol was adopted, applying the 2015 European Society of Cardiology Guidelines. Decision-making limits (99th percentile: 14 ng/L; delta change ≥ 30%) were agreed upon with the emergency department (ED) at the University Hospital of Siena in Siena, Italy. One-year requests (5177) for hs-cTnT serial determination were compared with the final International Classification of Diseases, 9th revision, clinical modifications diagnosis (contingency tables; receiver operating characteristic curves). RESULTS: The algorithm's capability to exclude or confirm ACS was verified by remarkable negative predictive value (97%) and high areas under the curve for the first troponin sampling (0.712), troponin sampling at 3 hours (0.789), and delta (0.744). The clinical utility for the general population-even those with comorbidities-accessing the ED was verified. Our data did not support a sex-differentiated cutoff utility because it would not have affected patient management. CONCLUSION: This methodology allowed us to confirm the effectiveness of our decision-making limits.
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Síndrome Coronario Agudo , Síndrome Coronario Agudo/diagnóstico , Biomarcadores , Servicio de Urgencia en Hospital , Femenino , Humanos , Masculino , Infarto del Miocardio , Troponina , Troponina TRESUMEN
Echis carinatus venom (EV) is a complex mixture of toxins that contribute to its lethality. EV proteolytic activity was analyzed by zymography, chromogenic assays, and SDS-PAGE. To understand the molecular mechanism of the envenomation, we investigated the in vitro effect of EV on human plasma proteins. We looked for EV protein substrates and their proteolytic fragments. We analyzed EV proteolytic activity on standard proteins such as prothrombin or fibrinogen. To set up the optimal EV:plasma protein ratio conditions, plasma was incubated with EV (treated plasma), depleted of abundant proteins, and subjected to SDS-PAGE. Samples from control and treated plasma were also analyzed by 2-DE/MALDI-TOF MS, leading to the identification of four classes of plasma proteins cleaved by EV: proteases, protease inhibitors, binding proteins, and transporters. EV mainly proteolyzes entire proteins but can also act on physiological fragments. In summary, the physiological effects of EV proteases involve other important processes in addition to blood coagulation; complement activation and hemoglobin metabolism are also affected. In particular, the cleavage of protease inhibitors appears to be the mechanism through which the venom neutralizes the body's defenses.
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Proteínas Sanguíneas , Proteoma , Venenos de Víboras , Animales , Proteínas Sanguíneas/química , Proteínas Sanguíneas/metabolismo , Electroforesis en Gel de Poliacrilamida/métodos , Humanos , Datos de Secuencia Molecular , Péptidos/análisis , Proteoma/análisis , Proteoma/efectos de los fármacos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Espectrometría de Masas en Tándem/métodos , Venenos de Víboras/química , Venenos de Víboras/farmacología , ViperidaeRESUMEN
Rett syndrome (RTT) is a leading cause of severe intellectual disability in females, caused by de novo loss-of function mutations in the X-linked methyl-CpG binding protein 2 (MECP2). To better investigate RTT disease progression/pathogenesis animal models of Mecp2 deficiency have been developed. Here, Mecp2 mouse models are employed to investigate the role of protein patterns in RTT. A proteome analysis was carried out in brain tissue from i) Mecp2 deficient mice at the pre-symptomatic and symptomatic stages and, ii) mice in which the disease phenotype was reversed by Mecp2 reactivation. Several proteins were shown to be differentially expressed in the pre-symptomatic (nâ¯=â¯18) and symptomatic (nâ¯=â¯20) mice. Mecp2 brain reactivated mice showed wild-type comparable levels of expression for twelve proteins, mainly related to proteostasis (nâ¯=â¯4) and energy metabolic pathways (nâ¯=â¯4). The remaining ones were found to be involved in redox homeostasis (nâ¯=â¯2), nitric oxide regulation (nâ¯=â¯1), neurodevelopment (nâ¯=â¯1). Ten out of twelve proteins were newly linked to Mecp2 deficiency. Our study sheds light on the relevance of the protein-regulation of main physiological process in the complex mechanisms leading from Mecp2 mutation to the RTT clinical phenotype. SIGNIFICANCE: We performed a proteomic study of a Mecp2stop/y mouse model for Rett syndrome (RTT) at the pre-symptomatic and symptomatic Mecp2 deficient mice stage and for the brain specific reactivated Mecp2 model. Our results reveal major protein expression changes pointing out to defects in proteostasis or energy metabolic pathways other than, to a lesser extent, in redox homeostasis, nitric oxide regulation or neurodevelopment. The Mecp2 mouse rescued model provides the possibility to select target proteins more susceptible to the Mecp2 gene mutation, potential and promising therapeutical targets.
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Encéfalo/metabolismo , Proteína 2 de Unión a Metil-CpG/fisiología , Mutación , Estrés Oxidativo , Proteoma/metabolismo , Síndrome de Rett/etiología , Animales , Biomarcadores/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Proteoma/análisis , Proteómica/métodos , Síndrome de Rett/patologíaRESUMEN
Serum protein electrophoresis is routinely used to identify pathologies involving dysproteinemia. The electropherogram mainly represents the most abundant serum proteins, one of which is the polymorphic haptoglobin (Hpt), characterized by a molecular heterogeneity with three major phenotypes (Hpt 1-1, 2-1, and 2-2). To improve the interpretation of electropherogram and possibly to extend its applicability, we aimed to explore the relationship between Hpt phenotypes (determined by immunoblotting) and protein profiles. Serum samples were separated by CZE with PROTEIN 6 and high-resolution methods. The PROTEIN 6 analysis showed significant associations between alpha2 zone profiles and Hpt phenotypes (chi-square=154.06, p<0.0001). The high-resolution method indicated significant differences between Hpt 2-2 and Hpt 1-1 peak mobilities, evidenced by receiver operating characteristic analysis, (area under the receiver operating characteristic curve=0.98, p<0.0001, standard error=0.01346, likelihood ratios=21.39), with 98.7% sensitivity, and 95.4% specificity. However, the structural heterogeneity of Hpt 2-1 made it difficult to relate with a particular profile. Thus, we developed an alternative approach that excluded the Hpt 1-1 or Hpt 2-2 phenotypes. This may prove to be a useful technique in clinical applications considering the involvement of Hpt 2-2 or Hpt 1-1 in various pathologies.
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Electroforesis Capilar/métodos , Haptoglobinas/análisis , Haptoglobinas/genética , Fenotipo , Polimorfismo GenéticoRESUMEN
Levodopa is the medication of choice for Parkinson's disease. The biological complexity of levodopa and of its main derivatives makes their determination important in the clinical field. The aim of this study was to develop an HPLC method for the simultaneous determination of serum concentrations of levodopa, dopamine, 3-O-methyldopa and alpha-methyldopa. We compared UV and fluorimetric detection of native and derivatised compounds. Though less sensitive than other methods, UV detection is important to exclude naturally fluorescent, interfering substances. Fluorimetric detection of derivatised compounds is more sensitive than UV detection. Since 3-O-methyldopa does not react with the derivatising agent 1,2-diphenylethylenediamine, it cannot be detected. For simultaneous determination of the four compounds after pharmacological treatment of patients we therefore advise fluorimetric detection of the native compound.
Asunto(s)
Cromatografía Líquida de Alta Presión/métodos , Dopamina/sangre , Levodopa/sangre , Metildopa/sangre , Tirosina/análogos & derivados , Fluorometría , Humanos , Tirosina/sangreRESUMEN
Rett syndrome (RTT) is a severe genetic disorder resulting from mutations in the X-linked methyl-CpG-binding protein 2 (MECP2) gene. Recently, a zebrafish carrying a mecp2-null mutation has been developed with the resulting phenotypes exhibiting defective sensory and thigmotactic responses, and abnormal motor behavior reminiscent of the human disease. Here, we performed a proteomic analysis to examine protein expression changes in mecp2-null vs. wild-type larvae and adult zebrafish. We found a total of 20 proteins differentially expressed between wild-type and mutant zebrafish, suggesting skeletal and cardiac muscle functional defects, a stunted glycolysis and depleted energy availability. This molecular evidence is directly linked to the mecp2-null zebrafish observed phenotype. In addition, we identified changes in expression of proteins critical for a proper redox balance, suggesting an enhanced oxidative stress, a phenomenon also documented in human patients and RTT murine models. The molecular alterations observed in the mecp2-null zebrafish expand our knowledge on the molecular cascade of events that lead to the RTT phenotype. BIOLOGICAL SIGNIFICANCE: We performed a proteomic study of a non-mammalian vertebrate model (zebrafish, Danio rerio) for Rett syndrome (RTT) at larval and adult stages of development. Our results reveal major protein expression changes pointing out to defects in energy metabolism, redox status imbalance, and muscle function, both skeletal and cardiac. Our molecular analysis grants the mecp2-null zebrafish as a valuable RTT model, triggering new research approaches for a better understanding of the RTT pathogenesis and phenotype expression. This non-mammalian vertebrate model of RTT strongly suggests a broad impact of Mecp2 dysfunction.