RESUMO
Mutations in the LRRK2 kinase are the most common cause of familial Parkinson's disease, and variants increase risk for the sporadic form of the disease. LRRK2 phosphorylates multiple RAB GTPases including RAB8A and RAB10. Phosphorylated RAB10 is recruited to centrosome-localized RILPL1, which may interfere with ciliogenesis in a disease-relevant context. Our previous studies indicate that the centrosomal accumulation of phosphorylated RAB8A causes centrosomal cohesion deficits in dividing cells, including in peripheral patient-derived cells. Here, we show that both RAB8 and RAB10 contribute to the centrosomal cohesion deficits. Pathogenic LRRK2 causes the centrosomal accumulation not only of phosho-RAB8 but also of phospho-RAB10, and the effects on centrosomal cohesion are dependent on RAB8, RAB10 and RILPL1. Conversely, the pathogenic LRRK2-mediated ciliogenesis defects correlate with the centrosomal accumulation of both phospho-RAB8 and phospho-RAB10. LRRK2-mediated centrosomal cohesion and ciliogenesis alterations are observed in patient-derived peripheral cells, as well as in primary astrocytes from mutant LRRK2 mice, and are reverted upon LRRK2 kinase inhibition. These data suggest that the LRRK2-mediated centrosomal cohesion and ciliogenesis defects are distinct cellular readouts of the same underlying phospho-RAB8/RAB10/RILPL1 nexus and highlight the possibility that either centrosomal cohesion and/or ciliogenesis alterations may serve as cellular biomarkers for LRRK2-related PD.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Centrossomo/metabolismo , Ciliopatias/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Ciliopatias/enzimologia , Ciliopatias/genética , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Fosforilação , Proteínas rab de Ligação ao GTP/genéticaRESUMO
OBJECTIVE: To evaluate the usefulness of ultrasound examination in patients with just a serological diagnosis of schistosomiasis but no other evidence of active infection. METHODS: 346 sub-Saharan patients with possible schistosomiasis that presented at a Tropical Medicine Unit between 2008 and 2019 were retrospectively selected. Possible schistosomiasis was considered in those patients with a positive serology for schistosomasis in the absence of direct microbiological isolates, hematuria and/or eosinophilia. Data from ultrasound examinations before and after treatment with praziquantel were collected and categorized following the World Health Organization-Niamey score to standardize the use of ultrasonography for the assessment of schistosomiasis-related morbidity. RESULTS: Ultrasound examinations were abnormal in only ten patients (2.89%). Main findings were focal thickening of the bladder wall (n = 6), ureteral dilatation (n = 3) and grade I hydronephrosis (n = 1). No malignant lesions, hepatic lesions nor hepatobiliary related disorders were found. After treatment, the S. haematobium global score (5 vs 3.4, p = 0.06) and the urinary bladder score (2 vs 1, p = 0.059) showed a trend towards improvement after treatment. In three patients the score after treatment dropped to 0, and in another three it remained the same although with signs of improvement. No worsening of the score was observed in any case. CONCLUSION: For those patients with a diagnosis of schistosomiasis based solely in a positive serology, the ultrasound examination could safely be spared due to the low prevalence of pathological findings and its response to treatment anyway.
Assuntos
Esquistossomose Urinária , África Subsaariana/epidemiologia , Humanos , Praziquantel , Estudos Retrospectivos , Esquistossomose Urinária/diagnóstico por imagem , Esquistossomose Urinária/tratamento farmacológico , UltrassonografiaRESUMO
Despite the considerable progress in strategies of myocardial protection, ischemic heart diseases (IHD) and consequent heart failure (HF) remain the main cause of mortality worldwide. Several procedures are used routinely to guarantee the prompt and successful reestablishment of blood flow to preserve the myocardial viability of infarcted hearts from ischemia injuries. However, ischemic heart reperfusion/revascularization triggers additional damages that occur when oxygen-rich blood re-enters the vulnerable myocardial tissue, which is a phenomenon known as ischemia and reperfusion (I/R) syndrome. Complications of I/R injuries provoke the adverse cardiac remodeling, involving inflammation, mishandling of Ca2+ homeostasis, apoptotic genes activation, cardiac myocytes loss, etc., which often progress toward HF. Therefore, there is an urgent need to develop new cardioprotective therapies for IHD and HF. Compelling evidence from animal studies and pilot clinical trials in HF patients suggest that urocortin (Ucn) isoforms, which are peptides associated with stress and belonging to the corticotropin releasing factor family, have promising potential to improve cardiovascular functions by targeting many signaling pathways at different molecular levels. This review highlights the current knowledge on the role of urocortin isoforms in cardioprotection, focusing on its acute and long-term effects.
Assuntos
Infarto do Miocárdio/genética , Isquemia Miocárdica/genética , Traumatismo por Reperfusão/genética , Urocortinas/genética , Apoptose/genética , Remodelamento Atrial/genética , Coração/fisiopatologia , Insuficiência Cardíaca/genética , Insuficiência Cardíaca/patologia , Humanos , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a promising therapeutic target for the treatment of Parkinson's disease (PD), and orally bioavailable, brain penetrant and highly potent LRRK2 kinase inhibitors are in early stages of clinical testing. Detection of LRRK2 phosphorylation, as well as phosphorylation of Rab10, a LRRK2 kinase substrate, have been proposed as target engagement biomarkers for LRRK2 inhibitor clinical trials. However, these readouts do not seem able to stratify patients based on enhanced LRRK2 kinase activity. Here, we describe a robust cell biological assay based on centrosomal cohesion alterations which were observed in peripheral blood mononuclear cell-derived lymphoblastoid cell lines (LCLs) from patients with G2019S LRRK2 mutations as compared with healthy controls, and could also be detected in a subset of sporadic PD patient samples. We suggest that LCLs may be a valuable resource for LRRK2 research, and that determination of centrosomal cohesion deficits may assist in the stratification of a subset of sporadic PD patients.
Assuntos
Centrossomo/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Leucócitos Mononucleares/metabolismo , Doença de Parkinson/metabolismo , Adulto , Idoso , Biomarcadores/metabolismo , Linhagem Celular Tumoral , Feminino , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , FosforilaçãoRESUMO
The Ischemic Heart Disease (IHD) is considered a clinical condition characterized by myocardial ischemia causing an imbalance between myocardial blood supply and demand, leading to morbidity and mortality across the worldwide. Prompt diagnostic and prognostic represents key factors for the treatment and reduction of the mortality rate. Therefore, one of the newest frontiers in cardiovascular research is related to non-coding RNAs (ncRNAs), which prompted a huge interest in exploring ncRNAs candidates for utilization as potential therapeutic targets for diagnostic and prognostic and/or biomarkers in IHD. However, there are undoubtedly many more functional ncRNAs yet to be discovered and characterized. Here we will discuss our current knowledge and we will provide insight on the roles and effects elicited by some ncRNAs related to IHD.
Assuntos
Isquemia Miocárdica , RNA não Traduzido , Biomarcadores , Humanos , Miocárdio/patologiaRESUMO
Mutations in leucine-rich repeat kinase 2 (LRRK2) comprise the most common cause of familial Parkinson's disease (PD), and sequence variants modify risk for sporadic PD. Previous studies indicate that LRRK2 interacts with microtubules (MTs) and alters MT-mediated vesicular transport processes. However, the molecular determinants within LRRK2 required for such interactions have remained unknown. Here, we report that most pathogenic LRRK2 mutants cause relocalization of LRRK2 to filamentous structures which colocalize with a subset of MTs, and an identical relocalization is seen upon pharmacological LRRK2 kinase inhibition. The pronounced colocalization with MTs does not correlate with alterations in LRRK2 kinase activity, but rather with increased GTP binding. Synthetic mutations which impair GTP binding, as well as LRRK2 GTP-binding inhibitors profoundly interfere with the abnormal localization of both pathogenic mutant as well as kinase-inhibited LRRK2. Conversely, addition of a non-hydrolyzable GTP analog to permeabilized cells enhances the association of pathogenic or kinase-inhibited LRRK2 with MTs. Our data elucidate the mechanism underlying the increased MT association of select pathogenic LRRK2 mutants or of pharmacologically kinase-inhibited LRRK2, with implications for downstream MT-mediated transport events.
Assuntos
Guanosina Trifosfato/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Ligação ao GTP/genética , Proteínas de Ligação ao GTP/metabolismo , Variação Genética , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Microtúbulos/genética , Microtúbulos/metabolismo , Mutação , Doença de Parkinson/genética , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Transdução de SinaisRESUMO
Voltage-dependent CaV1.2 L-type Ca2+ channels (LTCC) are the main route for calcium entry in vascular smooth muscle cells (VSMC). Several studies have also determined the relevant role of store-operated Ca2+ channels (SOCC) in vascular tone regulation. Nevertheless, the role of Orai1- and TRPC1-dependent SOCC in vascular tone regulation and their possible interaction with CaV1.2 are still unknown. The current study sought to characterize the co-activation of SOCC and LTCC upon stimulation by agonists, and to determine the possible crosstalk between Orai1, TRPC1, and CaV1.2. Aorta rings and isolated VSMC obtained from wild type or smooth muscle-selective conditional CaV1.2 knock-out (CaV1.2KO) mice were used to study vascular contractility, intracellular Ca2+ mobilization, and distribution of ion channels. We found that serotonin (5-HT) or store depletion with thapsigargin (TG) enhanced intracellular free Ca2+ concentration ([Ca2+]i) and stimulated aorta contraction. These responses were sensitive to LTCC and SOCC inhibitors. Also, 5-HT- and TG-induced responses were significantly attenuated in CaV1.2KO mice. Furthermore, hyperpolarization induced with cromakalim or valinomycin significantly reduced both 5-HT and TG responses, whereas these responses were enhanced with LTCC agonist Bay-K-8644. Interestingly, in situ proximity ligation assay revealed that CaV1.2 interacts with Orai1 and TRPC1 in untreated VSMC. These interactions enhanced significantly after stimulation of cells with 5-HT and TG. Therefore, these data indicate for the first time a functional interaction between Orai1, TRPC1, and CaV1.2 channels in VSMC, confirming that upon agonist stimulation, vessel contraction involves Ca2+ entry due to co-activation of Orai1- and TRPC1-dependent SOCC and LTCC.
Assuntos
Aorta/metabolismo , Canais de Cálcio Tipo L/metabolismo , Sinalização do Cálcio/fisiologia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Proteína ORAI1/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Aorta/citologia , Cálcio/metabolismo , Canais de Cálcio Tipo L/genética , Camundongos , Camundongos Knockout , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Proteína ORAI1/genética , Serotonina/metabolismo , Canais de Cátion TRPC/genética , Vasoconstrição/fisiologiaRESUMO
Mutations in the Leucine-Rich Repeat Kinase 2 (LRRK2) gene are intimately linked to both familial and sporadic Parkinson's disease. LRRK2 is a large protein kinase able to bind and hydrolyse GTP. A wealth of in vitro studies have established that the distinct pathogenic LRRK2 mutants differentially affect those enzymatic activities, either causing an increase in kinase activity without altering GTP binding/GTP hydrolysis, or displaying no change in kinase activity but increased GTP binding/decreased GTP hydrolysis. Importantly, recent studies have shown that all pathogenic LRRK2 mutants display increased kinase activity towards select kinase substrates when analysed in intact cells. To understand those apparently discrepant results, better insight into the cellular role(s) of normal and pathogenic LRRK2 is crucial. Various studies indicate that LRRK2 regulates numerous intracellular vesicular trafficking pathways, but the mechanism(s) by which the distinct pathogenic mutants may equally interfere with such pathways has largely remained elusive. Here, we summarize the known alterations in the catalytic activities of the distinct pathogenic LRRK2 mutants and propose a testable working hypothesis by which the various mutants may affect membrane trafficking events in identical ways by culminating in increased phosphorylation of select substrate proteins known to be crucial for membrane trafficking between specific cellular compartments.
Assuntos
GTP Fosfo-Hidrolases/metabolismo , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Microtúbulos/metabolismo , Transdução de Sinais , Animais , Biocatálise , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Modelos Biológicos , Mutação , FosforilaçãoRESUMO
Leucine-rich repeat kinase 2 (LRRK2) is a key player in the pathogenesis of Parkinson's disease. Mutations in LRRK2 are associated with increased kinase activity that correlates with cytotoxicity, indicating that kinase inhibitors may comprise promising disease-modifying compounds. However, before embarking on such strategies, detailed knowledge of the cellular deficits mediated by pathogenic LRRK2 in the context of defined and pathologically relevant kinase substrates is essential. LRRK2 has been consistently shown to impair various intracellular vesicular trafficking events, and recent studies have shown that LRRK2 can phosphorylate a subset of proteins that are intricately implicated in those processes. In light of these findings, we here review the link between cellular deficits in intracellular trafficking pathways and the LRRK2-mediated phosphorylation of those newly identified substrates.
Assuntos
Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/metabolismo , Doença de Parkinson/enzimologia , Vesículas Transportadoras/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Animais , Humanos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Modelos Biológicos , Mutação , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Fosforilação , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Proteínas rab de Ligação ao GTP/genéticaRESUMO
The goal of this study is to evaluate whether NT-proBNP plasma levels may help as a screening biomarker for monitoring right ventricular dilatation, pulmonary regurgitation and the onset of heart failure in patients with repaired Tetralogy of Fallot. Our single-centre observational prospective study involved 43 patients (15.1 years, SD = 8) with corrected Tetralogy of Fallot. Data collection included: clinical parameters (electrocardiogram, chest X-ray, NYHA scale, time since last surgery), biochemistry (NT-proBNP levels) and MRI values (ventricular volumetry, pulmonary flow assessment). Mean time since last surgery was 13.5 years (SD = 7.8). There was a statistically significant correlation between the NT-proBNP levels (187.4 pg/ml, SD = 154.9) and right ventricular dilatation for both the right ventricular end-diastolic volume (124.9 ml/m2, SD = 31.2) (Pearson = 0.19, p < 0.01) and end-systolic volume (56.1 ml/m2, SD = 18.8) (Pearson = 0.21, p < 0.01) and also with the pulmonary regurgitation fraction (36.5%, SD = 16, Pearson = 0.12, p < 0.01). No significant correlation was found between NT-proBNP and right ventricular ejection fraction (54.6%, SD = 10.6, Pearson = -0.07), left ventricular ejection fraction (59.9%, SD = 7.1, Pearson = -0.18) or any clinical parameters. The receiver operating curve analysis evidenced that a NT-proBNP cut-off value above 133.2 pg/ml predicted the presence of dilated right ventricular end-diastolic and end-systolic volumes over centile 95 (sensitivity 82 and 83% and specificity 93 and 79%, respectively). In conclusion, in patients with surgically corrected Tetralogy of Fallot, NT-proBNP levels correlate with right ventricular dilatation and the degree of pulmonary regurgitation. Ambulatory determination of NT-proBNP might be an easy, readily available and cost-effective alternative for MRI follow-up evaluation of these patients.
Assuntos
Imageamento por Ressonância Magnética , Peptídeo Natriurético Encefálico/sangue , Fragmentos de Peptídeos/sangue , Insuficiência da Valva Pulmonar/sangue , Volume Sistólico , Tetralogia de Fallot/cirurgia , Disfunção Ventricular Direita/sangue , Adolescente , Biomarcadores/sangue , Criança , Eletrocardiografia , Feminino , Humanos , Masculino , Estudos Prospectivos , Insuficiência da Valva Pulmonar/diagnóstico por imagem , Curva ROC , Disfunção Ventricular Direita/diagnóstico por imagem , Adulto JovemRESUMO
INTRODUCTION: Nearly two decades after leucine rich repeat kinase 2 (LRRK2) was discovered as a genetic determinant of Parkinson's disease (PD), LRRK2 has emerged a priority therapeutic target in PD and inhibition of its activity is hypothesized to be beneficial. AREAS COVERED: LRRK2 targeting agents, in particular kinase inhibitors and agents reducing LRRK2 expression show promise in model systems and have progressed to phase I and phase II clinical testing for PD. Several additional targeting strategies for LRRK2 are emerging, based on promoting specific 'healthy' LRRK2 quaternary structures, heteromeric complexes and conformations. EXPERT OPINION: It can be expected that LRRK2 targeting strategies may proceed to phase III clinical testing for PD in the next five years, allowing the field to discover the real clinical value of LRRK2 targeting strategies.
Assuntos
Antiparkinsonianos , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina , Doença de Parkinson , Patentes como Assunto , Inibidores de Proteínas Quinases , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/antagonistas & inibidores , Serina-Treonina Proteína Quinase-2 com Repetições Ricas em Leucina/genética , Humanos , Doença de Parkinson/tratamento farmacológico , Doença de Parkinson/fisiopatologia , Animais , Inibidores de Proteínas Quinases/farmacologia , Antiparkinsonianos/farmacologia , Terapia de Alvo Molecular , Desenvolvimento de MedicamentosRESUMO
INTRODUCTION AND OBJECTIVES: The current evaluation of acute heart failure (HF) does not allow an adequate prediction of its evolution. The electrical bioimpedance (BI) allows knowing the state of blood volume, until now only with fixed equipment. We have developed and validated a portable and wireless device to measure BI at the ankle (IVOL). The objective of the study is to know the long-term prognostic value of the point measurement of BI with IVOL in patients with acute HF. METHODS: A prospective cohort study of unselected patients admitted for acute HF in a tertiary hospital. The association between BI and different clinical, analytical and echocardiographic variables on admission and clinical evolution were analyzed. RESULTS: 76 patients were included (mean age 66.1 years, 71.1% men, 68.4% hypertensive, 34.2% diabetic, mean NT-ProBNP: 7,103 pg / ml). Of these, 52.6% with non-preserved left ventricular ejection fraction (LVEF) (<50%) and 56.6% with right ventricular (RV) dysfunction. 26.3% died during a mean follow-up of 35.8 months. Survival in patients with BI≤21,8Ω was lower, globally and in the subgroups of patients without preserved LVEF and with RV dysfunction, P<.008). In the multivariate analysis, a BI≥21.8Ω was an independent survival factor (HR: 0.242; 95% CI: 0.86-0.681; P=.007). CONCLUSIONS: BI values measured with IVOL may be an independent predictor of long-term mortality in patients hospitalized for acute HF. This prognostic value is maintained in patients without preserved LVEF function and with RV dysfunction.
Assuntos
Impedância Elétrica , Insuficiência Cardíaca , Humanos , Masculino , Insuficiência Cardíaca/fisiopatologia , Insuficiência Cardíaca/mortalidade , Insuficiência Cardíaca/diagnóstico , Feminino , Idoso , Prognóstico , Estudos Prospectivos , Doença Aguda , Pessoa de Meia-Idade , Tecnologia sem Fio/instrumentação , Idoso de 80 Anos ou mais , Seguimentos , Volume SistólicoRESUMO
Alterations induced by maternal immune activation (MIA) during gestation impact the subsequent neurodevelopment of progeny, a process that in humans, has been linked to the development of several neuropsychiatric conditions. To undertake a comprehensive examination of the molecular mechanisms governing MIA, we have devised an in vitro model based on neural stem cells (NSCs) sourced from fetuses carried by animals subjected to Poly I:C treatment. These neural progenitors demonstrate proliferative capacity and can be effectively differentiated into both neurons and glial cells. Transcriptomic, proteomic, and phosphoproteomic analyses conducted on these cellular models, in conjunction with counterparts from control treatments, revealed discernible shifts in the expression levels of a specific subset of proteins implicated in neuronal function. Noteworthy, we found an absence of congruence between these alterations at the transcriptomic level, suggesting that differences in protein translation contribute to the observed dysregulation. Furthermore, the phosphoproteomic data highlighted a discernible discrepancy in the basal phosphorylation of proteins between differentiated cells from both experimental groups, particularly within proteins associated with cytoskeletal architecture and synaptic functionality, notably those belonging to the MAP family. Observed alterations in MAP phosphorylation were found to potentially have functional consequences as they correlate with changes in neuronal plasticity and the establishment of neuronal synapses. Our data agrees with previous published observations and further underscore the importance of MAP2 phosphorylation state on its function and the impact that this protein has in neuronal structure and function.
RESUMO
BACKGROUND: Species hybridization represents a real concern in terms of parasite transmission, epidemiology and morbidity of schistosomiasis. It is greatly important to better understand the impact of species hybridization for the clinical management. METHODS: A prospective observational study was carried out in sub-Saharan migrants who were diagnosed with confirmed genitourinary schistosomiasis. A tailored protocol was applied, including Schistosoma serology, a specific urine LAMP tests for schistosomiasis and an ultrasound examination before treatment with praziquantel. A scheduled follow-up was performed at 3, 6 and 12 months to monitor treatment response, comparing patients carriers of Schistosoma hybrids with carriers of only genetically pure forms. RESULTS: A total of 31 male patients from West Africa were included in the study with a mean age of 26.5 years. Twelve (38.7 %) of the patients were carriers of Schistosoma hybrids. As compared with patients infected with S. haematobium alone, hybrid carriers had lower haemoglobin levels (13.8 g/dL [SD 1.8] vs 14.8 g/dL [SD 1.4], p = 0.04), a greater frequency of hematuria (100 % vs 52.6 %, p = 0.005), a higher ultrasound score (2.64, SD 2.20 vs 0.89, SD 0.99; p = 0.02). However, the presence of hybrids did not result in differences in clinical and analytical responses after treatment. CONCLUSIONS: The presence of Schistosoma hybrids seems to cause increased morbidity in infected individuals. However, it does not appear to result in differences in diagnostic tests or in clinical and analytical responses after treatment.
RESUMO
The carotid body (CB) is the major peripheral arterial chemoreceptor in mammals that mediates the acute hyperventilatory response to hypoxia. The CB grows in response to sustained hypoxia and also participates in acclimatisation to chronic hypoxaemia. Knowledge of CB physiology at the cellular level has increased considerably in recent times thanks to studies performed on lower mammals, and rodents in particular. However, the functional characteristics of human CB cells remain practically unknown. Herein, we use tissue slices or enzymatically dispersed cells to determine the characteristics of human CB cells. The adult human CB parenchyma contains clusters of chemosensitive glomus (type I) and sustentacular (type II) cells as well as nestin-positive progenitor cells. This organ also expresses high levels of the dopaminotrophic glial cell line-derived neurotrophic factor (GDNF). We found that GDNF production and the number of progenitor and glomus cells were preserved in the CBs of human subjects of advanced age. Moreover, glomus cells exhibited voltage-dependent Na(+), Ca(2+) and K(+) currents that were qualitatively similar to those reported in lower mammals. These cells responded to hypoxia with an external Ca(2+)-dependent increase of cytosolic Ca(2+) and quantal catecholamine secretion, as reported for other mammalian species. Interestingly, human glomus cells are also responsive to hypoglycaemia and together these two stimuli can potentiate each other's effects. The chemosensory responses of glomus cells are also preserved at an advanced age. These new data on the cellular and molecular physiology of the CB pave the way for future pathophysiological studies involving this organ in humans.
Assuntos
Potenciais de Ação , Corpo Carotídeo/citologia , Adolescente , Adulto , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Idoso , Cálcio/metabolismo , Sinalização do Cálcio , Corpo Carotídeo/metabolismo , Corpo Carotídeo/fisiologia , Hipóxia Celular , Células Cultivadas , Criança , Feminino , Glucose/metabolismo , Humanos , Canais Iônicos/metabolismo , Masculino , Pessoa de Meia-Idade , Nestina/genética , Nestina/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Potássio/metabolismo , Sódio/metabolismoRESUMO
OBJECTIVE: Human urotensin-II (UII) is considered the most potentendogenous vasoconstrictor discovered to date, although the precise mechanism activated downstream of its receptor UTS2R in blood vessels remains elusive. The aim of this study was to determine the role of the store operated Ca(2+) entry (SOCE) signaling pathway in UII-induced coronary artery vasoconstriction. METHODS AND RESULTS: We used a combination of isometric tension measurement, Ca(2+) imaging, pharmacology, and molecular approaches to study UII-mediated rat coronary artery vasoconstriction and intracellular Ca(2+) mobilization in coronary smooth muscle cells. We found that UII promoted dose-dependent vasoconstriction and elicited Ca(2+) and Mn(2+) influx, which were sensitive to classical SOCE inhibitors. In addition, knockdown of either STIM1 or Orai1 essentially inhibited UII-mediated SOCE and prevented UII but not high-KCL evoked contraction in transfected coronary artery. Moreover, we found that Ca(2+)-independent phospholipase A(2)ß was involved in UII effects and that is colocalized with STIM1 in different submembrane compartments. Importantly, STIM1 but not Orai1 downregulation inhibits significantly independent phospholipase A(2) activation. Furthermore, lysophosphatidylcholine, an independent phospholipase A(2) product, activated Orai1 but not STIM1-dependent contraction and SOCE. CONCLUSIONS: Here, we demonstrated that different critical players of SOCE signaling pathway are required for UII-induced vasoconstriction of rat coronary artery.
Assuntos
Transporte Biológico/fisiologia , Canais de Cálcio/metabolismo , Cálcio/metabolismo , Vasos Coronários/metabolismo , Glicoproteínas de Membrana/metabolismo , Urotensinas/metabolismo , Vasoconstrição/fisiologia , Animais , Células Cultivadas , Vasos Coronários/citologia , Líquido Intracelular/metabolismo , Masculino , Músculo Liso Vascular/citologia , Músculo Liso Vascular/metabolismo , Proteína ORAI1 , Ratos , Ratos Wistar , Transdução de Sinais , Molécula 1 de Interação Estromal , Vasoconstrição/efeitos dos fármacosRESUMO
INTRODUCTION: Imaging techniques, primarily ultrasound, are useful in the diagnosis and monitoring of patients with schistosomiasis in endemic areas. METHODS: Study of 219 patients treated in sub-Saharan Tropical Medicine Unit with a diagnosis of imported schistosomiasis by imaging techniques investigations including plain abdominal radiography and ultrasound. RESULTS: A total of 17.8% of patients who had an abdominal X-ray showed findings suggestive of schistosomiasis, in most cases bladder calcifications. In 73 patients (41%) ultrasound showed pathological findings, mainly diffuse or focal wall thickening (26 patients), nodular lesions (n=14), and pseudopolyps (n=8). One patient, who had a large bladder mass (9cm) and bilateral ureterohydronephrosis, was finally diagnosed with squamous cell carcinoma of the bladder. Ultrasound liver abnormalities were found in 10 patients, 4 with signs of portal hypertension, of which 3 had the characteristic periportal fibrosis in schistosomiasis. CONCLUSION: Imaging studies, especially abdominal and bladder ultrasound, are useful for diagnosis, the study of disease and monitoring of patients with schistosomiasis in non-endemic countries.
Assuntos
Emigrantes e Imigrantes , Esquistossomose Urinária/diagnóstico por imagem , Esquistossomose mansoni/diagnóstico por imagem , Adolescente , Adulto , África Subsaariana/etnologia , Calcinose/diagnóstico por imagem , Carcinoma de Células Escamosas/complicações , Carcinoma de Células Escamosas/diagnóstico por imagem , Comorbidade , Emigrantes e Imigrantes/estatística & dados numéricos , Feminino , Infecções por HIV/etnologia , Hepatite Viral Humana/etnologia , Humanos , Hidronefrose/diagnóstico por imagem , Hidronefrose/etiologia , Hipertensão Portal/diagnóstico por imagem , Hipertensão Portal/etiologia , Fígado/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Doenças Parasitárias/etnologia , Radiografia , Esquistossomose Urinária/complicações , Esquistossomose Urinária/etnologia , Esquistossomose mansoni/complicações , Esquistossomose mansoni/etnologia , Espanha/epidemiologia , Tuberculose/etnologia , Ultrassonografia , Bexiga Urinária/diagnóstico por imagem , Neoplasias da Bexiga Urinária/complicações , Neoplasias da Bexiga Urinária/diagnóstico por imagem , Adulto JovemRESUMO
The present protocol allows for quantification of inter-centrosome distances in G2 phase cells by confocal fluorescence microscopy to determine centrosome cohesion deficits. We describe transfection and immunofluorescence approaches followed by image acquisition and analysis of inter-centrosome distances. This protocol is for adherent A549 cells transiently overexpressing pathogenic LRRK2 and for immortalized murine embryonic fibroblasts endogenously expressing LRRK2 but is amenable to any other cultured cell type as well. For complete details on the use and execution of this protocol, please refer to Fdez et al.1 and Lara Ordóñez et al.2.
Assuntos
Centrossomo , Besouros , Animais , Camundongos , Humanos , Linhagem Celular , Células A549 , Microscopia ConfocalRESUMO
In high-rate activated sludge (HRAS) processes, reducing the solid retention time (SRT) minimizes COD oxidation and allows to obtain the maximum energy recovery. The aim of this research was to operate a pilot plant with an automatic control strategy to assure the HRAS process stability and high COD fractions removal at very low SRT. This study combines simulation and experimental tools (pilot plant 35 m3·d - 1) operating at SRT (0.2 d), HRT (0.6 h) and DO (0.5 mg·L - 1) treating high-strength raw wastewater, at 18-26°C, at variable flow. The research includes the effects of temperature, influent concentration and MLSS reactor concentration over the sCOD, cCOD and pCOD removal. The study points out that the best parameter to control the HRAS at a low SRT is not strictly the SRT but rather the reactor MLSS concentration: operating at 2,000±200mg·L - 1 assured a stable process despite the large influents variation. Low SVI values of 50-70ml·g - 1 indicated the good settling properties of the biomass. With only a 6.9% COD oxidation, a high organic matter removal (57±9% for COD and 56±10% for BOD5), was reached. The high removal efficiencies for pCOD (74%) compared to the (29%) for sCOD and (12%) for cCOD also confirmed the importance of settling efficiency and stability in the HRAS. The direct correlation between COD influent and COD removal makes advisable to use the HRAS as a replacement of the primary clarifier. The HRAS acted efficiently as a filter for COD and pCOD peak loads and, in a lesser extent, for BOD5, while sCOD peaks were not buffered. The adopted model presented a good fit for COD fractions except for pCOD when the temperature exceeds 23 °C.
Assuntos
Esgotos , Eliminação de Resíduos Líquidos , Reatores Biológicos , Águas Residuárias , TemperaturaRESUMO
Paracetamol is one of the most used pharmaceuticals worldwide, but due to its widespread use it is detected in various environmental matrices, such as surface and ground waters, sediments, soils or even plants, where it is introduced mainly from the discharge of wastewater and the use of sewage sludge as fertilizer in agriculture. Its accumulation in certain organisms can induce reproductive, neurotoxic or endocrine disorders, being therefore considered an emerging pollutant. This study reports on the isolation, from sewage sludge produced in wastewater treatment plants (WWTPs), of bacterial strains capable of degrading paracetamol. Up to 17 bacterial strains were isolated, but only two of them, identified as Pseudomonas stutzeri CSW02 and Pseudomonas extremaustralis CSW01, were able to degrade very high concentrations of paracetamol in solution as a sole carbon and energy source, and none of them had been previously described as paracetamol degraders. These bacteria showed the ability to degrade up to 500 mg L-1 of paracetamol in only 6 and 4 h, respectively, much quicker than any other paracetamol-degrader strain described in the literature. The two main paracetamol metabolites, 4-aminophenol and hydroquinone, which present high toxicity, were detected during the degradation process, although they disappeared very quickly for paracetamol concentrations up to 500 mg L-1. The IC50 of paracetamol for the growth of these two isolates was also calculated, indicating that P. extremaustralis CSW01 was more tolerant than S. stutzeri CSW02 to high concentrations of paracetamol and/or its metabolites in solution, and this is the reason for the much lower paracetamol degradation by S. stutzeri CSW02 at 2000-3000 mg L-1. These findings indicate that both bacteria are very promising candidates for their use in paracetamol bioremediation in water and sewage sludge.