RESUMO
The reprogramming of somatic cells to a spontaneously contracting cardiomyocyte-like state using defined transcription factors has proven successful in mouse fibroblasts. However, this process has been less successful in human cells, thus limiting the potential clinical applicability of this technology in regenerative medicine. We hypothesized that this issue is due to a lack of cross-species concordance between the required transcription factor combinations for mouse and human cells. To address this issue, we identified novel transcription factor candidates to induce cell conversion between human fibroblasts and cardiomyocytes, using the network-based algorithm Mogrify. We developed an automated, high-throughput method for screening transcription factor, small molecule, and growth factor combinations, utilizing acoustic liquid handling and high-content kinetic imaging cytometry. Using this high-throughput platform, we screened the effect of 4960 unique transcription factor combinations on direct conversion of 24 patient-specific primary human cardiac fibroblast samples to cardiomyocytes. Our screen revealed the combination of MYOCD, SMAD6, and TBX20 (MST) as the most successful direct reprogramming combination, which consistently produced up to 40% TNNT2+ cells in just 25 days. Addition of FGF2 and XAV939 to the MST cocktail resulted in reprogrammed cells with spontaneous contraction and cardiomyocyte-like calcium transients. Gene expression profiling of the reprogrammed cells also revealed the expression of cardiomyocyte associated genes. Together, these findings indicate that cardiac direct reprogramming in human cells can be achieved at similar levels to those attained in mouse fibroblasts. This progress represents a step forward towards the clinical application of the cardiac direct reprogramming approach.
Assuntos
Miócitos Cardíacos , Fatores de Transcrição , Humanos , Camundongos , Animais , Miócitos Cardíacos/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Regulação da Expressão Gênica , Perfilação da Expressão Gênica , Fibroblastos/metabolismo , Reprogramação Celular/genéticaRESUMO
OBJECTIVES: Soluble MER has emerged as a potential biomarker for delayed resolution of inflammation after myocardial injury and a therapeutic target to reduce cardiac-related morbidity and mortality in adults. The significance of soluble MER in pediatric populations, however, is unclear. We sought to investigate if soluble MER concentrations change in response to myocardial ischemia and reperfusion injury in pediatric patients. In parallel, we also sought to investigate for correlations between the change in soluble MER concentration and specific patient, bypass, and postoperative data. DESIGN: We quantified the change in plasma soluble MER concentration post- compared with precardiopulmonary bypass for each patient in a cohort of pediatric patients. Linear regression, correlation coefficients, and t tests were used to compare innate patient characteristics (i.e., sex, age, cyanotic vs acyanotic cardiac lesion), cardiac bypass data (i.e., total cardiac bypass time, total aortic cross-clamp time, perioperative steroid administration), and postcardiac bypass data (total postoperative ventilator days, total postoperative vasoactive medication days, and total postoperative ICU days) with change in soluble MER concentrations. SETTING: Whole blood samples were obtained intraoperatively at a single tertiary care children's hospital from April to October 2019. SUBJECTS: Our patient cohort included 24 pediatric patients ages ranging from birth to 19 years old with both cyanotic and acyanotic cardiac lesions. INTERVENTIONS: Retrospective analyses of pediatric blood specimens, as well as patient, bypass, and postoperative data, were performed. MEASUREMENTS AND MAIN RESULTS: We observed a statistically significant increase in soluble MER concentration post cardiac bypass in 17 of 24 patients (71%). CONCLUSIONS: Soluble MER concentrations increase with cardiopulmonary bypass-induced inflammation and myocardial ischemia and reperfusion injury in pediatric patients. The utility of soluble MER as a clinical biomarker to identify pediatric patients at risk for exacerbated postoperative outcomes after bypass-induced myocardial ischemia and reperfusion injury requires further investigation.
Assuntos
Isquemia Miocárdica , Traumatismo por Reperfusão , Adulto , Ponte Cardiopulmonar/efeitos adversos , Criança , Humanos , Inflamação/etiologia , Estudos RetrospectivosRESUMO
OBJECTIVES: Focusing on critically ill children with cardiac disease, we will review common causes of fluid perturbations, clinical recognition, and strategies to minimize and treat fluid-related complications. DATA SOURCE: MEDLINE and PubMed. CONCLUSIONS: Meticulous fluid management is vital in critically ill children with cardiac disease. Fluid therapy is important to maintain adequate blood volume and perfusion pressure in order to support cardiac output, tissue perfusion, and oxygen delivery. However, fluid overload and acute kidney injury are common and are associated with increased morbidity and mortality. Understanding the etiologies for disturbances in volume status and the pathophysiology surrounding those conditions is crucial for providing optimal care.
Assuntos
Injúria Renal Aguda/terapia , Estado Terminal/terapia , Hidratação/métodos , Terapia de Substituição Renal/métodos , Injúria Renal Aguda/complicações , Criança , Hidratação/efeitos adversos , HumanosRESUMO
Infection with the protozoan parasite Trypanosoma cruzi may lead to a potentially fatal cardiomyopathy known as Chagas heart disease. This disease is characterized by infiltration of the myocardium by mononuclear cells, including CD4+ T cells, together with edema, myofibrillary destruction, and fibrosis. A multifaceted systemic immune response develops that ultimately keeps parasitemia and tissue parasitosis low. T helper 1 and other pro-inflammatory T cell responses are effective at keeping levels of T. cruzi low in tissues and blood, but they may also lead to tissue inflammation when present chronically. The mechanism by which the inflammatory response is regulated in T. cruzi-infected individuals is complex, and the specific roles that Th17 and T regulatory (Treg) cells may play in that regulation are beginning to be elucidated. In this study, we found that depletion of Treg cells in T. cruzi-infected mice leads to reduced cardiac parasitosis and inflammation, accompanied by an augmented Th1 response early in the course of infection. This is followed by a downregulation of the Th1 response and increased Th17 response late in infection. The effect of Treg cell depletion on the Th1 and Th17 cells is not observed in mice immunized with T. cruzi in adjuvant. This suggests that Treg cells specifically regulate Th1 and Th17 cell responses during T. cruzi infection and may also be important for modulating parasite clearance and inflammation in the myocardium of T. cruzi-infected individuals.
Assuntos
Doença de Chagas/parasitologia , Coração/parasitologia , Inflamação/parasitologia , Linfócitos T Reguladores/fisiologia , Trypanosoma cruzi/fisiologia , Animais , Doença de Chagas/patologia , Imunização , Camundongos , Miocárdio/imunologia , Parasitemia/imunologia , Linfócitos T Reguladores/imunologia , Células Th17/imunologiaRESUMO
This review offers a critical-care perspective on the pathophysiology, monitoring, and management of acute heart failure syndromes in children. An in-depth understanding of the cardiovascular physiological disturbances in this population of patients is essential to correctly interpret clinical signs, symptoms and monitoring data, and to implement appropriate therapies. In this regard, the myocardial force-velocity relationship, the Frank-Starling mechanism, and pressure-volume loops are discussed. A variety of monitoring modalities are used to provide insight into the haemodynamic state, clinical trajectory, and response to treatment. Critical-care treatment of acute heart failure is based on the fundamental principles of optimising the delivery of oxygen and minimising metabolic demands. The former may be achieved by optimising systemic arterial oxygen content and the variables that determine cardiac output: heart rate and rhythm, preload, afterload, and contractility. Metabolic demands may be decreased by a number of ways including positive pressure ventilation, temperature control, and sedation. Mechanical circulatory support should be considered for refractory cases. In the near future, monitoring modalities may be improved by the capture and analysis of complex clinical data such as pressure waveforms and heart rate variability. Using predictive modelling and streaming analytics, these data may then be used to develop automated, real-time clinical decision support tools. Given the barriers to conducting multi-centre trials in this population of patients, the thoughtful analysis of data from multi-centre clinical registries and administrative databases will also likely have an impact on clinical practice.
Assuntos
Cuidados Críticos/métodos , Insuficiência Cardíaca/terapia , Pediatria , Respiração com Pressão Positiva/métodos , Doença Aguda , Pressão Sanguínea , Débito Cardíaco , Insuficiência Cardíaca/fisiopatologia , Frequência Cardíaca , Hemodinâmica , HumanosRESUMO
The flagellar calcium-binding protein (FCaBP) of Trypanosoma cruzi is localized to the flagellar membrane in all life cycle stages of the parasite. Myristoylation and palmitoylation of the N terminus of FCaBP are necessary for flagellar membrane targeting. Not all dually acylated proteins in T. cruzi are flagellar, however. Other determinants of FCaBP therefore likely contribute to flagellar specificity. We generated T. cruzi transfectants expressing the N-terminal 24 or 12 amino acids of FCaBP fused to GFP. Analysis of these mutants revealed that although amino acids 1-12 are sufficient for dual acylation and membrane binding, amino acids 13-24 are required for flagellar specificity and lipid raft association. Mutagenesis of several conserved lysine residues in the latter peptide demonstrated that these residues are essential for flagellar targeting and lipid raft association. Finally, FCaBP was expressed in the protozoan Leishmania amazonensis, which lacks FCaBP. The flagellar localization and membrane association of FCaBP in L. amazonensis suggest that the mechanisms for flagellar targeting, including a specific palmitoyl acyltransferase, are conserved in this organism.
Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Cílios/metabolismo , Membranas Intracelulares/metabolismo , Proteínas de Protozoários/metabolismo , Trypanosoma cruzi/metabolismo , Acilação/efeitos dos fármacos , Sequência de Aminoácidos , Proteínas de Ligação ao Cálcio/química , Sequência Conservada , Detergentes/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Leishmania/efeitos dos fármacos , Leishmania/metabolismo , Lisina/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ácido Mirístico/metabolismo , Palmitatos/metabolismo , Ligação Proteica/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteínas de Protozoários/química , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Trypanosoma cruzi/efeitos dos fármacosRESUMO
Many eukaryotic proteins are posttranslationally modified by the esterification of cysteine thiols to long-chain fatty acids. This modification, protein palmitoylation, is catalyzed by a large family of palmitoyl acyltransferases that share an Asp-His-His-Cys Cys-rich domain but differ in their subcellular localizations and substrate specificities. In Trypanosoma brucei, the flagellated protozoan parasite that causes African sleeping sickness, protein palmitoylation has been observed for a few proteins, but the extent and consequences of this modification are largely unknown. We undertook the present study to investigate T. brucei protein palmitoylation at both the enzyme and substrate levels. Treatment of parasites with an inhibitor of total protein palmitoylation caused potent growth inhibition, yet there was no effect on growth by the separate, selective inhibition of each of the 12 individual T. brucei palmitoyl acyltransferases. This suggested either that T. brucei evolved functional redundancy for the palmitoylation of essential palmitoyl proteins or that palmitoylation of some proteins is catalyzed by a noncanonical transferase. To identify the palmitoylated proteins in T. brucei, we performed acyl biotin exchange chemistry on parasite lysates, followed by streptavidin chromatography, two-dimensional liquid chromatography-tandem mass spectrometry protein identification, and QSpec statistical analysis. A total of 124 palmitoylated proteins were identified, with an estimated false discovery rate of 1.0%. This palmitoyl proteome includes all of the known palmitoyl proteins in procyclic-stage T. brucei as well as several proteins whose homologues are palmitoylated in other organisms. Their sequences demonstrate the variety of substrate motifs that support palmitoylation, and their identities illustrate the range of cellular processes affected by palmitoylation in these important pathogens.
Assuntos
Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Trypanosoma brucei brucei/metabolismo , Tripanossomíase Africana/parasitologia , Sequência de Aminoácidos , Humanos , Lipoilação , Espectrometria de Massas , Dados de Sequência Molecular , Proteínas de Protozoários/genética , Alinhamento de Sequência , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/crescimento & desenvolvimentoRESUMO
Dynamic post-translational modifications allow the rapid, specific, and tunable regulation of protein functions in eukaryotic cells. S-acylation is the only reversible lipid modification of proteins, in which a fatty acid, usually palmitate, is covalently attached to a cysteine residue of a protein by a zDHHC palmitoyl acyltransferase enzyme. Depalmitoylation is required for acylation homeostasis and is catalyzed by an enzyme from the alpha/beta hydrolase family of proteins usually acyl-protein thioesterase (APT1). The enzyme responsible for depalmitoylation in Trypanosoma brucei parasites is currently unknown. We demonstrate depalmitoylation activity in live bloodstream and procyclic form trypanosomes sensitive to dose-dependent inhibition with the depalmitoylation inhibitor, palmostatin B. We identified a homologue of human APT1 in Trypanosoma brucei which we named TbAPT-like (TbAPT-L). Epitope-tagging of TbAPT-L at N- and C- termini indicated a cytoplasmic localization. Knockdown or over-expression of TbAPT-L in bloodstream forms led to robust changes in TbAPT-L mRNA and protein expression but had no effect on parasite growth in vitro, or cellular depalmitoylation activity. Esterase activity in cell lysates was also unchanged when TbAPT-L was modulated. Unexpectedly, recombinant TbAPT-L possesses esterase activity with specificity for short- and medium-chain fatty acid substrates, leading to the conclusion, TbAPT-L is a lipase, not a depalmitoylase.
RESUMO
African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.
Assuntos
Proteínas de Ligação ao Cálcio/antagonistas & inibidores , Parasitemia/parasitologia , Proteínas de Protozoários/antagonistas & inibidores , Trypanosoma brucei brucei , Tripanossomíase Africana/parasitologia , Animais , Sítios de Ligação , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Proliferação de Células , Camundongos , Camundongos Endogâmicos BALB C , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Interferência de RNARESUMO
The protozoan parasite Trypanosoma cruzi, the etiologic agent of Chagas disease, is an obligate intracellular protozoan pathogen. Overlapping mechanisms ensure successful infection, yet the relationship between these cellular events and clinical disease remains obscure. This review explores the process of cell invasion from the perspective of cell surface interactions, intracellular signaling, modulation of the host cytoskeleton and endosomal compartment, and the intracellular innate immune response to infection.
Assuntos
Doença de Chagas/parasitologia , Trypanosoma cruzi/fisiologia , Animais , Cálcio/metabolismo , Membrana Celular/parasitologia , Doença de Chagas/imunologia , Doença de Chagas/patologia , Citoplasma/parasitologia , Citoesqueleto/parasitologia , Matriz Extracelular/parasitologia , Interações Hospedeiro-Parasita , Humanos , Insetos Vetores/parasitologia , Glicoproteínas de Membrana/metabolismo , Proteínas de Protozoários/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologiaRESUMO
While hyperlactatemia in postoperative cardiac surgery patients was once believed to solely reflect hypoperfusion, either from the accumulated "oxygen debt" during bypass or ongoing inadequate perfusion, our understanding of lactate generation, clearance, and management has evolved. A contemporary understanding of lactate balance is critical to the management of the postoperative patient with hyperlactatemia. In this review, we summarize the current understanding of lactate metabolism in pediatric patients following cardiac surgery and highlight two types of hyperlactatemia: type A, which is secondary to inadequate oxygen delivery and tissue hypoxia, and type B, which in postoperative pediatric cardiac surgery patients largely reflects increased glycolysis driven by the stress response. Both types may coexist; thus, it is imperative that providers first assess the patient for evidence of hypoperfusion. In patients with evidence of adequate perfusion, a type B component is often associated with a concomitant balanced (normal anion gap) metabolic acidosis and hyperglycemia. These patients will benefit from a more nuanced approach to their type B hyperlactatemia, as many will have a benign course and may be managed expectantly.
Assuntos
Acidose Láctica/metabolismo , Procedimentos Cirúrgicos Cardíacos , Hiperlactatemia/complicações , Hiperlactatemia/diagnóstico , Ácido Láctico/metabolismo , Complicações Pós-Operatórias/terapia , Biomarcadores , Feminino , Humanos , Hipóxia/terapia , Masculino , Oxigênio/metabolismo , Perfusão , Período Pós-OperatórioRESUMO
Human induced pluripotent stem cell (hiPSC) culture has become routine, yet the cost of pluripotent cell media, frequent medium changes, and the reproducibility of differentiation have remained restrictive. Here, we describe the formulation of a hiPSC culture medium (B8) as a result of the exhaustive optimization of medium constituents and concentrations, establishing the necessity and relative contributions of each component to the pluripotent state and cell proliferation. The reagents in B8 represent only 3% of the costs of commercial media, made possible primarily by the in-lab generation of three E. coli-expressed, codon-optimized recombinant proteins: fibroblast growth factor 2, transforming growth factor ß3, and neuregulin 1. We demonstrate the derivation and culture of 34 hiPSC lines in B8 as well as the maintenance of pluripotency long term (over 100 passages). This formula also allows a weekend-free feeding schedule without sacrificing capacity for differentiation.
Assuntos
Técnicas de Cultura de Células/economia , Técnicas de Cultura de Células/métodos , Células-Tronco Pluripotentes Induzidas/citologia , Bioensaio , Diferenciação Celular , Proliferação de Células , Células Cultivadas , HumanosRESUMO
Stem cell antigen-1 (Sca-1, Ly6A/E) is a glycosylphosphotidylinositol-anchored protein that identifies many tissue progenitor cells. We originally identified Sca-1 as a marker of myogenic precursor cells and subsequently demonstrated that Sca-1 regulates proliferation of activated myoblasts, suggesting an important role for Sca-1 in skeletal muscle homeostasis. Beyond its functional role in regulating proliferation, however, little is known about the mechanism(s) that drive Sca-1-mediated events. We now report that lipid microdomain organization is essential for normal myogenic differentiation, and that Sca-1 constitutively localizes to these domains during myoblast proliferation and differentiation. We also demonstrate that Sca-1 associates with insulin degrading enzyme (IDE), a catalytic protein responsible for the cleavage of mitogenic peptides, in differentiating myoblasts. We show that chemical inhibition of IDE as well as RNAi knockdown of IDE mRNA recapitulates the phenotype of Sca-1 interference, that is, sustained myoblast proliferation and delayed myogenic differentiation. These findings identify the first signaling protein that physically and functionally associates with Sca-1 in myogenic precursor cells, and suggest a potential pathway for Sca-1-mediated signaling. Future efforts to manipulate this pathway may lead to new strategies for augmenting the myogenic proliferative response, and ultimately muscle repair.
Assuntos
Antígenos Ly/metabolismo , Insulisina/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Mioblastos Esqueléticos/metabolismo , Transdução de Sinais/fisiologia , Animais , Western Blotting , Diferenciação Celular/fisiologia , Proliferação de Células , Células Cultivadas , Citometria de Fluxo , Imunofluorescência , Imunoprecipitação , Camundongos , Microscopia Confocal , Mioblastos Esqueléticos/citologia , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Cilia play important roles in cell signaling, facilitated by the unique lipid environment of a ciliary membrane containing high concentrations of sterol-rich lipid rafts. The African trypanosome Trypanosoma brucei is a single-celled eukaryote with a single cilium/flagellum. We tested whether flagellar sterol enrichment results from selective flagellar partitioning of specific sterol species or from general enrichment of all sterols. While all sterols are enriched in the flagellum, cholesterol is especially enriched. T. brucei cycles between its mammalian host (bloodstream cell), in which it scavenges cholesterol, and its tsetse fly host (procyclic cell), in which it both scavenges cholesterol and synthesizes ergosterol. We wondered whether the insect and mammalian life cycle stages possess chemically different lipid rafts due to different sterol utilization. Treatment of bloodstream parasites with cholesterol-specific methyl-ß-cyclodextrin disrupts both membrane liquid order and localization of a raft-associated ciliary membrane calcium sensor. Treatment with ergosterol-specific amphotericin B does not. The opposite results were observed with ergosterol-rich procyclic cells. Further, these agents have opposite effects on flagellar sterol enrichment and cell metabolism in the two life cycle stages. These findings illuminate differences in the lipid rafts of an organism employing life cycle-specific sterols and have implications for treatment.
Assuntos
Microdomínios da Membrana/química , Esteróis/análise , Trypanosoma brucei brucei/crescimento & desenvolvimento , Moscas Tsé-Tsé/parasitologia , Anfotericina B/farmacologia , Animais , Colesterol/análise , Ergosterol/análise , Flagelos/química , Estágios do Ciclo de Vida/efeitos dos fármacos , Microdomínios da Membrana/efeitos dos fármacos , Trypanosoma brucei brucei/química , Trypanosoma brucei brucei/efeitos dos fármacos , beta-Ciclodextrinas/farmacologiaRESUMO
African trypanosomiasis is caused by infection with the protozoan parasite Trypanosoma brucei During infection, this pathogen divides rapidly to high density in the bloodstream of its mammalian host in a manner similar to that of leukemia. Like all eukaryotes, T. brucei has a cell cycle involving the de novo synthesis of DNA regulated by ribonucleotide reductase (RNR), which catalyzes the conversion of ribonucleotides into their deoxy form. As an essential enzyme for the cell cycle, RNR is a common target for cancer chemotherapy. We hypothesized that inhibition of RNR by genetic or pharmacological means would impair parasite growth in vitro and prolong the survival of infected animals. Our results demonstrate that RNR inhibition is highly effective in suppressing parasite growth both in vitro and in vivo These results support drug discovery efforts targeting the cell cycle, not only for African trypanosomiasis but possibly also for other infections by eukaryotic pathogens.IMPORTANCE The development of drugs to treat infections with eukaryotic pathogens is challenging because many key virulence factors have closely related homologues in humans. Drug toxicity greatly limits these development efforts. For pathogens that replicate at a high rate, especially in the blood, an alternative approach is to target the cell cycle directly, much as is done to treat some hematologic malignancies. The results presented here indicate that targeting the cell cycle via inhibition of ribonucleotide reductase is effective at killing trypanosomes and prolonging the survival of infected animals.
Assuntos
Ciclo Celular/efeitos dos fármacos , Inibidores Enzimáticos/uso terapêutico , Hidroxiureia/uso terapêutico , Ribonucleotídeo Redutases/antagonistas & inibidores , Tripanossomicidas/uso terapêutico , Trypanosoma brucei brucei/efeitos dos fármacos , Tripanossomíase Africana/tratamento farmacológico , Animais , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/farmacologia , Camundongos , Ribonucleotídeo Redutases/metabolismo , Tripanossomicidas/farmacologia , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/genética , Trypanosoma brucei brucei/fisiologia , Tripanossomíase Africana/parasitologiaRESUMO
SIRT2 is a cytoplasmic sirtuin that plays a role in various cellular processes, including tumorigenesis, metabolism, and inflammation. Since these processes require iron, we hypothesized that SIRT2 directly regulates cellular iron homeostasis. Here, we have demonstrated that SIRT2 depletion results in a decrease in cellular iron levels both in vitro and in vivo. Mechanistically, we determined that SIRT2 maintains cellular iron levels by binding to and deacetylating nuclear factor erythroid-derived 2-related factor 2 (NRF2) on lysines 506 and 508, leading to a reduction in total and nuclear NRF2 levels. The reduction in nuclear NRF2 leads to reduced ferroportin 1 (FPN1) expression, which in turn results in decreased cellular iron export. Finally, we observed that Sirt2 deletion reduced cell viability in response to iron deficiency. Moreover, livers from Sirt2-/- mice had decreased iron levels, while this effect was reversed in Sirt2-/- Nrf2-/- double-KO mice. Taken together, our results uncover a link between sirtuin proteins and direct control over cellular iron homeostasis via regulation of NRF2 deacetylation and stability.
Assuntos
Ferro/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Processamento de Proteína Pós-Traducional , Sirtuína 2/fisiologia , Acetilação , Animais , Proteínas de Transporte de Cátions/metabolismo , Epigênese Genética , Expressão Gênica , Células HEK293 , Células Hep G2 , Homeostase , Humanos , Fígado/metabolismo , Camundongos Knockout , Estabilidade Proteica , Ativação TranscricionalRESUMO
Low cardiac output syndrome frequently complicates the post-operative care of infants and children following cardiac surgery. The onset of low cardiac output follows a predictable course in the hours following cardiopulmonary bypass, as myocardial performance declines in the face of an elevated demand for cardiac output. When demand outstrips supply, shock ensues, and early recognition and intervention can decrease mortality. Multifactorial in etiology, this article will discuss the pathophysiology of low cardiac output syndrome, including myocardial depression following bypass, altered cardiac loading conditions, and inflammation driving a hypermetabolic state. Contributions from altered neurohormonal, thyroid, and adrenal axes will also be discussed. Sources included the clinical experiences of four cardiac intensivists, supported throughout by primary sources and relevant reviews obtained through PubMed searches and from seminal textbooks in the field. This article addresses the second of eight topics comprising the special issue entitled "Pharmacologic strategies with afterload reduction in low cardiac output syndrome after pediatric cardiac surgery".
Assuntos
Baixo Débito Cardíaco/fisiopatologia , Procedimentos Cirúrgicos Cardíacos/efeitos adversos , Complicações Pós-Operatórias/fisiopatologia , Débito Cardíaco/fisiologia , Baixo Débito Cardíaco/epidemiologia , Baixo Débito Cardíaco/etiologia , Ponte Cardiopulmonar/efeitos adversos , Criança , Humanos , Lactente , Miocárdio/patologia , Complicações Pós-Operatórias/epidemiologiaRESUMO
We report the case of a child with both propionic acidemia and cyanotic congenital heart disease. The presence of an underlying inborn error of metabolism confounded the management of this patient in the postoperative period, resulting in therapeutic misdirection until the true etiology of hyperlactemia was recognized.
Assuntos
Ponte Cardiopulmonar/métodos , Cardiopatias Congênitas/cirurgia , Complicações Pós-Operatórias/etiologia , Acidemia Propiônica/complicações , Acidose Láctica/diagnóstico , Acidose Láctica/etiologia , Diagnóstico Diferencial , Cardiopatias Congênitas/complicações , Humanos , Recém-Nascido , Masculino , Complicações Pós-Operatórias/diagnóstico , Acidemia Propiônica/diagnósticoRESUMO
UNLABELLED: Sphingolipids are important constituents of cell membranes and also serve as mediators of cell signaling and cell recognition. Sphingolipid metabolites such as sphingosine-1-phosphate and ceramide regulate signaling cascades involved in cell proliferation and differentiation, autophagy, inflammation, and apoptosis. Little is known about how sphingolipids and their metabolites function in single-celled eukaryotes. In the present study, we investigated the role of sphingosine kinase (SPHK) in the biology of the protozoan parasite Trypanosoma brucei, the agent of African sleeping sickness. T. brucei SPHK (TbSPHK) is constitutively but differentially expressed during the life cycle of T. brucei. Depletion of TbSPHK in procyclic-form T. brucei causes impaired growth and attenuation in the G1/S phase of the cell cycle. TbSPHK-depleted cells also develop organelle positioning defects and an accumulation of tyrosinated α-tubulin at the elongated posterior end of the cell, known as the "nozzle" phenotype, caused by other molecular perturbations in this organism. Our studies indicate that TbSPHK is involved in G1-to-S cell cycle progression, organelle positioning, and maintenance of cell morphology. Cytotoxicity assays using TbSPHK inhibitors revealed a favorable therapeutic index between T. brucei and human cells, suggesting TbSPHK to be a novel drug target. IMPORTANCE: Trypanosoma brucei is a single-celled parasite that is transmitted between humans and other animals by the tsetse fly. T. brucei is endemic in sub-Saharan Africa, where over 70 million people and countless livestock are at risk of developing T. brucei infection, called African sleeping sickness, resulting in economic losses of ~$35 million from the loss of cattle alone. New drugs for this infection are sorely needed and scientists are trying to identify essential enzymes in the parasite that can be targets for new therapies. One possible enzyme target is sphingosine kinase, an enzyme involved in the synthesis of lipids important for cell surface integrity and regulation of cell functions. In this study, we found that sphingosine kinase is essential for normal growth and structure of the parasite, raising the possibility that it could be a good target for new chemotherapy for sleeping sickness.
Assuntos
Ciclo Celular , Proliferação de Células , Microtúbulos/metabolismo , Biogênese de Organelas , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Trypanosoma brucei brucei/fisiologia , Sequência de Aminoácidos , Transporte Biológico , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Trypanosoma brucei brucei/enzimologia , Trypanosoma brucei brucei/crescimento & desenvolvimento , Trypanosoma brucei brucei/metabolismoRESUMO
OBJECTIVE: The objective of this study was to characterize the natural history of metabolic uncoupling (type B hyperlactemia and hyperglycemia) following cardiopulmonary bypass (CPB), and to determine the impact of insulin therapy on time to lactate normalization in patients without low cardiac output. DESIGN: The design used was a retrospective cohort study. SETTING: The study was set in a pediatric cardiac intensive care unit in a tertiary-care urban children's hospital. PATIENTS: All patients were aged ≤21 years admitted between 2007 and 2013 following cardiac surgery involving CPB with empiric intraoperative corticosteroids. ELIGIBILITY CRITERIA: simultaneous hyperlactemia (≥3.5 mEq/L) and hyperglycemia (≥200 mg/dL) within 48 hours after bypass. EXCLUSION CRITERIA: Exclusion criteria were evidence of low cardiac output state, diabetes or postoperative steroid administration. INTERVENTIONS: Characteristics were compared between those treated with insulin and those who were not (controls). OUTCOME MEASURES: Outcome measures used were time from admission to onset of hyperglycemia and hyperlactemia and time to resolution. Clinical outcomes included duration of mechanical ventilation, length of stay, unplanned readmission/reoperation, hypoglycemia and death. RESULTS: Of the 1345 patients receiving CPB, 132 (9.8%) met inclusion criteria. Seventy-eight (59%) were treated with insulin, leaving 54 controls. Patient characteristics, surgical complexity and time to onset of hyperglycemia and hyperlactemia were similar between groups. The insulin group had a shorter duration of hyperglycemia. There was no significant difference between groups in time to lactate normalization, ventilator days, length of stay, readmission and reoperation rates. Hypoglycemia (<60 mg/dL) occurred in three patients. CONCLUSIONS: In children with metabolic uncoupling after CPB, insulin use did not shorten the time to lactate normalization or alter clinical outcomes. These findings suggest that type B hyperlactemia with hyperglycemia after CPB will resolve spontaneously and does not warrant specific treatment.