RESUMO
Health care costs now comprise nearly one-fifth of the United States' gross domestic product, with the last 25 years marked by rising administrative costs, a lack of labor productivity growth, and rising patient and physician dissatisfaction. Policy experts have responded with a series of reforms that have - ironically - increased patient and physician administrative burden with little meaningful effect on cost and quality. Artificial intelligence (AI), a topic of great consternation, can serve as the "wheat thresher" for health care delivery, empowering and freeing both patients and physicians by decreasing administrative burden and improving labor productivity. In this Viewpoint, we discuss three principal areas where AI poses an unprecedented opportunity to reduce cost, improve care, and markedly enhance the patient and physician experience: (1) automation of administrative process, (2) augmentation of clinical practice, and (3) automation of elements of clinical practice.
Assuntos
Inteligência Artificial , Médicos , Humanos , Estados Unidos , Atenção à SaúdeRESUMO
The mitochondrial ATP dependent matrix protease, Lon, is involved in the maintenance of mitochondrial DNA nucleoids and degradation of abnormal or misfolded proteins. The Lon protease regulates mitochondrial Tfam (mitochondrial transcription factor A) level and thus modulates mitochondrial DNA (mtDNA) content. We have previously shown that hypoxic stress induces the PKA-dependent phosphorylation of cytochrome c oxidase (CcO) subunits I, IVi1, and Vb and a time-dependent reduction of these subunits in RAW 264.7 murine macrophages subjected to hypoxia and rabbit hearts subjected to ischemia/reperfusion. Here, we show that Lon is involved in the preferential turnover of phosphorylated CcO subunits under hypoxic/ischemic stress. Induction of Lon protease occurs at 6 to 12 h of hypoxia and this increase coincides with lower CcO subunit contents. Over-expression of flag-tagged wild type and phosphorylation site mutant Vb and IVi1 subunits (S40A and T52A, respectively) caused marked degradation of wild type protein under hypoxia while the mutant proteins were relatively resistant. Furthermore, the recombinant purified Lon protease degraded the phosphorylated IVi1 and Vb subunits, while the phosphorylation-site mutant proteins were resistant to degradation. 3D structural modeling shows that the phosphorylation sites are exposed to the matrix compartment, accessible to matrix PKA and Lon protease. Hypoxic stress did not alter CcO subunit levels in Lon depleted cells, confirming its role in CcO turnover. Our results therefore suggest that Lon preferentially degrades the phosphorylated subunits of CcO and plays a role in the regulation of CcO activity in hypoxia and ischemia/reperfusion injury.
Assuntos
Proteases Dependentes de ATP/metabolismo , Hipóxia Celular/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias Cardíacas/enzimologia , Proteínas Mitocondriais/metabolismo , Isquemia Miocárdica/enzimologia , Proteases Dependentes de ATP/química , Proteases Dependentes de ATP/genética , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Humanos , Masculino , Camundongos , Proteínas Mitocondriais/química , Proteínas Mitocondriais/genética , Modelos Moleculares , Fosforilação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Subunidades Proteicas , Células RAW 264.7 , Interferência de RNA , RNA Interferente Pequeno/genética , Coelhos , Proteínas Recombinantes/metabolismoRESUMO
Malaria control in Somaliland depends on the effective identification of potential malaria vectors, particularly those that may be invasive. The malaria vector Anopheles stephensi has been detected in multiple countries in the Horn of Africa (HOA), but data on its geographic distribution and population genetic diversity are incomplete. We implemented a vector surveillance program and performed molecular analysis of Anopheles in three urban areas in Somaliland. Our study confirmed the presence of both the invasive An. stephensi and the long-established HOA malaria vector Anopheles arabiensis. Further analysis of An. stephensi genetic diversity revealed three cytochrome oxidase I (COI) haplotypes, all of which have been observed in other countries in East Africa and one also observed in South Asia. We also detected the knockdown resistance (kdr) L1014F mutation, which is associated with pyrethroid resistance; this finding supports the need for further assessment of the potential for insecticide resistance. The detection of multiple haplotypes previously observed in other regions of East Africa indicates that An. stephensi is an established population in Somaliland and likely shares its origin with other newly identified An. stephensi populations in East Africa. The detection of genetic diversity in An. stephensi in Somaliland provides a basis for future studies on the history of the species in the region and its dispersal throughout East Africa.
Assuntos
Anopheles , Inseticidas , Malária , Animais , Anopheles/genética , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Mosquitos Vetores/genética , MutaçãoRESUMO
Anopheles stephensi is a malaria vector that has been recently introduced into East Africa, where it threatens to increase malaria disease burden. The use of insecticides, especially pyrethroids, is still one of the primary malaria vector control strategies worldwide. The knockdown resistance (kdr) mutation in the IIS6 transmembrane segment of the voltage-gated sodium channel (vgsc) is one of the main molecular mechanisms of pyrethroid resistance in Anopheles. Extensive pyrethroid resistance in An. stephensi has been previously reported in Ethiopia. Thus, it is important to determine whether or not the kdr mutation is present in An. stephensi populations in Ethiopia to inform vector control strategies. In the present study, the kdr locus was analyzed in An. stephensi collected from ten urban sites (Awash Sebat Kilo, Bati, Dire Dawa, Degehabur, Erer Gota, Godey, Gewane, Jigjiga, Semera, and Kebridehar) situated in Somali, Afar, and Amhara regions, and Dire Dawa Administrative City, to evaluate the frequency and evolution of kdr mutations and the association of the mutation with permethrin resistance phenotypes. Permethrin is one of the pyrethroid insecticides used for vector control in eastern Ethiopia. DNA extractions were performed on adult mosquitoes from CDC light trap collections and those raised from larval and pupal collections. PCR and targeted sequencing were used to analyze the IIS6 transmembrane segment of the vgsc gene. Of 159 An. stephensi specimens analyzed from the population survey, nine (5.7%) carried the kdr mutation (L1014F). An. stephensi with kdr mutations were only observed from Bati, Degehabur, Dire Dawa, Gewane, and Semera. We further selected randomly twenty resistant and twenty susceptible An. stephensi mosquitoes from Dire Dawa post-exposure to permethrin and investigated the role of kdr in pyrethroid resistance by comparing the vgsc gene in the two populations. We found no kdr mutations in the permethrin-resistant mosquitoes. Population genetic analysis of the sequences, including neighboring introns, revealed limited evidence of non-neutral evolution (e.g., selection) at this locus. The low kdr mutation frequency detected and the lack of kdr mutation in the permethrin-resistant mosquitoes suggest the existence of other molecular mechanisms of pyrethroid resistance in eastern Ethiopian An. stephensi.
Assuntos
Anopheles , Inseticidas , Malária , Piretrinas , Animais , Anopheles/genética , Etiópia , Genética Populacional , Resistência a Inseticidas/genética , Inseticidas/farmacologia , Malária/prevenção & controle , Mosquitos Vetores/genética , Mutação , Permetrina , Piretrinas/farmacologiaRESUMO
BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in the Horn of Africa (HOA) raises concerns about the impact of this mosquito on malaria transmission in the region. Analysis of An. stephensi genetic diversity and population structure can provide insight into the history of the mosquito in the HOA to improve predictions of future spread. We investigated the genetic diversity of An. stephensi in eastern Ethiopia, where detection suggests a range expansion into this region, in order to understand the history of this invasive population. METHODS: We sequenced the cytochrome oxidase subunit I (COI) and cytochrome B gene (CytB) in 187 An. stephensi collected from 10 sites in Ethiopia in 2018. Population genetic, phylogenetic, and minimum spanning network analyses were conducted for Ethiopian sequences. Molecular identification of blood meal sources was also performed using universal vertebrate CytB sequencing. RESULTS: Six An. stephensi COI-CytB haplotypes were observed, with the highest number of haplotypes in the northeastern sites (Semera, Bati, and Gewana towns) relative to the southeastern sites (Kebridehar, Godey, and Degehabur) in eastern Ethiopia. We observed population differentiation, with the highest differentiation between the northeastern sites compared to central sites (Erer Gota, Dire Dawa, and Awash Sebat Kilo) and the southeastern sites. Phylogenetic and network analysis revealed that the HOA An. stephensi are more genetically similar to An. stephensi from southern Asia than from the Arabian Peninsula. Finally, molecular blood meal analysis revealed evidence of feeding on cows, goats, dogs, and humans, as well as evidence of multiple (mixed) blood meals. CONCLUSION: We show that An. stephensi is genetically diverse in Ethiopia and with evidence of geographical structure. Variation in the level of diversity supports the hypothesis for a more recent introduction of An. stephensi into southeastern Ethiopia relative to the northeastern region. We also find evidence that supports the hypothesis that HOA An. stephensi populations originate from South Asia rather than the Arabian Peninsula. The evidence of both zoophagic and anthropophagic feeding support the need for additional investigation into the potential for livestock movement to play a role in vector spread in this region.
Assuntos
Anopheles/genética , Variação Genética , Malária/transmissão , Mosquitos Vetores/genética , Animais , Citocromos b/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Etiópia , Genética Populacional , Haplótipos , FilogeniaRESUMO
BACKGROUND: The recent detection of the South Asian malaria vector Anopheles stephensi in Ethiopia and other regions in the Horn of Africa has raised concerns about its potential impact on malaria transmission. We report here the findings of a survey for this species in eastern Ethiopia using both morphological and molecular methods for species identification. METHODS: Adult and larval/pupal collections were conducted at ten sites in eastern Ethiopia and Anopheles specimens were identified using standard morphological keys and genetic analysis. RESULTS: In total, 2231 morphologically identified An. stephensi were collected. A molecular approach incorporating both PCR endpoint assay and sequencing of portions of the internal transcribed spacer 2 (ITS2) and cytochrome c oxidase subunit 1 (cox1) loci confirmed the identity of the An. stephensi in most cases (119/124 of the morphologically identified An. stephensi confirmed molecularly). Additionally, we observed Aedes aegypti larvae and pupae at many of the An. stephensi larval habitats. CONCLUSIONS: Our findings show that An. stephensi is widely distributed in eastern Ethiopia and highlight the need for further surveillance in the southern, western and northern parts of the country and throughout the Horn of Africa.
Assuntos
Anopheles/fisiologia , Malária/transmissão , Mosquitos Vetores/fisiologia , Aerossóis , Animais , Estudos Transversais , Etiópia/epidemiologia , Habitação/classificação , Inseticidas/administração & dosagem , Malária/epidemiologia , Reação em Cadeia da Polimerase , Estações do AnoRESUMO
We have mapped the sites of ischemia/reperfusion-induced phosphorylation of cytochrome c oxidase (CcO) subunits in rabbit hearts by using a combination of Blue Native gel/Tricine gel electrophoresis and nano-LC-MS/MS approaches. We used precursor ion scanning combined with neutral loss scanning and found that mature CcO subunit I was phosphorylated at tandem Ser115/Ser116 positions, subunit IVi1 at Thr52 and subunit Vb at Ser40. These sites are highly conserved in mammalian species. Molecular modeling suggests that phosphorylation sites of subunit I face the inter membrane space while those of subunits IVi1 and Vb face the matrix side.
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Complexo IV da Cadeia de Transporte de Elétrons/química , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Eletroforese em Gel de Poliacrilamida , Glicina/análogos & derivados , Glicina/química , Coração , Dados de Sequência Molecular , Fosforilação , Conformação Proteica , Estrutura Terciária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Coelhos , Serina/metabolismo , Espectrometria de Massas por Ionização por Electrospray , Treonina/metabolismoRESUMO
Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Estresse Oxidativo , Animais , Antioxidantes/farmacologia , Hipóxia Celular/efeitos dos fármacos , Linhagem Celular , Respiração Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/genética , Ativação Enzimática/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Mutação , Isquemia Miocárdica/enzimologia , Isquemia Miocárdica/metabolismo , Isquemia Miocárdica/patologia , Estresse Oxidativo/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/enzimologia , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologiaRESUMO
Protein kinase A (PKA) activation has been implicated in early-phase ischemic preconditioning. We recently found that during ischemia PKA activation causes inactivation of cytochrome-c oxidase (CcO) and contributes to myocardial damage due to ischemia-reperfusion. It may be that beta-adrenergic stimulation during ischemia via endogenous catecholamine release activates PKA. Thus beta-adrenergic stimulation may mediate both myocardial protection and damage during ischemia. The present studies were designed to determine the role of the beta(1)-adrenergic receptor (beta(1)-AR) in myocardial ischemic damage and ischemic preconditioning. Langendorff-perfused rabbit hearts underwent 30-min ischemia by anterior coronary artery ligation followed by 2-h reperfusion. Occlusion-reperfusion damage was evaluated by delineating the nonperfused volume of myocardium at risk and volume of myocardial necrosis after 2-h reperfusion. In some hearts ischemic preconditioning was accomplished by two 5-min episodes of global low-flow ischemia separated by 10 min before coronary occlusion-reperfusion. Orthogonal electrocardiograms were recorded, and coronary flow was monitored by a drip count. Three hearts from each experimental group were used to determine mitochondrial CcO and aconitase activities. Two-hour reperfusion after occlusion caused an additional decrease in CcO activity vs. that after 30-min occlusion alone. Blocking the beta(1)-AR during occlusion-reperfusion reversed CcO activity depression and preserved myocardium at risk for necrosis. Similarly, mitochondrial aconitase activity exhibited a parallel response after occlusion-reperfusion as well as for the other interventions. Furthermore, classic ischemic preconditioning had no effect on CcO depression. However, blocking the beta(1)-AR during preconditioning eliminated the cardioprotection. If the beta(1)-AR was blocked after preconditioning, the myocardium was preserved. Interestingly, in both of the latter cases the depression in CcO activity was reversed. Thus the beta(1)-AR plays a dual role in myocardial ischemic damage. Our findings may lead to therapeutic strategies for preserving myocardium at risk for infarction, especially in coronary reperfusion intervention.
Assuntos
Agonistas de Receptores Adrenérgicos beta 1 , Precondicionamento Isquêmico Miocárdico/métodos , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Receptores Adrenérgicos beta 1/metabolismo , Animais , Masculino , CoelhosRESUMO
We have investigated the effects of hypoxia and myocardial ischemia/reperfusion on the structure and function of cytochrome c oxidase (CcO). Hypoxia (0.1% O(2) for 10 h) and cAMP-mediated inhibition of CcO activity were accompanied by hyperphosphorylation of subunits I, IVi1, and Vb and markedly increased reactive O(2) species production by the enzyme complex in an in vitro system that uses reduced cytochrome c as an electron donor. Both subunit phosphorylation and enzyme activity were effectively reversed by 50 nm H89 or 50 nm myristoylated peptide inhibitor (MPI), specific inhibitors of protein kinase A, but not by inhibitors of protein kinase C. In rabbit hearts subjected to global and focal ischemia, CcO activity was inhibited in a time-dependent manner and was accompanied by hyperphosphorylation as in hypoxia. Additionally, CcO activity and subunit phosphorylation in the ischemic heart were nearly completely reversed by H89 or MPI added to the perfusion medium. Hyperphosphorylation of subunits I, IVi1, and Vb was accompanied by reduced subunit contents of the immunoprecipitated CcO complex. Most interestingly, both H89 and MPI added to the perfusion medium dramatically reduced the ischemia/reperfusion injury to the myocardial tissue. Our results pointed to an exciting possibility of using CcO activity modulators for controlling myocardial injury associated with ischemia and oxidative stress conditions.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Hipóxia , Isquemia/patologia , Miocárdio/patologia , Animais , Monóxido de Carbono , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/química , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Immunoblotting , Imunoprecipitação , Isoquinolinas/farmacologia , Macrófagos/metabolismo , Masculino , Camundongos , Mitocôndrias/metabolismo , Monócitos/metabolismo , Isquemia Miocárdica , Estresse Oxidativo , Oxigênio/metabolismo , Peptídeos/química , Perfusão , Fosforilação , Proteína Quinase C/metabolismo , Coelhos , Espécies Reativas de Oxigênio , Traumatismo por Reperfusão , Sulfonamidas/farmacologia , Fatores de TempoRESUMO
The effects of 20-min ligations of the anterior branch of the left coronary artery were studied in Langendorff-perfused rabbit hearts with 92 microM D-sotalol added to the perfusate to induce long QT intervals and triggered arrhythmias. Epicardial electrograms, a left ventricular endocardial monophasic action potential, and simulated X and Y lead electrocardiograms were used to characterize ventricular conduction and recovery. In contrast to previous work showing that global ischemia eliminated triggered activity, coronary occlusion did not alter its mean incidence. Although the anatomic distribution of earliest sites of epicardial activation by triggered beats was altered, triggered beats still appeared on the epicardial surface in the nonperfused regions. Coronary occlusion had a small and variable effect on epicardial conduction velocity but caused a significantly greater percent shortening of epicardial activation-recovery intervals in the nonperfused region of hearts given D-sotalol than in control hearts. In hearts given D-sotalol, preconditioning significantly attenuated the shortening of epicardial activation-recovery intervals in response to coronary occlusion. However, preconditioning had no effect on the mean incidence of triggered activity during coronary occlusion. Thus, the persistence of triggered activity and the shortened myocardial recovery time associated with coronary occlusion could contribute to increasing the likelihood of occurrence of malignant ventricular arrhythmias. Preconditioning by attenuating the shortening of recovery would be anti-arrhythmic.