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In order to accurately detect the temperature of molten aluminum and overcome the adverse influence of high temperature and corrosiveness on the sensing results, a temperature detection system based on a multi-node sapphire fiber sensor was proposed and developed. Through the structural parameter design of the fiber sensor, the scheme of utilizing the 0.7 mm diameter fiber and 0.5 mm groove was formulated. Simulation and analysis were carried out to determine the ultrasonic response distribution of the signal passing through the whole fiber sensor. The results indicate that the system is capable of distinguishing test signals from various positions and temperatures. Following the completion of the static calibration, the temperature of the molten aluminum was observed in real-time, and the data of the temperature measurements conducted at the two groove locations were compared. According to the obtained results, the test accuracy was greater than 1 degree Celsius and the temperature test stability was good, laying a solid foundation for the potential development of temperature measurement devices.
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CD8+ T cells play a critical role during adaptive immune response, which often change locations and expand or contract in numbers under different states. In the past, many attempts to develop CD8+T cells that express luciferase in vivo have involved the use of viral transduction, which has drawbacks of hardly tracked via detection of luciferase signal in untouched natural states. Here, we generate a transgenic mouse model via CRISPR-mediated genome editing, C57BL/6-CD8aem(IRES-AkaLuci-2A-EGFP) knock-in mice(CD8a-Aka mice), as a novel tool for non-invasive imaging of CD8+ T cells, which expressed a highly sensitive luciferase-Akaluciferase. Our study offers a convenient and robust tool for understanding fundamental CD8+ T cell biology in experimental applications and preclinical translational studies.
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Linfocitos T CD8-positivos/metabolismo , Sistemas CRISPR-Cas , Neoplasias del Colon/diagnóstico por imagen , Efecto Fundador , Edición Génica/métodos , Genoma , Ratones Transgénicos/genética , Animales , Antígenos CD8/genética , Antígenos CD8/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteína 9 Asociada a CRISPR/genética , Proteína 9 Asociada a CRISPR/metabolismo , Línea Celular Tumoral , Neoplasias del Colon/genética , Neoplasias del Colon/inmunología , Diagnóstico por Imagen/métodos , Técnicas de Sustitución del Gen , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Xenoinjertos , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Ratones Transgénicos/inmunología , ARN Guía de Kinetoplastida/genética , ARN Guía de Kinetoplastida/metabolismo , Cigoto/inmunología , Cigoto/metabolismoRESUMEN
PURPOSE: Testosterone affects male development, maturation and aging but limited data exist on testosterone effects on the juvenile genitourinary system. We hypothesized that testosterone has bladder and kidney developmental effects, and investigated this in juvenile male rats. MATERIALS AND METHODS: To examine the testosterone effect 21-day-old prepubertal male Wistar rats were divided into 3 groups of 12 each, including sham orchiectomy as controls, and bilateral orchiectomy with vehicle and bilateral orchiectomy with testosterone. Starting at age 28 days (week 0) testosterone enanthate (5 mg/100 gm) or vehicle was injected weekly. Testosterone was measured at study week 0 before injection, and at weeks 1, 6 and 16. Whole bladders and kidneys were evaluated for androgen receptor, bladder collagen-to-smooth muscle ratio, and renal morphometry and immunohistochemistry. RESULTS: Testosterone was not detectable at week 0 in all groups. It remained undetectable at weeks 1, 6 and 16 in the orchiectomy plus vehicle group. Testosterone levels were physiological in controls and rats with orchiectomy plus testosterone but levels were higher in the latter than in the former group. Rats with orchiectomy plus testosterone had increased bladder-to-body and kidney-to-body weight ratios (p<0.01 and <0.05, respectively), and decreased collagen-to-smooth muscle ratio than the orchiectomy plus vehicle and control groups. Rats with orchiectomy plus testosterone had a lower renal total glomerular count (p<0.01) but increased androgen receptor density. CONCLUSIONS: In juvenile male rats testosterone was associated with increased bladder and renal mass, and increased bladder smooth muscle. Testosterone associated kidneys also appeared to have fewer but larger glomeruli. These data support an important role for sex hormones in structural and functional development of the bladder and kidney.
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Riñón/citología , Testosterona/análogos & derivados , Vejiga Urinaria/citología , Andrógenos/farmacología , Animales , Riñón/efectos de los fármacos , Riñón/crecimiento & desarrollo , Masculino , Tamaño de los Órganos/efectos de los fármacos , Ratas , Ratas Wistar , Testosterona/farmacología , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/crecimiento & desarrolloRESUMEN
We presented a 31-year-old female patient with a history of hypertension and severe pulsing headache for about 3 months. The patient had pulsing headaches over the past 3 months with increased very high blood pressure (BP = 220/130 mmHg), sometimes with blurred vision, nausea and vomiting, with no known pathological conditions in her medical history or family background. A digital subtraction angiography confirmed tight stenosis (90%) in the middle segment of the right renal artery. Balloon angioplasty with a stent was the treatment of choice. Blood pressure dropped to normal after treatment.
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Angioplastia de Balón , Displasia Fibromuscular/complicaciones , Displasia Fibromuscular/terapia , Hipertensión Maligna/complicaciones , Hipertensión Maligna/terapia , Obstrucción de la Arteria Renal/complicaciones , Obstrucción de la Arteria Renal/terapia , Adulto , Femenino , Displasia Fibromuscular/fisiopatología , Cefalea/complicaciones , Cefalea/fisiopatología , Cefalea/terapia , Humanos , Hipertensión Maligna/fisiopatología , Arteria Renal/fisiopatología , Obstrucción de la Arteria Renal/fisiopatología , StentsAsunto(s)
Arbovirus/fisiología , Genoma Viral , Insectos Vectores/virología , Mamíferos/virología , Animales , HumanosRESUMEN
OBJECTIVE: To investigate the myocardial proliferation/regeneration capacities at different time points and at different parts of the heart post acute myocardial infarction (AMI) in rats. METHODS: A total of 64 adult Sprague-Dawley (SD) rats were randomly divided into AMI group (left anterior descending coronary ligation, n = 44) and sham-operated group (n = 20). Rats were sacrificed on day 3, 5, 7, 14 and 60 respectively post operation (n = 5-9 at each time point) and ventricular tissues were harvested. 5-Bromo-2-deoxyUridine (Brdu, 50 mg/kg) was injected intraperitoneally at 12 and 24 hours before sacrifice. Morphological and pathological changes of the myocardium were observed after HE staining. Brdu-positive and c-kit and Brdu double-positive cardiomyocytes were analyzed post immunohistochemistry and immunofluorescence staining. Striated structure of new cells was detected by PTAH staining. Alpha-sarcomeric actin antibody was used to identify new cardiomyocytes. RESULT: Brdu-positive cardiomyocytes at border zone and non-ischemic zone were significantly increased at 5 days post AMI and peaked at 7th day post operation (Border zone, AMI: 1.26% ± 0.15% vs.Sham: 0.22% ± 0.06%, P < 0.01; right ventricle, AMI: 0.75% ± 0.12% vs.Sham: 0.18% ± 0.07%, P < 0.01). There was no significant difference between the two groups on the 60th post-operation day. Brdu-positive cells were 1.7-fold higher in infarct border zone than in the right ventricular area of AMI rats on the 7th post operation day (1.26% ± 0.15%, vs.0.75% ± 0.12%, P < 0.01) and was 1.4-fold higher on the 14th post operation day (0.77% ± 0.09%, vs.0.54% ± 0.11%, P < 0.01). PTAH staining evidenced myocardial striated structure inside the new cells. Immunofluorescent assay showed that parts of Brdu positive cells were myocardial actin positive, and the c-kit and Brdu double-positive myocardial cells were also observed. Most nuclei of tehse new cardiomyocytes were small and round-shaped. CONCLUSIONS: Myocardial proliferation/regeneration increased significantly after AMI in rats, especially around the infarct border zone. The myocardial proliferation/regeneration was time-dependent. Parts of the new cardiomyocytes had some characteristics of cardiac stem cells. This study suggests that myocardial proliferation/regeneration may be activated after acute myocardial injury.
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Proliferación Celular , Infarto del Miocardio/patología , Miocitos Cardíacos/patología , Animales , Masculino , Ratas , Ratas Sprague-Dawley , RegeneraciónRESUMEN
Background: Early myocardial calcifications secondary to fulminant myocarditis (FM) are rare, and their natural evolution and effects on cardiac function are poorly understood. Here, we followed the patient for 4 years to observe the development of cardiac calcification and its impact on heart function. Case summary: A 16-year-old man was hospitalized with a fever and cough for 1 day. The patient was previously healthy and had no history of heart disease or specific family conditions. The patient was positive for anti-Epstein-Barr virus IgG and IgM. The computed tomography (CT) scan showed no coronary lesions. Cardiogenic shock and recurrent ventricular fibrillation developed on the third day after admission, and the patient received rescue therapy such as endotracheal intubation, defibrillation, extracorporeal membrane oxygenation, and corticosteroids. On the 13th day of admission, a CT scan revealed significant calcification in the left ventricular wall. The patient was discharged after 30 days in the hospital. After discharge, his left ventricular calcification peaked at 6 months and gradually subsided after that, and his left ventricular function slowly returned to normal at 12 months. Discussion: In younger patients, myocardial calcifications secondary to FM may occur as early as 13 days and affect cardiac function. After proper treatment and rehabilitation, the patient's myocardial calcification can gradually subside and the cardiac function can gradually recover. For FM patients, timely and comprehensive intensive treatment, including heart, lung, and kidney replacement therapy and early administration of hormone preparations, may be beneficial to the early recovery of patients.
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The quality of financial decision-making is very important to the future development of an enterprise, but it is often affected by the completeness of useful information for decision-making and the subjective factors of decision makers, and is often unstable. With the development of computer technology, the financial decision support system came into being, which improved the quality of financial decision to some extent. However, although the existing financial decision support system has achieved dataization to a certain extent, it still faces problems such as artificial leadership, insufficient intelligence, and poor decision-making efficiency, and cannot fully meet the needs of decision-makers. The explosion of artificial intelligence technology in recent years has provided potential improvements to financial decision support systems. In this article, we conduct a detailed analysis of the deficiencies in the current financial decision support system, build the mechanism and implementation path of the financial decision support system under artificial intelligence, and design a digital and intelligent financial decision support system. At the same time, we apply the proposed financial decision support system to the financial practice of X enterprise. Through the questionnaire survey, it is found that through the comprehensive application of artificial intelligence technology, the new system has a higher degree of intelligence than the existing system, and its construction can effectively improve the timeliness and accuracy of financial decision-making, while reducing the cost of financial decision-making. It is conducive to promoting the integration of management accounting and financial accounting.
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Inteligencia Artificial , InteligenciaRESUMEN
Coronavirus disease 2019 (COVID-19) has spread, at an unprecedented speed and scale, into a global pandemic, infecting more than 29 million cases worldwide across 215 countries and territories and killing more than 930,000 individuals. There is evidence that preexisting cardiac disease can render individuals vulnerable. A large number of patients with COVID-19 present with preexisting cardiovascular disease or develop new-onset cardiac dysfunction during the course of the illness. Therefore, particular attention should be given to cardiovascular protection during COVID-19 treatment. This review highlights recent advances in our understanding of the interaction between COVID-19 and the cardiovascular system, with special attention to the virological, pathological, and immunological characteristics of COVID-19, acute myocardial injury, myocarditis, arrhythmias, coronary artery disease, heart function, and the possible mechanisms.
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PURPOSE: To create a reliable rat model with small renal cortical scars and evaluate the accuracy and sensitivity of dynamic contrast-enhanced MRI in detecting the kinds of lesions that are associated with reflux nephropathy. MATERIALS AND METHODS: In 16 rats, three unilateral renal cortical lesions were created using either electrocautery or pure alcohol with the contralateral kidney serving as control. MRI on a 1.5 Tesla GE Signa was performed 10-14 days after surgery. After bolus injection of 0.2 mM/Kg Gd-DTPA, sequential MRI acquisitions were performed using a 4-inch quadrature birdcage coil. Renal and scar volumes and pathology were compared after scanning and killing. RESULTS: Of the 48 points of injury, 40 (83%) in the 16 rats were detected grossly. Under microscopy, 36 injuries (75%) were detected on mid-kidney cross-sections. The average lesion was 4.2 mm(3) corresponding to 0.5% of the kidney volume. Using pathological findings as the gold standard, the sensitivity and specificity of scar detection using MRI was 69% and 93%, respectively. CONCLUSION: A rat model was created to demonstrate the sensitivity of dynamic contrast-enhanced MRI for detecting renal scars. Alcohol and electrocautery created reliable renal scars that were confirmed pathologically. MRI detected these lesions that averaged 4.2 mm(3) (0.5% total renal volume) with sensitivity and specificity of 69% and 93%, respectively.
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Cicatriz/patología , Modelos Animales de Enfermedad , Gadolinio DTPA , Aumento de la Imagen/métodos , Enfermedades Renales/patología , Imagen por Resonancia Magnética/métodos , Animales , Medios de Contraste , Femenino , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
Phage PhiC31 integrase-mediated gene delivery is believed to be safer than using retroviral vectors since the protein confines its insertion of the target gene to a limited number of sites in mammalian genomes. To evaluate its safety in human cells, it is important to understand the interactions between this integrase and cellular proteins. Here we show that PhiC31 integrase interacts with TTRAP as presented by yeast two-hybrid and co-immunoprecipitation assays. Reducing the expression of endogenous TTRAP can increase the efficiency of PhiC31 integrase-mediated integration. A possible effect of interaction between PhiC31 integrase and TTRAP was highlighted by the fact that PhiC31 integrase inhibited the NFkappaB activation mediated by IL-1 in a dose-dependent manner. Because low dose of PhiC31 integrase can mediate considerable recombination events, we suggest that low dose of PhiC31 integrase be used when this integrase is applied in human cells.
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Bacteriófagos/enzimología , Integrasas/metabolismo , FN-kappa B/metabolismo , Péptidos y Proteínas Asociados a Receptores de Factores de Necrosis Tumoral/metabolismo , Bacteriófagos/efectos de los fármacos , Células HeLa , Humanos , Interleucina-1/farmacología , Unión Proteica/efectos de los fármacos , Recombinación Genética/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Integración Viral/efectos de los fármacosRESUMEN
PML nuclear body (PML NB) is an important macromolecular nuclear structure that is involved in many essential aspects of cellular function. Tens of proteins have been found in PML NBs, and promyelocytic leukemia protein (PML) has been proven to be essential for the formation of this structure. Here, we showed that TRAF and TNF receptor-associated protein (TTRAP) was a novel PML NBs-associated protein. TTRAP colocalized with three important PML NBs-associated proteins, PML, DAXX and Sp100 in the typical fashion of PML NBs. By yeast mating assay, TTRAP was identified to interact with these PML NBs-associated proteins. The transcription and expression of TTRAP could be induced by IFN-gamma, representing another common feature of PML NBs-associated proteins. These results would not only be important for understanding PML NBs but also be helpful in studying the TTRAP function in the future.
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Núcleo Celular/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Antígenos Nucleares/metabolismo , Autoantígenos/metabolismo , Línea Celular , Proteínas Co-Represoras , Proteínas de Unión al ADN , Humanos , Interferón gamma/farmacología , Chaperonas Moleculares , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas , Proteína de la Leucemia Promielocítica , Factores de Transcripción/genética , Transcripción Genética , LevadurasRESUMEN
OBJECTIVE: An endothelial nicotinic acetylcholine receptor (nAChR) participates in atherogenesis and tumorigenesis by promoting neovascularization. To date, the mechanisms of nAChR-mediated angiogenesis and their relationship to angiogenic factors, eg, VEGF and bFGF, are unknown. METHODS AND RESULTS: Nicotine induced dose-dependent human microvascular endothelial cell (HMVEC) migration, a key angiogenesis event, to an extent which was equivalent in magnitude to bFGF (10 ng/mL) but less than for VEGF (10 ng/mL). Unexpectedly, nAChR antagonism not only abolished nicotine-induced HMVEC migration but also abolished migration induced by bFGF and attenuated migration induced by VEGF. Transcriptional profiling identified gene expression programs which were concordantly regulated by all 3 angiogens (nicotine, VEGF, and bFGF), a notable feature of which includes corepression of thioredoxin-interacting protein (TXNIP), endogenous inhibitor of the redox regulator thioredoxin. Furthermore, TXNIP repression by all 3 angiogens induced thioredoxin activity. Silencing thioredoxin by small interference RNA abrogated all angiogen-induced migration while silencing TXNIP strongly induced HMVEC migration. Interestingly, nAChR antagonism abrogates growth factor (VEGF and bFGF)-mediated induction of thioredoxin activity. CONCLUSIONS: Nicotine promotes angiogenesis via stimulation of nAChR-dependent endothelial cell migration. Furthermore, growth factor-induced HMVEC migration, a key angiogenesis event, requires nAChR activation--an effect mediated in part by nAChR-dependent regulation of thioredoxin activity.
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Movimiento Celular/efectos de los fármacos , Endotelio Vascular/citología , Factor 2 de Crecimiento de Fibroblastos/fisiología , Receptores Nicotínicos/fisiología , Factor A de Crecimiento Endotelial Vascular/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Movimiento Celular/fisiología , Células Cultivadas , Relación Dosis-Respuesta a Droga , Endotelio Vascular/efectos de los fármacos , Endotelio Vascular/fisiología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Neovascularización Fisiológica/efectos de los fármacos , Neovascularización Fisiológica/fisiología , Nicotina/farmacología , Agonistas Nicotínicos/farmacología , ARN Interferente Pequeño/farmacología , Receptores Nicotínicos/genética , Tiorredoxinas/genética , Tiorredoxinas/metabolismoRESUMEN
Several epidemiological and clinical studies have shown that females with ST-segment elevation myocardial infarction (STEMI) have a higher mortality than males following primary percutaneous coronary intervention (PPCI). Many analyses of sex-based differences following STEMI have revealed conflicting results. Currently, more and more elderly patients with STEMI have undergone emergency interventional therapy. From January 2014 to December 2016, a total of 337 elderly patients with STEMI were enrolled in this study from two chest pain centers, and all patients underwent PPCI. Patients were divided into two groups: elderly females (n=117, mean age 73.4±9.6 years) and elderly males (n=220, mean age 71.7±8.6 years). The prevalence of diabetes was higher in females than in males (29.1% vs. 19.6%,P<0. 01). Typical ischemic chest pain was lower in females than in males (45.3% vs 57.3%, P<0.01). The number of nonsmokers was also significantly higher in females than in males (5.1% vs. 52.3%,P<0. 01). Serum creatinine (sCr) levels (87.6±17.4 umol/L vs 99.5±20.2 umol/L,P<0.01) and body mass index (23.8±2.7 vs 27.3±3.1, P<0.01) were lower in females than in males. The incidences of major adverse cardiac events (MACE) in-hospital showed no significantly difference (P>0.05) between the two groups. However, the cumulative MACE showed a significant difference between the two groups in the 12-month follow-up (16.8% in male vs 12.8% in female, P = 0.04). Our results suggest that the PPCI is safe and effective in elderly female STEMI patients. The cumulative MACE in females are not higher than in males. PPCI are helpful in elderly STEMI patients.
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Both nitric oxide and asymmetric dimethylarginine (ADMA) play a critical role in the regulation of cerebral blood flow, though their neuroprotective and cytotoxic effects are still under investigation. In this study, we found that nitrate/nitrite (NOx) levels in plasma, ischemic brain tissue, and cerebrospinal fluid (CSF) increased significantly 24h after 2h transient middle cerebral artery occlusion (MCAO) in rats. ADMA levels were unchanged in plasma, but decreased significantly in CSF 24h following MCAO. The CSF ADMA/NOx ratio decreased markedly following ischemia. Rats protected by expression of the chaperonin GroEL or its folding deficient mutant D87K had lower plasma NOx levels at 24h reperfusion. ADMA, NO, and their ratio in CSF merit further study as biomarkers for ischemic brain injury.
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Arginina/análogos & derivados , Isquemia Encefálica/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Circulación Cerebrovascular/fisiología , Chaperonina 60/metabolismo , Óxido Nítrico/sangre , Animales , Arginina/sangre , Arginina/líquido cefalorraquídeo , Biomarcadores/análisis , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Encéfalo/fisiopatología , Isquemia Encefálica/fisiopatología , Circulación Cerebrovascular/efectos de los fármacos , Chaperonina 60/genética , Citoprotección/genética , Terapia Genética/métodos , Infarto de la Arteria Cerebral Media/tratamiento farmacológico , Infarto de la Arteria Cerebral Media/metabolismo , Infarto de la Arteria Cerebral Media/fisiopatología , Masculino , Óxido Nítrico/líquido cefalorraquídeo , Ratas , Ratas Sprague-Dawley , Transfección/métodos , Regulación hacia Arriba/fisiologíaRESUMEN
BACKGROUND: This study was designed to determine whether overexpression of the enzyme dimethylarginine dimethylaminohydrolase (DDAH) could enhance angiogenesis by reducing levels of the endogenous nitric oxide synthase (NOS) inhibitor asymmetric dimethylarginine (ADMA). METHODS AND RESULTS: In DDAH1 transgenic (TG) and wild-type mice (each n=42), the role of DDAH overexpression on angiogenesis was studied by use of the disk angiogenesis system and a murine model of hindlimb ischemia (each n=21). After surgery, animals were treated with either PBS or the NOS inhibitors ADMA or N(omega)-nitro-L-arginine methyl ester (L-NAME; each 250 micromol x kg(-1) x d(-1)) by use of osmotic minipumps (each n=7). L-NAME was chosen to study an inhibitor that is not degraded by DDAH. Neovascularization in the disk angiogenesis system was impaired by both NOS inhibitors; however, TG animals were resistant to the effects of ADMA on neovascularization. Similarly, TG mice were more resistant to the inhibitory effect of ADMA on angioadaptation (angiogenesis and arteriogenesis) after hindlimb ischemia, as assessed by fluorescent microsphere studies and postmortem microangiograms. Enhanced neovascularization and limb perfusion in TG mice were associated with reduced plasma and tissue ADMA levels and enhanced tissue NOS enzyme activity. CONCLUSIONS: We describe a novel mechanism by which DDAH regulates postnatal neovascularization. Therapeutic manipulation of DDAH expression or activity may represent a novel approach to restore tissue perfusion.
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Amidohidrolasas/fisiología , Neovascularización Fisiológica/fisiología , Amidohidrolasas/genética , Animales , Arginina/análogos & derivados , Arginina/sangre , Arginina/farmacocinética , Arginina/farmacología , Inducción Enzimática , Femenino , Miembro Posterior/irrigación sanguínea , Humanos , Isquemia/fisiopatología , Flujometría por Láser-Doppler , Ratones , Ratones Transgénicos , Microesferas , Músculo Esquelético/irrigación sanguínea , NG-Nitroarginina Metil Éster/farmacología , Nitratos/sangre , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Nitritos/sangre , Prótesis e Implantes , Proteínas Recombinantes de Fusión/fisiologíaRESUMEN
BACKGROUND: NO is a major regulator of cardiovascular physiology that reduces vascular and cardiac contractility. Accumulating evidence indicates that endogenous inhibitors may regulate NOS. The NOS inhibitors asymmetric dimethylarginine (ADMA) and N-monomethylarginine are metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). This study was designed to determine if increased expression of DDAH could reduce tissue and plasma levels of the NOS inhibitors and thereby increase NO synthesis. METHODS AND RESULTS: We used gene transfer and transgenic approaches to overexpress human DDAH I in vitro and in vivo. The overexpression of DDAH in cultured endothelial cells in vitro induced a 2-fold increase in NOS activity and NO production. In the hDDAH-1 transgenic mice, we observed approximately 2-fold increases in tissue NOS activity and urinary nitrogen oxides, associated with a 2-fold reduction in plasma ADMA. The systolic blood pressure of transgenic mice was 13 mm Hg lower than that of wild-type controls (P<0.05). The systemic vascular resistance and cardiac contractility were decreased in response to the increase in NO production. CONCLUSIONS: DDAH I overexpression increases NOS activity in vitro and in vivo. The hDDAH-1 transgenic animal exhibits a reduced systolic blood pressure, systemic vascular resistance, and cardiac stroke volume. This study provides compelling evidence that the elaboration and metabolism of endogenous ADMA plays an important role in regulation of NOS activity.
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Amidohidrolasas/metabolismo , Arginina/análogos & derivados , Óxido Nítrico/biosíntesis , Amidohidrolasas/genética , Animales , Arginina/sangre , Células Cultivadas , Endotelio/metabolismo , Hemodinámica , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico Sintasa/metabolismo , TransfecciónRESUMEN
UNLABELLED: Codon pair bias (CPB), which has been observed in all organisms, is a neglected genomic phenomenon that affects gene expression. CPB results from synonymous codons that are paired more or less frequently in ORFeomes regardless of codon bias. The effect of an individual codon pair change is usually small, but when it is amplified by large-scale genome recoding, strikingly altered biological phenotypes are observed. The utility of codon pair bias in the development of live attenuated vaccines was recently demonstrated by recodings of poliovirus (a positive-strand RNA virus) and influenza virus (a negative-strand segmented RNA virus). Here, the L gene of vesicular stomatitis virus (VSV), a nonsegmented negative-sense RNA virus, was partially recoded based on codon pair bias. Totals of 858 and 623 silent mutations were introduced into a 5'-terminal segment of the viral L gene (designated L1) to create sequences containing either overrepresented or underrepresented codon pairs, designated L1(sdmax) and L1(min), respectively. Analysis revealed that recombinant VSV containing the L1(min) sequence could not be recovered, whereas the virus with the sdmax sequence showed a modest level of attenuation in cell culture. More strikingly, in mice the L1(sdmax) virus was almost as immunogenic as the parental strain but highly attenuated. Taken together, these results open a new road to attain a balance between VSV virulence and immunogenicity, which could serve as an example for the attenuation of other negative-strand, nonsegmented RNA viruses. IMPORTANCE: Vesicular stomatitis virus (VSV) is the prototypic rhabdovirus in the order Mononegavirales. A wide range of human pathogens belong to this family. Using a unique computer algorithm and large-scale genome synthesis, we attempted to develop a live attenuated vaccine strain for VSV, which could be used as an antigen delivery platform for humans. Recombinant VSVs with distinct codon pair biases were rationally designed, constructed, and analyzed in both cell culture and an animal model. One such recombinant virus, L1(sdmax), contained extra overrepresented codon pairs in its L gene open reading frame (ORF) and showed promise as an effective vaccine candidate because of a favorable balance between virulence and immunogenicity. Our study not only contributes to the understanding of the underlying mechanism of codon pair bias but also may facilitate the development of live attenuated vaccines for other viruses in the order Mononegavirales.
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Ingeniería de Proteínas , ARN Polimerasa Dependiente del ARN/metabolismo , Mutación Silenciosa , Vesiculovirus/inmunología , Vesiculovirus/fisiología , Proteínas Virales/metabolismo , Vacunas Virales/inmunología , Animales , Diseño Asistido por Computadora , Masculino , Ratones Endogámicos BALB C , ARN Polimerasa Dependiente del ARN/genética , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/genética , Vacunas Atenuadas/inmunología , Vacunas Atenuadas/aislamiento & purificación , Vesiculovirus/genética , Vesiculovirus/crecimiento & desarrollo , Proteínas Virales/genética , Vacunas Virales/administración & dosificación , Vacunas Virales/genética , Vacunas Virales/aislamiento & purificación , VirulenciaRESUMEN
PURPOSE: The incidence of pregnancies complicated by hyperglycemia and hypertension is increasing along with associated morbidities to mother and offspring. The high fructose diet is a well-studied model that induces hyperglycemia and hypertension in male rodents, but may not affect females. We hypothesized that the physiologic stress of pregnancy may alter metabolic responses to dietary fructose. MATERIALS AND METHODS: In this study female Sprague-Dawley rats were divided into two gestational dietary groups: (1) 60% carbohydrate standard rat chow (Pregnant-S-controls) and (2) 60% fructose enriched chow (Pregnant-F). Body weight, blood pressure, blood glucose, triglycerides, and insulin were measured in pregnancy and during the post-partum period. Maternal organ weight and histological changes were also assessed after delivery. RESULTS: By midpregnancy Pregnant-F rats had increased weight, elevated blood pressure, higher fasting glucose, and elevated triglycerides compared with Pregnant-S rats. Both groups demonstrated elevated gestational insulin levels with signs of insulin resistance (increased HOMA-IR). Pregnant-F rats showed significant histopathologic hepatic steatosis and renal tubular changes characterized by tubular dilation and glomerulosclerosis. CONCLUSION: Our study provides a model in which dietary change during pregnancy can be examined. We demonstrate, moreover, that high dietary fructose ingestion in pregnant rats may result in profound systemic and pathologic changes not appreciated during routine pregnancy.