RESUMEN
The Three Prime Repair EXonuclease I (TREX1) is critical for degrading post-apoptosis DNA. Mice expressing catalytically inactive TREX1 (TREX1 D18N) develop lupus-like autoimmunity due to chronic sensing of undegraded TREX1 DNA substrates, production of the inflammatory cytokines, and the inappropriate activation of innate and adaptive immunity. This study aimed to investigate Thelper (Th) dysregulation in the TREX1 D18N model system as a potential mechanism for lupus-like autoimmunity. Comparison of immune cells in secondary lymphoid organs, spleen and peripheral lymph nodes (LNs) between TREX1 D18N mice and the TREX1 null mice revealed that the TREX1 D18N mice exhibit a Th1 bias. Additionally, the T-follicular helper cells (Tfh) and the germinal celter (GC) B cells were also elevated in the TREX1 D18N mice. Targeting Bcl6, a lineage-defining transcription factor for Tfh and GC B cells, with a commercially available Bcl6 inhibitor, FX1, attenuated Tfh, GC, and Th1 responses, and rescued TREX1 D18N mice from autoimmunity. The study presents Tfh and GC B-cell responses as potential targets in autoimmunity and that Bcl6 inhibitors may offer therapeutic approach in TREX1-associated or other lupus-like diseases.
Asunto(s)
Autoinmunidad , Centro Germinal , Animales , Diferenciación Celular , ADN , Modelos Animales de Enfermedad , Exodesoxirribonucleasas , Ratones , Ratones Noqueados , Fosfoproteínas , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismo , Células T Auxiliares Foliculares , Linfocitos T Colaboradores-InductoresRESUMEN
OBJECTIVE: Maternal morbidity and mortality is a global concern despite advances in medical care and technology and improved economic resources of nations worldwide. The primary objective of our study was to describe racial/ethnic disparities in severe maternal morbidity by using admission to an intensive care unit (ICU) as a marker. The secondary objective was to evaluate associations between patient characteristics, including obstetric outcomes, and severe maternal morbidity. METHODS: This retrospective cohort study used a large inpatient database to identify pregnancy and postpartum hospitalizations in Hawai'i from January 2012 through September 2017. We evaluated associations between sociodemographic and clinical characteristics and race/ethnicity by using χ2 tests. We used multivariable logistic regression to assess associations between race/ethnicity and ICU admission. We used a post hoc analysis to assess associations between ICU admission and obstetric outcomes by race/ethnicity. RESULTS: After adjustment, we found a significantly higher ICU admission rate among Asian (adjusted odds ratio [aOR] = 1.30; 95% CI, 1.04-1.62; P = .02), Filipino (aOR = 1.45; 95% CI, 1.17-1.79; P < .001), and Native Hawaiian/Other Pacific Islander (aOR = 1.39; 95% CI, 1.15-1.68; P < .001) women compared with non-Hispanic White women. Multiple clinical characteristics and outcomes were associated with ICU admission, such as preexisting chronic conditions and pregnancy-induced hypertensive disorders. CONCLUSION: We found that severe maternal morbidity represented by ICU admission is higher among Asian, Filipino, and Native Hawaiian/Other Pacific Islander women than among non-Hispanic White women in Hawai'i. Our findings reemphasize the need for health care providers to be vigilant in caring for members of racial/ethnic minority groups and managing their comorbidities.
Asunto(s)
Etnicidad , Complicaciones del Embarazo , Cuidados Críticos , Femenino , Hawaii/epidemiología , Hospitalización , Humanos , Grupos Minoritarios , Periodo Posparto , Embarazo , Complicaciones del Embarazo/epidemiología , Estudios RetrospectivosRESUMEN
The Coronavirus disease 2019 (COVID-19) caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) pandemic has had a rapid and deadly onset, spreading quickly throughout the world. Pregnant patients have had high mortality rates, perinatal losses, and Intensive Care Unit (ICU) admissions from acute respiratory syndrome Coronavirus (SARS-CoV) and Middle East respiratory syndrome Coronavirus (MERS-CoV) in the past. Potentially, a surge of patients may require hospitalization and ICU care beyond the capacity of the health care system. This article is to provide institutional guidance on how to prepare an obstetric hospital service for a pandemic, mass casualty, or natural disaster by identifying a care model and resources for a large surge of critically ill pregnant patients over a short time. We recommend a series of protocols, education, and simulation training, with a structured and tiered approach to match the needs for the patients, for hospitals specialized in obstetrics.
Asunto(s)
Betacoronavirus , Infecciones por Coronavirus/complicaciones , Cuidados Críticos , Obstetricia/métodos , Pandemias , Neumonía Viral/complicaciones , Complicaciones Infecciosas del Embarazo/virología , COVID-19 , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/terapia , Enfermedad Crítica/terapia , Planificación en Desastres , Servicios Médicos de Urgencia/ética , Servicios Médicos de Urgencia/organización & administración , Femenino , Maternidades , Humanos , Admisión y Programación de Personal , Neumonía Viral/epidemiología , Neumonía Viral/terapia , Embarazo , Complicaciones Infecciosas del Embarazo/terapia , SARS-CoV-2 , Capacidad de ReacciónRESUMEN
BACKGROUND: Advanced cervical cancer during pregnancy is an extremely rare event. We describe a case of at least stage IIIB cervical squamous cell carcinoma during pregnancy. This may possibly represent the longest gestation from time of diagnosis to delivery in a case of advanced cervical cancer, with potentially the most advanced gestational age at delivery and a relatively favorable outcome in the current literature.Case: A 29-year-old female at 20 0/7 weeks of gestation with at least stage IIIB squamous cell carcinoma of the cervix flew from Micronesia to Hawaii for oncologic treatment. After consultation with gynecologic oncology and maternal-fetal medicine, she opted to continue the pregnancy and began neoadjuvant chemotherapy with carboplatin and paclitaxel. At 33 2/7 weeks of gestation, she was admitted for preterm prelabor rupture of membranes and immediately underwent a cesarean delivery for heavy vaginal bleeding. Postpartum, she underwent cisplatin chemotherapy with concurrent radiation therapy. After 6 cycles of chemotherapy, the patient's cancer had progressed to the point that hospice was recommended. She died 11 months after initial presentation. CONCLUSION: Advanced cervical cancer during pregnancy requires individualized treatment, shared decision making, and a multidisciplinary team approach. If the pregnancy is continued, antepartum chemotherapy should be strongly considered. Maternal prognoses tend to be poor, but neonatal outcomes appear to be favorable.
RESUMEN
Differences in contraceptive method use have been noted among women of different races, but studies describing contraceptive method use among Native Hawaiian women have not been published. To examine method choice in this group, the authors conducted a database review of the Hawai'i State Department of Health Title X program. Reviewed were client visit records (CVRs) that health care providers completed for women who were ages 15-44 years, avoiding pregnancy, not currently pregnant, and using a contraceptive method (N=54 513). Because a patient could have had several visits during the study period, the contraceptive method chosen at the last visit was selected for analysis. Statistical analyses included descriptive statistics, bivariate analyses, and logistic regression. The proportion of Native Hawaiian women who selected a highly-effective method of contraception (HEC), defined as an intrauterine device, implant, or permanent contraception, was higher than the proportion of non-Native Hawaiian women who selected an HEC. Overall, 15.4% of Native Hawaiian women during the study period chose HEC, compared to 8.8% of non-Native Hawaiian women. In a logistic regression analysis, Native Hawaiian women ages 15-29 were 1.46 times more likely to use HEC (95% CI: 1.35-1.58) than non-Native Hawaiian women ages 15-29, and Native Hawaiian women ages 30-44 were 1.69 times more likely to use HEC (95% CI: 1.53-1.87) than non-Native Hawaiian women in the same age group. Because Native Hawaiian women are reported to have higher rates of unintended pregnancy in the state compared to other racial groups, additional research exploring contraceptive non-use and pregnancy intention are needed.
Asunto(s)
Conducta de Elección , Conducta Anticonceptiva/etnología , Anticoncepción/métodos , Efectividad Anticonceptiva , Nativos de Hawái y Otras Islas del Pacífico , Adolescente , Adulto , Anticonceptivos Femeninos , Femenino , Hawaii , Humanos , Dispositivos Intrauterinos , Modelos Logísticos , Aceptación de la Atención de Salud , Embarazo , Embarazo no Planeado , Esterilización Tubaria , Adulto JovenRESUMEN
Autoimmunity can result when cells fail to properly dispose of DNA. Mutations in the three-prime repair exonuclease 1 (TREX1) cause a spectrum of human autoimmune diseases resembling systemic lupus erythematosus. The cytosolic dsDNA sensor, cyclic GMP-AMP synthase (cGAS), and the stimulator of IFN genes (STING) are required for pathogenesis, but specific cells in which DNA sensing and subsequent type I IFN (IFN-I) production occur remain elusive. In this study, we demonstrate that TREX1 D18N catalytic deficiency causes dysregulated IFN-I signaling and autoimmunity in mice. Moreover, we show that bone marrow-derived cells drive this process. We identify both innate immune and, surprisingly, activated T cells as sources of pathological IFN-α production. These findings demonstrate that TREX1 enzymatic activity is crucial to prevent inappropriate DNA sensing and IFN-I production in immune cells, including normally low-level IFN-α-producing cells. These results expand our understanding of DNA sensing and innate immunity in T cells and may have relevance to the pathogenesis of human disease caused by TREX1 mutation.
Asunto(s)
Exodesoxirribonucleasas/genética , Lupus Eritematoso Sistémico/genética , Fosfoproteínas/genética , Linfocitos T/inmunología , Animales , Autoantígenos/inmunología , Autoinmunidad , Células Cultivadas , ADN/inmunología , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata , Interferón-alfa/metabolismo , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nucleotidiltransferasas/metabolismoRESUMEN
The temporal trend of hospitalizations, cost, and outcomes associated with preeclampsia with severe features have been inadequately studied. The publicly available Healthcare Cost and Utilization Project (HCUP) National Inpatient Sample (NIS) database was accessed to examine the temporal trend of total number of discharges, age, death, and mean charges per admission associated with preeclampsia with severe features. Eleven-year temporal trends (2004 to 2014) of these measures were compared using linear regression and run charts using the statistical process control rule. From 2004 to 2014, the total number of discharges related to preeclampsia with severe features increased both for Hawai'i and the U.S. (United States) (Hawai'i: 104 to 231; U.S.: 35,082 to 55,235; both P<.0001). The corresponding rates of discharges per 100,000 population also both increased (Hawai'i: 8.2 to 16.3; U.S.: 12.0 to 17.3; both P<.0001). Comparing the temporal trends between Hawai'i and the U.S., Hawai'i had a significantly higher average annual increase in the rate of incidence than the national level (an annual increase rate of 9.2% in Hawai'i vs 4.2% nationally; P=.0004). The cost of hospitalization for preeclampsia with severe features also showed an increased trend for both Hawai'i and the U.S. (Hawai'i: 33.1% increase, P=.0005; U.S.: 41.1% increase, P<.0001). In the U.S., in-hospital mortality rates associated with this condition decreased from 0.09% in 2004 to 0.02% in 2014 (P=.03). In conclusion, the number of discharges related to preeclampsia with severe features increased over an 11-year period in Hawai'i and the U.S., and the rate of increase was higher in Hawai'i than the U.S. Maternal mortality rates from this condition also declined over the study period.
Asunto(s)
Costos de Hospital/estadística & datos numéricos , Hospitalización/estadística & datos numéricos , Preeclampsia/mortalidad , Bases de Datos Factuales , Femenino , Hawaii/epidemiología , Humanos , Preeclampsia/economía , Embarazo , Índice de Severidad de la Enfermedad , Estados UnidosRESUMEN
Anaemia is commonly observed in chronic inflammatory conditions, including systemic lupus erythematosus (SLE), where â¼50% of patients display clinical signs of anaemia. Mutation at the aspartate residue 18 of the three prime repair exonuclease 1 (TREX1) gene causes a monogenic form of cutaneous lupus in humans and the genetically precise TREX1 D18N mice recapitulate a lupus-like disease. TREX1 degrades single- and double-stranded DNA (dsDNA), and the link between failed DNA degradation by nucleases, including nucleoside-diphosphate kinases (NM23H1/H2) and Deoxyribonuclease II (DNase II), and anaemia prompted our studies to investigate whether TREX1 dysfunction contributes to anaemia. Utilizing the TREX1 D18N mice we demonstrate that (1) TREX1 mutant mice develop normocytic normochromic anaemia and (2) TREX1 exonuclease participates in the degradation of DNA originating from erythroblast nuclei during definitive erythropoiesis. Gene expression, hematocrit, hemoglobin, immunohistochemistry (IHC) and flow cytometry were used to quantify dysfunctional erythropoiesis. An altered response to induced anaemia in the TREX1 D18N mice was determined through IHC, flow cytometry, and interferon-stimulated gene (ISG) expression analysis of the liver, spleen and erythroblastic islands (EBIs). IHC, flow cytometry, and ISG expression studies were performed in vitro to determine the role of TREX1 in the degradation of erythroblast DNA within EBIs. The TREX1 D18N mice exhibit altered erythropoiesis including a 20% reduction in hematocrit, 10-20 fold increased erythropoietic gene expression levels in the spleen and phenotypic signs of normocytic normochromic anaemia. Anaemia in TREX1 D18N mice is accompanied by increased erythropoietin (Epo), normal hepcidin levels and the TREX1 D18N mice display an inappropriate response to anaemic challenge. Enhanced ISG expression results from failed processing and subsequent sensing of undegraded erythroblast DNA in EBIs. TREX1 participates in the degradation of erythroblast DNA in the EBI and TREX1 D18N mice exhibit a normocytic normochromic anaemia.
RESUMEN
Many regulated epigenetic elements and base lesions found in genomic DNA can both directly impact gene expression and play a role in disease processes. However, due to their noncanonical nature, they are challenging to assess with conventional technologies. Here, we present a new approach for the targeted detection of diverse modified bases in DNA. We first use enzymatic components of the DNA base excision repair pathway to install an individual affinity label at each location of a selected modified base with high yield. We then probe the resulting material with a solid-state nanopore assay capable of discriminating labeled DNA from unlabeled DNA. The technique features exceptional modularity via selection of targeting enzymes, which we establish through the detection of four DNA base elements: uracil, 8-oxoguanine, T:G mismatch, and the methyladenine analog 1,N6-ethenoadenine. Our results demonstrate the potential for a quantitative nanopore assessment of a broad range of base modifications.
Asunto(s)
Técnicas Biosensibles/métodos , Daño del ADN , ADN/análisis , Nanoporos , Neoplasias/genética , Adenina/análogos & derivados , Disparidad de Par Base , ADN/genética , Reparación del ADN , Epigénesis Genética , Guanina/análogos & derivados , Guanina/análisis , Humanos , Modelos Moleculares , Nanoporos/ultraestructura , Nanotecnología/métodos , Uracilo/análisisRESUMEN
The objective of this study was to assess racial-ethnic differences in the prevalence of postpartum hemorrhage (PPH) among Native Hawaiians and other Pacific Islanders (NHOPI), Asians, and Whites. We performed a retrospective study on statewide inpatient data for delivery hospitalizations in Hawai'i between January 1995 and December 2013. A total of 243,693 in-hospital delivery discharges (35.0% NHOPI, 44.0% Asian, and 21.0% White) were studied. Among patients with PPH, there were more NHOPI (37.1%) and Asians (47.6%), compared to Whites (15.3%). Multivariable logistic regression was used to assess the impact of maternal race-ethnicity on the prevalence of PPH after adjusting for delivery type, labor induction, prolonged labor, multiple gestation, gestational hypertension, gestational diabetes, preeclampsia, chorioamnionitis, placental abruption, placenta previa, obesity, and period with different diagnostic criteria for preeclampsia. In the multivariable analyses, NHOPI (adjusted odds ratio [aOR], 1.40; 95% confidence interval [CI], 1.32-1.48) and Asians (aOR, 1.45; 95% CI, 1.37-1.53) were more likely to have PPH compared to Whites. In the secondary analyses of 12,142 discharges with PPH, NHOPI and Asians had higher prevalence of uterine atony than Whites (NHOPI: 77.2%, Asians: 73.9% vs Whites: 65.1%, P < .001 for both comparisons).
Asunto(s)
Etnicidad/estadística & datos numéricos , Disparidades en Atención de Salud/etnología , Hemorragia Posparto/epidemiología , Prevalencia , Adulto , Parto Obstétrico/normas , Parto Obstétrico/estadística & datos numéricos , Femenino , Hawaii/epidemiología , Disparidades en Atención de Salud/estadística & datos numéricos , Humanos , Nativos de Hawái y Otras Islas del Pacífico/estadística & datos numéricos , Oportunidad Relativa , Hemorragia Posparto/etnología , Embarazo , Estudios RetrospectivosRESUMEN
Objective To assess differences in the rates of preeclampsia among a multiethnic population in Hawaii. Methods We performed a retrospective study on statewide inpatient data for delivery hospitalizations in Hawaii between January 1995 and December 2013. Multivariable logistic regression was used to assess the impact of maternal race/ethnicity on the rates of preeclampsia after adjusting for age, multiple gestation, multiparity, chronic hypertension, pregestational diabetes, obesity and smoking. Results A total of 271,569 hospital discharges for delivery were studied. The rates of preeclampsia ranged from 2.0 % for Chinese to 4.6 % for Filipinos. Preeclampsia rates were higher among Native Hawaiians who are age <35 and non-obese (OR 1.54; 95 % CI 1.43-1.66), age ≥35 and non-obese (OR 2.31; 95 % CI 2.00-2.68), age ≥35 and obese (OR 1.80; 95 % CI 1.24-2.60); other Pacific Islanders who are age <35 and non-obese (OR 1.40; 95 % CI 1.27-1.54), age ≥35 and non-obese (OR 2.18; 95 % CI 1.79-2.64), age ≥35 and obese (OR 1.68; 95 % CI 1.14-2.49); and Filipinos who are age <35 and non-obese (OR 1.55; 95 % CI 1.43-1.67), age ≥35 and non-obese (OR 2.26; 95 % CI 1.97-2.60), age ≥35 and obese (OR 1.64; 95 % CI 1.04-2.59) compared to whites. Pregestational diabetes (OR 3.41; 95 % CI 3.02-3.85), chronic hypertension (OR 5.98; 95 % CI 4.98-7.18), and smoking (OR 1.19; 95 % CI 1.07-1.33) were also independently associated with preeclampsia. Conclusions for Practice In Hawaii, Native Hawaiians, other Pacific Islanders and Filipinos have a higher risk of preeclampsia compared to whites. For these high-risk ethnic groups, more frequent monitoring for preeclampsia may be needed.
Asunto(s)
Pueblo Asiatico/estadística & datos numéricos , Hospitalización/estadística & datos numéricos , Nativos de Hawái y Otras Islas del Pacífico/estadística & datos numéricos , Preeclampsia/etnología , Población Blanca/estadística & datos numéricos , Adulto , Femenino , Hawaii/epidemiología , Humanos , Salud de las Minorías , Embarazo , Estudios Retrospectivos , Factores de Riesgo , Adulto JovenRESUMEN
The TREX1 gene encodes a potent DNA exonuclease, and mutations in TREX1 cause a spectrum of lupus-like autoimmune diseases. Most lupus patients develop autoantibodies to double-stranded DNA (dsDNA), but the source of DNA antigen is unknown. The TREX1 D18N mutation causes a monogenic, cutaneous form of lupus called familial chilblain lupus, and the TREX1 D18N enzyme exhibits dysfunctional dsDNA-degrading activity, providing a link between dsDNA degradation and nucleic acid-mediated autoimmune disease. We determined the structure of the TREX1 D18N protein in complex with dsDNA, revealing how this exonuclease uses a novel DNA-unwinding mechanism to separate the polynucleotide strands for single-stranded DNA (ssDNA) loading into the active site. The TREX1 D18N dsDNA interactions coupled with catalytic deficiency explain how this mutant nuclease prevents dsDNA degradation. We tested the effects of TREX1 D18N in vivo by replacing the TREX1 WT gene in mice with the TREX1 D18N allele. The TREX1 D18N mice exhibit systemic inflammation, lymphoid hyperplasia, vasculitis, and kidney disease. The observed lupus-like inflammatory disease is associated with immune activation, production of autoantibodies to dsDNA, and deposition of immune complexes in the kidney. Thus, dysfunctional dsDNA degradation by TREX1 D18N induces disease in mice that recapitulates many characteristics of human lupus. Failure to clear DNA has long been linked to lupus in humans, and these data point to dsDNA as a key substrate for TREX1 and a major antigen source in mice with dysfunctional TREX1 enzyme.
Asunto(s)
Eritema Pernio/enzimología , Eritema Pernio/genética , Daño del ADN , ADN/metabolismo , Exodesoxirribonucleasas/genética , Inflamación/patología , Lupus Eritematoso Cutáneo/enzimología , Lupus Eritematoso Cutáneo/genética , Fosfoproteínas/genética , Alelos , Animales , Anticuerpos/inmunología , Autoinmunidad/inmunología , Secuencia de Bases , Eritema Pernio/patología , ADN/química , ADN/genética , Exodesoxirribonucleasas/química , Humanos , Lupus Eritematoso Cutáneo/patología , Ratones , Datos de Secuencia Molecular , Mutación/genética , Conformación de Ácido Nucleico , Fenotipo , Fosfoproteínas/química , Biosíntesis de ProteínasRESUMEN
Background. Rupture of a splenic artery aneurysm is rare complication of pregnancy that is associated with a significant maternal and fetal mortality. Case. A multiparous female presented in the third trimester with hypotension, tachycardia, and altered mental status. A ruptured splenic artery aneurysm was discovered at the time of laparotomy and cesarean delivery. The patient made a full recovery following resection of the aneurysm. The neonate survived but suffered severe neurologic impairment. Conclusion. The diagnosis of ruptured splenic artery aneurysm should be considered in a pregnant woman presenting with signs of intra-abdominal hemorrhage. Early intervention by a multidisciplinary surgical team is key to preserving the life of the mother and fetus.
RESUMEN
TREX1 is an autonomous 3'-exonuclease that degrades DNA to prevent inappropriate immune activation. The TREX1 protein is composed of 314 amino acids; the N-terminal 242 amino acids contain the catalytic domain, and the C-terminal region (CTR) localizes TREX1 to the cytosolic compartment. In this study, we show that TREX1 modification by ubiquitination is controlled by a highly conserved sequence in the CTR to affect cellular localization. Transfection of TREX1 deletion constructs into human cells demonstrated that this sequence is required for ubiquitination at multiple lysine residues through a "non-canonical" ubiquitin linkage. A proteomic approach identified ubiquilin 1 as a TREX1 CTR-interacting protein, and this interaction was verified in vitro and in vivo. Cotransfection studies indicated that ubiquilin 1 localizes TREX1 to cytosolic punctate structures dependent upon the TREX1 CTR and lysines within the TREX1 catalytic core. Several TREX1 mutants linked to the autoimmune diseases Aicardi-Goutières syndrome and systemic lupus erythematosus that exhibit full catalytic function were tested for altered ubiquitin modification and cellular localization. Our data show that these catalytically competent disease-causing TREX1 mutants exhibit differential levels of ubiquitination relative to WT TREX1, suggesting a novel mechanism of dysfunction. Furthermore, these differentially ubiquitinated disease-causing mutants also exhibit altered ubiquilin 1 co-localization. Thus, TREX1 post-translational modification indicates an additional mechanism by which mutations disrupt TREX1 biology, leading to human autoimmune disease.
Asunto(s)
Exodesoxirribonucleasas/química , Exodesoxirribonucleasas/metabolismo , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ubiquitinación , Proteínas Adaptadoras Transductoras de Señales , Enfermedades Autoinmunes del Sistema Nervioso/metabolismo , Proteínas Relacionadas con la Autofagia , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , Células HEK293 , Humanos , Lupus Eritematoso Sistémico/metabolismo , Lisina/metabolismo , Proteínas Mutantes/metabolismo , Malformaciones del Sistema Nervioso/metabolismo , Unión Proteica , Procesamiento Proteico-Postraduccional , Transporte de Proteínas , Relación Estructura-ActividadRESUMEN
A thorough characterization of the transcriptome and proteome of endogenous podocytes has been hampered by low cell yields during isolation. Here we describe a double fluorescent reporter mouse model combined with an optimized bead perfusion protocol and efficient single cell dissociation to yield more than 500,000 podocytes per mouse allowing for global, unbiased downstream applications. Combining mRNA and miRNA transcriptional profiling with quantitative proteomic analyses revealed programs of highly specific gene regulation tightly controlling cytoskeleton, cell differentiation, endosomal transport, and peroxisome function in podocytes. Strikingly, the analyses further predict that these podocyte-specific gene regulatory networks are accompanied by alternative splicing of respective genes. Thus, our 'omics' approach will facilitate the discovery and integration of novel gene, protein, and organelle regulatory networks that deepen our systematic understanding of podocyte biology.
Asunto(s)
Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Luminiscentes/biosíntesis , Podocitos/metabolismo , Proteómica , Transducción de Señal , Empalme Alternativo , Animales , Separación Celular , Biología Computacional , Perfilación de la Expresión Génica/métodos , Genes Reporteros , Genotipo , Proteínas Luminiscentes/genética , Espectrometría de Masas , Ratones , Ratones Transgénicos , MicroARNs/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Proteómica/métodos , Transducción de Señal/genéticaRESUMEN
DOI: 10.1002/jssc.201200537 Europium(III) derivative formed by reaction of the europium ion with a fluorinated ß-diketonate reagent and a neutral donor. The resulting non-polar, thermally stable, and luminescent derivative partitions into cyclohexane upon formation and can be subjected to high-resolution capillary gas chromatography. This approach is used to separate derivatives of lanthanides that are adjacent to one another in the periodic table. In addition, an on-fiber SPME derivatization/concentration GC/MS method that uses a fiber preloaded with the same ß-diketonate reagent is described for the analysis of beryllium(II) ion.
RESUMEN
Previous studies describe derivatization of metal ions followed by analysis using gas chromatography, usually on packed columns. In many of these studies, stable and volatile derivatives were formed using fluorinated ß-diketonate reagents. This paper extends previous work by investigating separations of the derivatives on small-diameter capillary gas chromatography columns and exploring on-fiber, solid-phase microextraction derivatization techniques for beryllium. The ß-diketonate used for these studies was 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Derivatization of lanthanides also required addition of a neutral donor, dibutyl sulfoxide, in addition to 1,1,1,2,2,6,6,7,7,7-decafluoro-3,5-heptanedione. Unoptimized separations on a 100-µm i.d. capillary column proved capable of rapid separations (within 15 min) of lanthanide derivatives that are adjacent to one another in the periodic table. Full-scan mass spectra were obtained from derivatives containing 5 ng of each lanthanide. Studies also developed a simple on-fiber solid-phase microextraction derivatization of beryllium. Beryllium could be analyzed in the presence of other alkali earth elements (Ba(II) and Sr(II)) without interference. Extension of the general approach was demonstrated for several additional elements (i.e. Cu(II), Cr(III), and Ga(III)).
RESUMEN
The purpose of this study was to perform a preliminary investigation of compound-specific isotope analysis (CSIA) of diesel fuels to evaluate whether the technique could distinguish diesel samples from different sources/locations. The ability to differentiate or correlate diesel samples could be valuable for discovering fuel tax evasion schemes or for environmental forensic studies. Two urea adduction-based techniques were used to isolate the n-alkanes from the fuel. Both carbon isotope ratio (δ(13)C) and hydrogen isotope ratio (δD) values for the n-alkanes were then determined by CSIA in each sample. The samples investigated had δ(13)C values that ranged from -30.1 to -26.8, whereas δD values ranged from -83 to -156. Plots of δD versus δ(13)C with sample n-alkane points connected in order of increasing carbon number gave well-separated clusters with characteristic shapes for each sample. Principal components analysis (PCA) with δ(13)C, δD, or combined δ(13)C and δD data was applied to extract the maximum information content. PCA scores plots could clearly differentiate the samples, thereby demonstrating the potential of this approach for distinguishing (e.g., fingerprinting) fuel samples using δ(13)C and δD values.
Asunto(s)
Fraccionamiento Químico/métodos , Cromatografía de Gases y Espectrometría de Masas/métodos , Gasolina/análisis , Alcanos/análisis , Alcanos/aislamiento & purificación , InyeccionesRESUMEN
The goal of this study is to extend sampling by the field and laboratory emission cell (FLEC) dynamic headspace technique to applications that target nonvolatile residues. On-matrix derivatization of residues to render analytes stable and more volatile is explored to achieve this goal. Results show that on-matrix derivatizations of nerve agent hydrolysis products (monoalkyl methylphosphonic acids and methylphosphonic acid [MPA]) with diazomethane were successful on glass and painted wallboard (at the 10-µg level). It also was successful on the more difficult concrete (at the 500-µg level) and carpet (at the 20-µg level), substrates that cannot be successfully sampled using swipe techniques. Analysis of additional chemical warfare (CW)-associated residues can be approached by on-matrix derivatization with trifluoroacetic anhydride (TFAA). For example, amines (used as stabilizers or present as decomposition products of the nerve agent VX) or thiodiglycol (hydrolysis product of sulfur mustard) could be sampled as their TFAA derivatives from glass, painted wallboard, and concrete (at the 40-µg level), as well as carpet (at the 80-µg level) surfaces. Although the amine and thiodiglycol are semi-volatile and could be sampled directly, derivatization improves the recovery and chromatographic behavior of these analytes.