RESUMO
BACKGROUND: Complete reporting of seroepidemiologic studies is critical to their utility in evidence synthesis and public health decision making. The Reporting of Seroepidemiologic studies-SARS-CoV-2 (ROSES-S) guideline is a checklist that aims to improve reporting in SARS-CoV-2 seroepidemiologic studies. Adherence to the ROSES-S guideline has not yet been evaluated. OBJECTIVES: This study aims to evaluate the completeness of SARS-CoV-2 seroepidemiologic study reporting by the ROSES-S guideline during the COVID-19 pandemic, determine whether guideline publication was associated with reporting completeness, and identify study characteristics associated with reporting completeness. METHODS: A random sample from the SeroTracker living systematic review database was evaluated. For each reporting item in the guideline, the percentage of studies that were adherent was calculated, as well as median and interquartile range (IQR) adherence across all items and by item domain. Beta regression analyses were used to evaluate predictors of adherence to ROSES-S. RESULTS: One hundred and ninety-nine studies were analyzed. Median adherence was 48.1% (IQR 40.0%-55.2%) per study, with overall adherence ranging from 8.8% to 72.7%. The laboratory methods domain had the lowest median adherence (33.3% [IQR 25.0%-41.7%]). The discussion domain had the highest median adherence (75.0% [IQR 50.0%-100.0%]). Reporting adherence to ROSES-S before and after guideline publication did not significantly change. Publication source (p < 0.001), study risk of bias (p = 0.001), and sampling method (p = 0.004) were significantly associated with adherence. CONCLUSIONS: Completeness of reporting in SARS-CoV-2 seroepidemiologic studies was suboptimal. Publication of the ROSES-S guideline was not associated with changes in reporting practices. Authors should improve adherence to the ROSES-S guideline with support from stakeholders.
Assuntos
COVID-19 , Fidelidade a Diretrizes , SARS-CoV-2 , Humanos , COVID-19/epidemiologia , Estudos Soroepidemiológicos , SARS-CoV-2/imunologia , Fidelidade a Diretrizes/estatística & dados numéricos , PandemiasRESUMO
BACKGROUND: In Canada, nutrition policy, as outlined in the Nutrition for Healthy Term Infants recommendations, includes a daily vitamin D supplement of 10 µg (400 IU) for breastfed infants and young children to support adequate vitamin D status. OBJECTIVES: This study aimed to report on adherence to vitamin D supplementation recommendations for breastfed infants (≤12 months); and for children breastfed >12 mo. METHODS: Canadian Community Health Survey (paired-cycles 2015/2016 and 2017/2018) maternal experiences data for infants born 2012-2018 who received any breastmilk formed the sample (n = 7079). Whether the infant was given a vitamin D supplement (yes/no) and the frequency (daily/almost every day, 1-2/wk, or <1/wk) were surveyed. Weighted data (95% CI) were summarized according to breastfeeding history (exclusive to 6 mo and continuing; partial to 6 mo and continuing; and stopped ≤6 mo). Correlates of supplement adherence were explored using logistic regression. RESULTS: Overall, 87.1% (95% CI: 85.9%, 88.3%) of participants reported giving their infant (≤12 mo) a vitamin D supplement, and of these, 83.3% (95% CI: 81.9%, 84.7%) did so daily/almost every day, 12.4% (95% CI: 11.1%, 13.7%) did so 1-2/wk, and 4.3% (95% CI: 3.6%, 5.0%) did so <1/wk. Lower adjusted odds of adherence were observed among participants reporting: stopped breastfeeding ≤6 mo, lower education or income, recent immigration, and overweight prepregnancy body mass index; higher odds of adherence were observed in the western provinces. Regarding mothers of children >12 mo and breastfed (n = 2312), 58.0% (95% CI: 54.9%, 61.1%) gave a vitamin D supplement daily/almost every day. CONCLUSIONS: Adherence to providing a vitamin D supplement to breastfed infants is high in Canada. Nonetheless, we estimate that â¼27% of mothers are nonadherent to daily/almost every day administration of a vitamin D supplement and that adherence declines in children breastfed >12 mo. Further promotion to support uptake of the current guidance may be necessary, particularly for parents of recent immigration or lower socioeconomic status.
Assuntos
Aleitamento Materno , Suplementos Nutricionais , Vitamina D , Humanos , Lactente , Vitamina D/administração & dosagem , Canadá , Feminino , Masculino , Adulto , Recém-Nascido , Inquéritos Epidemiológicos , Pré-Escolar , Deficiência de Vitamina D/prevenção & controleRESUMO
Aneurysmal subarachnoid hemorrhage is a devastating condition causing significant morbidity and mortality. While outcomes from subarachnoid hemorrhage have improved in recent years, there continues to be significant interest in identifying therapeutic targets for this disease. In particular, there has been a shift in emphasis toward secondary brain injury that develops in the first 72 hours after subarachnoid hemorrhage. This time period of interest is referred to as the early brain injury period and comprises processes including microcirculatory dysfunction, blood-brain-barrier breakdown, neuroinflammation, cerebral edema, oxidative cascades, and neuronal death. Advances in our understanding of the mechanisms defining the early brain injury period have been accompanied by improved imaging and nonimaging biomarkers for identifying early brain injury, leading to the recognition of an elevated clinical incidence of early brain injury compared with prior estimates. With the frequency, impact, and mechanisms of early brain injury better defined, there is a need to review the literature in this area to guide preclinical and clinical study.
Assuntos
Lesões Encefálicas , Hemorragia Subaracnóidea , Humanos , Hemorragia Subaracnóidea/complicações , Incidência , Microcirculação , Barreira Hematoencefálica , Lesões Encefálicas/complicaçõesRESUMO
Objectives: Little is known about the impact of elite sport participation on long-term athlete health. We aimed to: (1) describe musculoskeletal, mental health, reproductive/endocrine and cardiovascular characteristics in retired elite female athletes and compare to the general population and (2) explore athletes' perceptions of their elite sport participation and its impact on health. Methods: A 136-item online questionnaire was disseminated to Canadian elite female rowing and rugby athletes >18 years old, >2 years retired from elite competition. Matched general population data were obtained from Statistics Canada when available. Results: Seventy-four (24% response rate) athletes (average age 45 (±9) years; retired 15 (±9) years) completed the questionnaire (30 rowing, 44 rugby athletes). During their career, 63 athletes (85%) experienced a hip/groin, knee, foot/ankle injury, or low back pain, with 42 (67%) reporting ongoing symptoms. Athletes 35-54 years reported worse knee symptoms and quality of life compared with the general population (symptom: p=0.197; d=1.15 [0.66, 1.63]; quality of life: p=0.312 d=1.03 [0.54, 1.51]) while other hip, knee and foot/ankle outcome scores were similar. Retired athletes had lower odds of anxiety (OR=0.155 [95% CI0.062 to 0.384]), greater lifetime/ever odds of amenorrhea (OR=6.10 [95%CI 2.67 to 13.96]) and gave birth when older (p<0.05). Fifty-nine (79%) recalled witnessing or experiencing at least one form of harassment/abuse during their career. Sixty athletes (81%) rated their current health as above average or excellent and 61 (82%) would compete at the same level again if given the choice. Conclusion: These novel insights can inform future preventative efforts to promote positive elite sport-related outcomes for current, former and future female athletes.
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OBJECTIVE: The role of hemorrhage volume in risk of vasospasm, delayed cerebral ischemia (DCI), and poor outcomes after aneurysmal subarachnoid hemorrhage (SAH) is well established. However, the relative contribution of blood within individual compartments is unclear. We present an automated technique for measuring not only total but also volumes of blood in each major compartment after SAH. METHODS: We trained convolutional neural networks to identify compartmental blood (cisterns, sulci, and ventricles) from baseline computed tomography scans of patients with SAH. We compared automated blood volumes against traditional markers of bleeding (modified Fisher score [mFS], Hijdra sum score [HSS]) in 190 SAH patients for prediction of vasospasm, DCI, and functional status (modified Rankin Scale) at hospital discharge. RESULTS: Combined cisternal and sulcal volume was better correlated with mFS and HSS than cisternal volume alone (ρ = 0.63 vs. 0.58 and 0.75 vs. 0.70, P < 0.001). Only blood volume in combined cisternal plus sulcal compartments was independently associated with DCI (OR 1.023 per mL, 95% CI 1.002-1.048), after adjusting for clinical factors while ventricular blood volume was not. Total and specifically sulcal blood volume was strongly associated with poor outcome (OR 1.03 per mL, 1.01-1.06, P = 0.006 and OR 1.04, 1.00-1.08 for sulcal) as was HSS (OR 1.06 per point, 1.00-1.12, P = 0.04), while mFS was not (P = 0.24). CONCLUSIONS: An automated imaging algorithm can measure the volume of bleeding after SAH within individual compartments, demonstrating cisternal plus sulcal (and not ventricular) blood contributes to risk of DCI/vasospasm. Automated blood volume was independently associated with outcome, while qualitative grading was not.
Assuntos
Doenças do Sistema Nervoso Autônomo , Isquemia Encefálica , Hemorragia Subaracnóidea , Vasoespasmo Intracraniano , Humanos , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/diagnóstico por imagem , Infarto Cerebral/complicações , Isquemia Encefálica/etiologia , Isquemia Encefálica/complicações , Volume Sanguíneo , Tomografia Computadorizada por Raios X/métodos , Doenças do Sistema Nervoso Autônomo/complicaçõesRESUMO
Functional connectivity (FC) is a sensitive metric that provides a readout of whole cortex coordinate neural activity in a mouse model. We examine the impact of experimental SAH modeled through endovascular perforation, and the effectiveness of subsequent treatment on FC, through three key questions: 1) Does the endovascular perforation model of SAH induce deficits in FC; 2) Does exposure to hypoxic conditioning provide protection against these FC deficits and, if so, is this neurovascular protection SIRT1-mediated; and 3) does treatment with the SIRT1 activator resveratrol alone provide protection against these FC deficits? Cranial windows were adhered on skull-intact mice that were then subjected to either sham or SAH surgery and either left untreated or treated with hypoxic post-conditioning (with or without EX527) or resveratrol for 3 days. Mice were imaged 3 days post-SAH/sham surgery, temporally aligned with the onset of major SAH sequela in mice. Here we show that the endovascular perforation model of SAH induces global and network-specific deficits in FC by day 3, corresponding with the time frame of DCI in mice. Hypoxic conditioning provides SIRT1-mediated protection against these network-specific FC deficits post-SAH, as does treatment with resveratrol. Conditioning-based strategies provide multifaceted neurovascular protection in experimental SAH.
Assuntos
Sirtuína 1 , Hemorragia Subaracnóidea , Animais , Modelos Animais de Doenças , Camundongos , Camundongos Endogâmicos C57BL , Resveratrol/farmacologia , Sirtuína 1/metabolismo , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/tratamento farmacológico , Hemorragia Subaracnóidea/metabolismoRESUMO
BACKGROUND AND PURPOSE: Early brain injury may be a more significant contributor to poor outcome after aneurysmal subarachnoid hemorrhage (aSAH) than vasospasm and delayed cerebral ischemia. However, studying this process has been hampered by lack of a means of quantifying the spectrum of injury. Global cerebral edema (GCE) is the most widely accepted manifestation of early brain injury but is currently assessed only through subjective, qualitative or semi-quantitative means. Selective sulcal volume (SSV), the CSF volume above the lateral ventricles, has been proposed as a quantitative biomarker of GCE, but is time-consuming to measure manually. Here we implement an automated algorithm to extract SSV and evaluate the age-dependent relationship of reduced SSV on early outcomes after aSAH. METHODS: We selected all adults with aSAH admitted to a single institution with imaging within 72 hours of ictus. Scans were assessed for qualitative presence of GCE. SSV was automatically segmented from serial CTs using a deep learning-based approach. Early SSV was the lowest SSV from all early scans. Modified Rankin Scale score of 4 to 6 at hospital discharge was classified as a poor outcome. RESULTS: Two hundred forty-four patients with aSAH were included. Sixty-five (27%) had GCE on admission while 24 developed it subsequently within 72 hours. Median SSV on admission was 10.7 mL but frequently decreased, with minimum early SSV being 3.0 mL (interquartile range, 0.3-11.9). Early SSV below 5 mL was highly predictive of qualitative GCE (area under receiver-operating-characteristic curve, 0.90). Reduced early SSV was an independent predictor of poor outcome, with a stronger effect in younger patients. CONCLUSIONS: Automated assessment of SSV provides an objective biomarker of GCE that can be leveraged to quantify early brain injury and dissect its impact on outcomes after aSAH. Such quantitative analysis suggests that GCE may be more impactful to younger patients with SAH.
Assuntos
Algoritmos , Lesões Encefálicas/diagnóstico por imagem , Lesões Encefálicas/etiologia , Líquido Cefalorraquidiano/diagnóstico por imagem , Hemorragia Subaracnóidea/complicações , Lesões Encefálicas/patologia , Humanos , Hemorragia Subaracnóidea/diagnóstico por imagem , Hemorragia Subaracnóidea/patologia , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: Delayed cerebral ischemia (DCI) after aneurysmal subarachnoid hemorrhage (SAH) has been identified as an independent predictor of poor outcome in numerous studies. OBJECTIVE: To investigate the potential protective role of inhalational anesthetics against angiographic vasospasm, DCI, and neurologic outcome in SAH patients. METHODS: After Institutional Review Board approval, data were collected retrospectively for SAH patients who received general anesthesia for aneurysm repair between January 1st, 2010 and May 31st, 2018. Primary outcomes were angiographic vasospasm, DCI, and neurologic outcome as measured by modified Rankin scale at hospital discharge. Univariate and logistic regression analysis were performed to identify independent predictors of these outcomes. RESULTS: The cohort included 390 SAH patients with an average age of 56 ± 15 (mean ± SD). Multivariate logistic regression analysis identified inhalational anesthetic only technique, Hunt-Hess grade, age, anterior circulation aneurysm and average intraoperative mean blood pressure as independent predictors of angiographic vasospasm. Inhalational anesthetic only technique and modified Fishers grade were identified as independent predictors of DCI. No impact on neurological outcome at time of discharge was noted. CONCLUSION: Our data provide additional evidence that inhalational anesthetic conditioning in SAH patients affords protection against angiographic vasospasm and new evidence that it exerts a protective effect against DCI. When coupled with similar results from preclinical studies, our data suggest further investigation into the impact of inhalational anesthetic conditioning on SAH patients, including elucidating the most effective dosing regimen, defining the therapeutic window, determining whether a similar protective effect against early brain injury, and on long-term neurological outcome exists.
Assuntos
Anestésicos Inalatórios/uso terapêutico , Pressão Arterial/efeitos dos fármacos , Isquemia Encefálica/epidemiologia , Hemorragia Subaracnóidea/complicações , Vasoespasmo Intracraniano/epidemiologia , Adulto , Idoso , Isquemia Encefálica/etiologia , Isquemia Encefálica/prevenção & controle , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Fatores de Risco , Vasoespasmo Intracraniano/etiologiaRESUMO
Delayed cerebral ischemia (DCI) is identified as one of the significant contributors to poor patient outcome after aneurysmal subarachnoid hemorrhage (SAH). We previously reported that a supratherapeutic dose of isoflurane conditioning (2%) provided robust protection against SAH-induced DCI. The aim of our current study is to compare the efficacy of the supratherapeutic dose of isoflurane to that typically used to establish general anesthesia or sedation. After IRB approval for animal studies, ten to fourteen-week-old wild-type male mice (C57BL/6) were divided into five groups - sham, SAH alone, or SAH with isoflurane conditioning (0.5%, 1%, and 2%). Conditioning was performed with one-hour of isoflurane initiated one-hour after induction of SAH via endovascular perforation technique. Vasospasm measurement in the middle cerebral artery was assessed 72 h after SAH. Neurological assessment was performed at baseline and for next three days after SAH. It was identified that all tested doses of isoflurane conditioning (0.5%, 1%, and 2%) significantly attenuated large artery vasospasm and markedly improved neurological deficits following SAH. No significant differences in neurovascular outcome were noted between the three doses of isoflurane conditioning. Our data show that isoflurane dosing typically used for general anesthesia (1%) or sedation (0.5%) provide similar levels of DCI protection in SAH as that provided by a supratherapeutic dose (2%). This result has important implications for future translational studies. Additional studies examining the therapeutic potential of anesthetic conditioning for SAH are therefore warranted.
Assuntos
Isquemia Encefálica/prevenção & controle , Isoflurano/farmacologia , Hemorragia Subaracnóidea/tratamento farmacológico , Animais , Isquemia Encefálica/tratamento farmacológico , Isquemia Encefálica/fisiopatologia , Infarto Cerebral , Modelos Animais de Doenças , Isoflurano/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ultrassonografia Doppler Transcraniana , Vasoespasmo IntracranianoRESUMO
Background Delayed cerebral ischemia remains a common and profound risk factor for poor outcome after subarachnoid hemorrhage (SAH). The aim of our current study is to define the role of endothelial nitric oxide synthase (eNOS) in isoflurane conditioning-induced neurovascular protection after SAH. Methods and Results Ten- to 14-week-old male wild-type mice (C57BL/6) as controls and eNOS knockout male mice (strain # 002684) were obtained for the study. Animals underwent either sham surgery, SAH surgery, or SAH with isoflurane conditioning. Anesthetic post conditioning was performed with isoflurane 2% for 1 hour, 1 hour after SAH. Normothermia was maintained with the homeothermic blanket. In a separate cohort, nitric oxide synthase was inhibited by a pan nitric oxide synthase inhibitor, L-nitroarginine methyl ester. Vasospasm measurement was assessed 72 hours after SAH and neurological function was assessed daily. Isoflurane-induced changes in the eNOS protein expression were measured. eNOS protein expression was significantly increased by isoflurane conditioning in naïve mice as well as mice subjected to SAH. Vasospasm of the middle cerebral artery and neurological deficits were evident following SAH versus sham surgery, both in wild-type mice and eNOS knockout mice. Isoflurane conditioning attenuated vasospasm and neurological deficits in wild-type mice. This delayed cerebral ischemia protection was lost in L-nitroarginine methyl ester -administered mice and eNOS knockout mice. Conclusions Our data indicate isoflurane conditioning provides robust protection against SAH-induced vasospasm and neurological deficits, and that this delayed cerebral ischemia protection is critically mediated via isoflurane-induced augmentation of eNOS.
Assuntos
Isquemia Encefálica , Endotélio Vascular/metabolismo , Óxido Nítrico Sintase Tipo III/metabolismo , Anestésicos Inalatórios/farmacologia , Animais , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Isquemia Encefálica/prevenção & controle , Modelos Animais de Doenças , Pós-Condicionamento Isquêmico , Precondicionamento Isquêmico , Isoflurano/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuroproteção , Hemorragia Subaracnóidea/complicações , Hemorragia Subaracnóidea/metabolismoRESUMO
The World Health Organization classification of lymphoma recommends the subdivision of follicular lymphoma (FL) into 3 grades (FL1-3) based on the average number of centroblasts per high-power field in the neoplastic follicles, but does not recognize a form of FL characterized by a predominance of large cleaved cells (centrocytes) without enough centroblasts to meet the World Health Organization criteria for FL3. We have classified such cases as follicular large cleaved cell lymphoma (FLC) and, herein, describe the pathologic and clinical features of 72 cases of this entity. The features of FLC include a follicular growth pattern with pale follicles at low magnification and frequent follicular and/or interfollicular fibrosis. Cytologically, the cells are predominantly large cleaved cells with moderately coarse to fine chromatin, absent or inconspicuous nucleoli, and small to moderate amounts of pale cytoplasm. The mean nuclear diameter of the large cleaved cells was 10.1µ, approximately twice that of small lymphocytes and similar to centroblasts. The t(14;18) was present in 83% of the cases, and a high proportion expressed BCL2 (84%), BCL6 (100%), and CD10 (88%) and had high Ki67 proliferation (81%). The clinical features of patients with FLC were similar to those with other types of FL, and survival was excellent with anthracycline-based chemotherapy plus rituximab. FLC is a variant of follicular lymphoma which should be recognized in future lymphoma classifications because the diagnosis of FLC may be important for the selection of therapy.
Assuntos
Linfócitos B/patologia , Linfócitos/patologia , Linfoma Folicular/patologia , Rituximab/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linfócitos B/efeitos dos fármacos , Diagnóstico Diferencial , Feminino , Humanos , Linfócitos/efeitos dos fármacos , Linfoma Folicular/diagnóstico , Linfoma Folicular/mortalidade , Masculino , Pessoa de Meia-Idade , Translocação Genética/genéticaRESUMO
Anti-picornaviral antisense agents are part of a broader group of nucleic acid-based molecules developed for sequence-specific inhibition of translation and/or transcription of the target sequence through induced nuclease activity or physical hindrance. Three types of nucleic acid-based gene silencing molecules can be distinguished, including DNA-base antisense oligonucleotides (ASO), nucleic acid enzymes (ribozyme and DNAzyme) and double-stranded small interfering RNA (siRNA or microRNA). These antisense DNA and RNA molecules have been widely studied for gene functional studies and therapeutic purposes. In this review, we focus on drug development using ASO and siRNA strategies to inhibit picornavirus infections. The picornavirus genome organization and life cycle is described, followed by discussion of design considerations, chemical modifications and drug delivery approaches. Recent studies using antisense against picornavirus are reviewed. Finally, we compare the advantages and disadvantages of the antisense agents with those of other therapeutics, taking into consideration their limitations which need to be overcome to achieve the final goal of clinical application.
Assuntos
Antivirais/uso terapêutico , DNA Antissenso/uso terapêutico , Genoma Viral , Infecções por Picornaviridae/tratamento farmacológico , Picornaviridae/efeitos dos fármacos , Picornaviridae/genética , RNA Antissenso/uso terapêutico , Antivirais/administração & dosagem , DNA Antissenso/administração & dosagem , Humanos , Lipossomos , Oligonucleotídeos Antissenso/uso terapêutico , Picornaviridae/crescimento & desenvolvimento , Proteoma , RNA Antissenso/administração & dosagem , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/uso terapêuticoRESUMO
Coxsackievirus B3 (CVB3) is a positive, single-stranded RNA virus. The secondary structure of the 3' untranslated region (3'UTR) of CVB3 RNA consists of three stem-loops and is followed by a poly(A) tail sequence. These stem-loop structures have been suggested to participate in the regulation of viral replication through interaction with cellular proteins that are yet to be identified. In this study, by competitive UV cross-linking using mutated 3'UTR probes we have demonstrated that the poly(A) tail is essential for promoting HeLa cell protein interactions with the 3'UTR because deletion of this sequence abolished most of the protein interactions. Unexpectedly, mutations that disrupted the tertiary loop-loop interactions without affecting the stem-loops did not apparently affect these protein interactions, indicating that secondary structure rather than the high-order structure may play a major role in recruiting these RNA binding proteins. Among the observed 3'UTR RNA binding proteins, we have confirmed a 52 kDa protein as the human La autoantigen by using purified recombinant protein and a polyclonal La antibody. This protein can interact with both the 3' and 5'UTRs independently of the poly(A) tail. Further analysis by two-stage UV cross-linking, we found that the 3' and 5'UTR sequences may share the same binding site on the La protein.
Assuntos
Regiões 3' não Traduzidas/metabolismo , Regiões 5' não Traduzidas/metabolismo , Autoantígenos/metabolismo , Enterovirus Humano B/metabolismo , Ribonucleoproteínas/metabolismo , Regiões 3' não Traduzidas/química , Regiões 5' não Traduzidas/química , Autoantígenos/genética , Sequência de Bases , Sítios de Ligação , Reagentes de Ligações Cruzadas , Enterovirus Humano B/química , Enterovirus Humano B/genética , Células HeLa , Humanos , Poli A/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Ribonucleoproteínas/genética , Antígeno SS-BRESUMO
We examined the role of S-linked palmitoylation of human apolipoprotein (apo) B in the assembly and secretion of very low density lipoproteins using recombinant human apoB48. There are four free cysteine residues (Cys(1085), Cys(1396), Cys(1478), and Cys(1635)) within apoB48 that potentially can be palmitoylated. All four cysteine residues were substituted with serine by site-specific mutagenesis. The mutant protein was expressed in transfected rat hepatoma McA-RH7777 cells. Metabolic labeling of the stably transfected cells with iodopalmitic acid analog showed that the mutant apoB48 lacked palmitoylation. The lack of palmitoylation had little impact on the ability of apoB48 to assemble and secrete very low density lipoproteins or high density lipoproteins. Immunocytochemistry experiments using confocal microscopy failed to reveal any major alterations in the intracellular distribution of the mutant apoB48 at steady state. Pulse-chase analysis combined with subcellular fractionation showed no apparent deficiency in the movement of the mutant apoB48 protein from the endoplasmic reticulum to cis/medial Golgi. However, the mutant apoB48 lacking palmitoylation showed retarded movement toward the distal Golgi and increased association (>2-fold) with the membranes of the secretory compartments. A marginal decrease (by 15-20%) in secretion efficiency as compared with that of wild type apoB48 was also observed. These results suggest that lack of palmitoylation may influence the partitioning of apoB48 between microsomal membranes and microsomal lumen, but it does not compromise the ability of apoB48 to assemble lipoproteins.
Assuntos
Apolipoproteínas B/química , Lipoproteínas VLDL/química , Lipoproteínas VLDL/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Apolipoproteína B-48 , Apolipoproteínas B/metabolismo , Linhagem Celular , Cisteína/química , Relação Dose-Resposta a Droga , Retículo Endoplasmático/metabolismo , Complexo de Golgi/metabolismo , Humanos , Imuno-Histoquímica , Lipoproteínas/metabolismo , Microscopia de Fluorescência , Mutagênese Sítio-Dirigida , Mutação , Palmitatos/farmacologia , Ácido Palmítico/química , Ácido Palmítico/metabolismo , Plasmídeos/metabolismo , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico , Ratos , Serina/química , Fatores de Tempo , Transfecção , UltracentrifugaçãoRESUMO
Familial hypobetalipoproteinemia (FHBL), an autosomal co-dominant disorder, is associated with reduced plasma concentrations (<5th percentile for age and sex) of apolipoprotein (apo) B and beta-migrating lipoproteins. To date, only mutations in APOB encoding prematurely truncated apoB have been found in FHBL. We discovered a novel APOB gene mutation, namely R463W, in an extended Christian Lebanese FHBL kindred. Heterozygotes for R463W had the typical FHBL phenotype, whereas homozygotes had barely detectable apoB-100. The effect of the R463W mutation on apoB secretion was examined using transfected McA-RH7777 cells that expressed one of two recombinant human apoBs, namely B48 and B17. In both cases, the mutant proteins (B48RW and B17RW) were retained within the endoplasmic reticulum and were secreted poorly compared with their wild-type counterparts. Pulse-chase analysis showed that secretion efficiencies of B48RW and B17RW were, respectively, 45 and 40% lower than those of the wild-types. Substitution of Arg(463) with Ala in apoB-17 (B17RA) decreased secretion efficiency by approximately 50%, but substitution with Lys (B17RK) had no effect on secretion, indicating that the positive charge was important. Molecular modeling of apoB predicted that Arg(463) was in close proximity to Glu(756) and Asp(456). Substitution of Glu(756) with Gln (B17EQ) had no effect on secretion, but substitution of Asp(456) with Asn (B17DN) decreased secretion to the same extent as B17RW. In co-transfection experiments, the mutant B17RW showed increased binding to microsomal triglyceride transfer protein as compared with wild-type B17. Thus, the naturally occurring R463W mutant reveals a key local domain governing assembly and secretion of apoB-containing lipoproteins.
Assuntos
Apolipoproteínas B/genética , Hipobetalipoproteinemias/genética , Mutação de Sentido Incorreto , Mutação Puntual , Substituição de Aminoácidos , Apolipoproteína B-100 , Apolipoproteína B-48 , Apolipoproteínas B/química , Arginina , Sequência de Bases , Feminino , Triagem de Portadores Genéticos , Humanos , Masculino , Modelos Moleculares , Oligodesoxirribonucleotídeos/química , Linhagem , Conformação ProteicaRESUMO
We determined the role of N-linked glycosylation of apolipoprotein B (apoB) in the assembly and secretion of lipoproteins using transfected rat hepatoma McA-RH7777 cells expressing human apoB-17, apoB-37, and apoB-50, three apoB variants with different ability to recruit neutral lipids. Substituting Asn residue with Gln at the single glycosylation site within apoB-17 (N(158)) decreased its secretion efficiency to a level equivalent to that of wild-type apoB-17 treated with tunicamycin, but had little effect on its synthesis or intracellular distribution. When selective N-to-Q substitution was introduced at one or more of the five N-linked glycosylation sites within apoB-37 (N(158), N(956), N(1341), N(1350), and N(1496)), secretion efficiency of apoB-37 from transiently transfected cells was variably affected. When all five N-linked glycosylation sites were mutated within apoB-37, the secretion efficiency and association with lipoproteins were decreased by >50% as compared with wild-type apoB-37. Similarly, mutant apoB-50 with all of its N-linked glycosylation sites mutagenized showed decreased secretion efficiency and decreased lipoprotein association in both d < 1.02 and d > 1.02 g/ml fractions. The inability of mutant apoB-37 and apoB-50 to associate with very low-density lipoproteins was attributable to impaired assembly and was not due to the limitation of lipid availability. The decreased secretion of mutant apoB-17 and apoB-37 was not accompanied by accumulation within the cells, suggesting that the proportion of mutant apoB not secreted was rapidly degraded. However unlike apoB-17 or apoB-37, accumulation of mutant apoB-50 was observed within the endoplasmic reticulum and Golgi compartments. These data imply that the N-glycans at the amino terminus of apoB play an important role in the assembly and secretion of lipoproteins containing the carboxyl terminally truncated apoB.
Assuntos
Apolipoproteínas B/química , Apolipoproteínas B/metabolismo , Lipoproteínas VLDL/biossíntese , Lipoproteínas VLDL/metabolismo , Oligossacarídeos/metabolismo , Substituição de Aminoácidos/genética , Apolipoproteínas B/genética , Asparagina/metabolismo , Glutamina/metabolismo , Humanos , Relação Estrutura-Atividade , Tunicamicina/farmacologiaRESUMO
The development of high throughput genomic and bioinformatic analysis tools, coupled with established molecular techniques, has allowed new insights into the pathogenesis of infectious diseases. In humans, coxasackievirus B3 (CVB3) is the primary etiological agent of viral myocarditis, an inflammatory disease process involving the heart muscle. Early host cellular survival and apoptotic mechanisms during viral infections, as well as immune events, affect myocarditis progression and outcome. Therefore, our laboratory has been keenly interested in infectomics, defined here as the transcriptional events of both virus and host. We first elucidated up- or downregulated transcriptional activities in CVB3-infected hearts by mRNA differential display. Further characterization of these regulated genes including Nip21, IP10, and IGTPase, and study of their role in CVB3-infection are underway. In further dissection of the stages of myocarditis-peak viremia, inflammatory infiltration and tissue repair-we used cDNA microarrays to probe differential gene expression in the myocardium following virus infection. Following virus infection, there are global decreases in metabolic and mitochondrial genes, increases in signaling genes and distinctive patterns in other functional groups. To establish early gene expression profiles in infected cells by themselves, we also used oligonucleotide arrays in an in vitro model of CVB3 infection. Notably, we have found increased expression of transcription factors c-fos and c-jun down-stream of extracellular signal-related kinase, a pathway which is crucial for virus replication and pathogenesis. Our investigations based on gene profiling following CVB3 infection have thus far been fruitful in providing new experimental leads. High throughput genetic analysis has allowed us to simultaneously try on greater than 12,000 potential genetic "glass slippers." Our in vitro experimental plan has enabled us to chart prominent patterns of gene expression, analyzed by novel bioinformatic approaches, and to separate varied and potentially significant gene expression events.