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Bottom-of-sulcus dysplasia (BOSD) is increasingly recognized as a cause of drug-resistant, surgically-remediable, focal epilepsy, often in seemingly MRI-negative patients. We describe the clinical manifestations, morphological features, localization patterns and genetics of BOSD, with the aims of improving management and understanding pathogenesis. We studied 85 patients with BOSD diagnosed between 2005-2022. Presenting seizure and EEG characteristics, clinical course, genetic findings and treatment response were obtained from medical records. MRI (3 T) and 18F-FDG-PET scans were reviewed systematically for BOSD morphology and metabolism. Histopathological analysis and tissue genetic testing were performed in 64 operated patients. BOSD locations were transposed to common imaging space to study anatomical location, functional network localization and relationship to normal MTOR gene expression. All patients presented with stereotyped focal seizures with rapidly escalating frequency, prompting hospitalization in 48%. Despite 42% patients having seizure remissions, usually with sodium channel blocking medications, most eventually became drug-resistant and underwent surgery (86% seizure-free). Prior developmental delay was uncommon but intellectual, language and executive dysfunction were present in 24%, 48% and 29% when assessed preoperatively, low intellect being associated with greater epilepsy duration. BOSDs were missed on initial MRI in 68%, being ultimately recognized following repeat MRI, 18F-FDG-PET or image postprocessing. MRI features were grey-white junction blurring (100%), cortical thickening (91%), transmantle band (62%), increased cortical T1 signal (46%) and increased subcortical FLAIR signal (26%). BOSD hypometabolism was present on 18F-FDG-PET in 99%. Additional areas of cortical malformation or grey matter heterotopia were present in eight patients. BOSDs predominated in frontal and pericentral cortex and related functional networks, mostly sparing temporal and occipital cortex, and limbic and visual networks. Genetic testing yielded pathogenic mTOR pathway variants in 63% patients, including somatic MTOR variants in 47% operated patients and germline DEPDC5 or NPRL3 variants in 73% patients with familial focal epilepsy. BOSDs tended to occur in regions where the healthy brain normally shows lower MTOR expression, suggesting these regions may be more vulnerable to upregulation of MTOR activity. Consistent with the existing literature, these results highlight (i) clinical features raising suspicion of BOSD; (ii) the role of somatic and germline mTOR pathway variants in patients with sporadic and familial focal epilepsy associated with BOSD; and (iii) the role of 18F-FDG-PET alongside high-field MRI in detecting subtle BOSD. The anatomical and functional distribution of BOSDs likely explain their seizure, EEG and cognitive manifestations and may relate to relative MTOR expression.
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Epilepsia Refractaria , Epilepsias Parciales , Síndromes Epilépticos , Malformaciones del Desarrollo Cortical , Humanos , Fluorodesoxiglucosa F18 , Malformaciones del Desarrollo Cortical/genética , Epilepsias Parciales/diagnóstico por imagen , Epilepsias Parciales/genética , Epilepsias Parciales/patología , Epilepsia Refractaria/diagnóstico por imagen , Epilepsia Refractaria/genética , Epilepsia Refractaria/cirugía , Imagen por Resonancia Magnética/métodos , Convulsiones/complicaciones , Serina-Treonina Quinasas TOR , Proteínas Activadoras de GTPasa/genéticaRESUMEN
Childhood apraxia of speech (CAS), the prototypic severe childhood speech disorder, is characterized by motor programming and planning deficits. Genetic factors make substantive contributions to CAS aetiology, with a monogenic pathogenic variant identified in a third of cases, implicating around 20 single genes to date. Here we aimed to identify molecular causation in 70 unrelated probands ascertained with CAS. We performed trio genome sequencing. Our bioinformatic analysis examined single nucleotide, indel, copy number, structural and short tandem repeat variants. We prioritised appropriate variants arising de novo or inherited that were expected to be damaging based on in silico predictions. We identified high confidence variants in 18/70 (26%) probands, almost doubling the current number of candidate genes for CAS. Three of the 18 variants affected SETBP1, SETD1A and DDX3X, thus confirming their roles in CAS, while the remaining 15 occurred in genes not previously associated with this disorder. Fifteen variants arose de novo and three were inherited. We provide further novel insights into the biology of child speech disorder, highlighting the roles of chromatin organization and gene regulation in CAS, and confirm that genes involved in CAS are co-expressed during brain development. Our findings confirm a diagnostic yield comparable to, or even higher, than other neurodevelopmental disorders with substantial de novo variant burden. Data also support the increasingly recognised overlaps between genes conferring risk for a range of neurodevelopmental disorders. Understanding the aetiological basis of CAS is critical to end the diagnostic odyssey and ensure affected individuals are poised for precision medicine trials.
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Apraxias , Trastornos del Habla , Niño , Humanos , Trastornos del Habla/genética , Apraxias/genética , Mapeo Cromosómico , Causalidad , Encéfalo , N-Metiltransferasa de Histona-LisinaRESUMEN
PRKACA and PRKACB code for two catalytic subunits (Cα and Cß) of cAMP-dependent protein kinase (PKA), a pleiotropic holoenzyme that regulates numerous fundamental biological processes such as metabolism, development, memory, and immune response. We report seven unrelated individuals presenting with a multiple congenital malformation syndrome in whom we identified heterozygous germline or mosaic missense variants in PRKACA or PRKACB. Three affected individuals were found with the same PRKACA variant, and the other four had different PRKACB mutations. In most cases, the mutations arose de novo, and two individuals had offspring with the same condition. Nearly all affected individuals and their affected offspring shared an atrioventricular septal defect or a common atrium along with postaxial polydactyly. Additional features included skeletal abnormalities and ectodermal defects of variable severity in five individuals, cognitive deficit in two individuals, and various unusual tumors in one individual. We investigated the structural and functional consequences of the variants identified in PRKACA and PRKACB through the use of several computational and experimental approaches, and we found that they lead to PKA holoenzymes which are more sensitive to activation by cAMP than are the wild-type proteins. Furthermore, expression of PRKACA or PRKACB variants detected in the affected individuals inhibited hedgehog signaling in NIH 3T3 fibroblasts, thereby providing an underlying mechanism for the developmental defects observed in these cases. Our findings highlight the importance of both Cα and Cß subunits of PKA during human development.
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Anomalías Múltiples/genética , Disfunción Cognitiva/genética , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/genética , Dedos/anomalías , Mutación de Línea Germinal , Defectos de los Tabiques Cardíacos/genética , Polidactilia/genética , Dedos del Pie/anomalías , Anomalías Múltiples/diagnóstico , Anomalías Múltiples/patología , Adolescente , Adulto , Animales , Secuencia de Bases , Disfunción Cognitiva/diagnóstico , Disfunción Cognitiva/patología , AMP Cíclico/metabolismo , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/química , Subunidades Catalíticas de Proteína Quinasa Dependientes de AMP Cíclico/deficiencia , Femenino , Dedos/patología , Regulación del Desarrollo de la Expresión Génica , Defectos de los Tabiques Cardíacos/diagnóstico , Defectos de los Tabiques Cardíacos/patología , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Holoenzimas/química , Holoenzimas/deficiencia , Holoenzimas/genética , Humanos , Recién Nacido , Masculino , Ratones , Modelos Moleculares , Mosaicismo , Células 3T3 NIH , Linaje , Polidactilia/diagnóstico , Polidactilia/patología , Estructura Secundaria de Proteína , Dedos del Pie/patologíaRESUMEN
Febrile seizures represent the most common type of pathological brain activity in young children and are influenced by genetic, environmental and developmental factors. In a minority of cases, febrile seizures precede later development of epilepsy. We conducted a genome-wide association study of febrile seizures in 7635 cases and 83 966 controls identifying and replicating seven new loci, all with P < 5 × 10-10. Variants at two loci were functionally related to altered expression of the fever response genes PTGER3 and IL10, and four other loci harboured genes (BSN, ERC2, GABRG2, HERC1) influencing neuronal excitability by regulating neurotransmitter release and binding, vesicular transport or membrane trafficking at the synapse. Four previously reported loci (SCN1A, SCN2A, ANO3 and 12q21.33) were all confirmed. Collectively, the seven novel and four previously reported loci explained 2.8% of the variance in liability to febrile seizures, and the single nucleotide polymorphism heritability based on all common autosomal single nucleotide polymorphisms was 10.8%. GABRG2, SCN1A and SCN2A are well-established epilepsy genes and, overall, we found positive genetic correlations with epilepsies (rg = 0.39, P = 1.68 × 10-4). Further, we found that higher polygenic risk scores for febrile seizures were associated with epilepsy and with history of hospital admission for febrile seizures. Finally, we found that polygenic risk of febrile seizures was lower in febrile seizure patients with neuropsychiatric disease compared to febrile seizure patients in a general population sample. In conclusion, this largest genetic investigation of febrile seizures to date implicates central fever response genes as well as genes affecting neuronal excitability, including several known epilepsy genes. Further functional and genetic studies based on these findings will provide important insights into the complex pathophysiological processes of seizures with and without fever.
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Epilepsia , Convulsiones Febriles , Anoctaminas/genética , Niño , Preescolar , Epilepsia/genética , Fiebre/complicaciones , Fiebre/genética , Estudio de Asociación del Genoma Completo , Humanos , Canal de Sodio Activado por Voltaje NAV1.1/genética , Convulsiones Febriles/genéticaRESUMEN
Ring chromosomes occur when the ends of normally rod-shaped chromosomes fuse. In ring chromosome 20 (ring 20), intellectual disability and epilepsy are usually present, even if there is no deleted coding material; the mechanism by which individuals with complete ring chromosomes develop seizures and other phenotypic abnormalities is not understood. We investigated altered gene transcription as a contributing factor by performing RNA-sequencing (RNA-seq) analysis on blood from seven patients with ring 20, and 11 first-degree relatives (all parents). Geographic analysis did not identify altered expression in peritelomeric or other specific chromosome 20 regions. RNA-seq analysis revealed 97 genes potentially differentially expressed in ring 20 patients. These included one epilepsy gene, NPRL3, but this finding was not confirmed on reverse transcription Droplet Digital polymerase chain reaction analysis. Molecular studies of structural chromosomal anomalies such as ring chromosome are challenging and often difficult to interpret because many patients are mosaic, and there may be genome-wide chromosomal instability affecting gene expression. Our findings nevertheless suggest that peritelomeric altered transcription is not the likely pathogenic mechanism in ring 20. Underlying genetic mechanisms are likely complex and may involve differential expression of many genes, the majority of which may not be located on chromosome 20.
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Epilepsia Refractaria/genética , Perfilación de la Expresión Génica , Expresión Génica/genética , Discapacidad Intelectual/genética , Cromosomas en Anillo , Adulto , Niño , Familia , Femenino , Proteínas Activadoras de GTPasa/genética , Ontología de Genes , Humanos , Masculino , RNA-Seq , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
BACKGROUND: Determination of blood donor hemoglobin (Hb) levels is a pre-requisite to ensure donor safety and blood product quality. We aimed to identify Hb measurement practices across blood donation services and to what extent differences associate with low-Hb deferral rates. METHODS: An online survey was performed among Biomedical Excellence for Safer Transfusion (BEST) Collaborative members, extended with published data. Multivariable negative-binomial regression models were built to estimate adjusted associations of minimum donation intervals, Hb cut-offs (high, ≥13.5 g/dL in men or ≥ 12.5 g/dL in women, vs. lower values), iron monitoring (yes/no), providing or prescribing iron supplementation (yes/no), post-versus pre-donation Hb measurement and geographical location (Asian vs. rest), with low-Hb deferral rates. RESULTS: Data were included from 38 blood services. Low-Hb deferral rates varied from 0.11% to 8.81% among men and 0.84% to 31.85% among women. Services with longer minimum donation intervals had significantly lower deferral rates among both women (rate ratio, RR 0.53, 95%CI 0.33-0.84) and men (RR 0.53, 95%CI 0.31-0.90). In women, iron supplementation was associated with lower Hb deferral rates (RR 0.47, 95%CI 0.23-0.94). Finally, being located in Asia was associated with higher low-Hb deferral rates; RR 9.10 (95%CI 3.89-21.27) for women and 6.76 (95%CI 2.45-18.68) for men. CONCLUSION: Differences in Hb measurement and eligibility criteria, particularly longer donation intervals and iron supplementation in women, are associated with variations in low-Hb deferral rates. These insights could help improve both blood donation service efficiency and donor care.
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Donantes de Sangre/estadística & datos numéricos , Hemoglobinas/metabolismo , Transfusión Sanguínea/métodos , Selección de Donante , Femenino , Pruebas Hematológicas , Humanos , Hierro/metabolismo , Encuestas y Cuestionarios , Factores de TiempoRESUMEN
Variants in RORB have been reported in eight individuals with epilepsy, with phenotypes ranging from eyelid myoclonia with absence epilepsy to developmental and epileptic encephalopathies. We identified novel RORB variants in 11 affected individuals from four families. One was from whole genome sequencing and three were from RORB screening of three epilepsy cohorts: developmental and epileptic encephalopathies (n = 1021), overlap of generalized and occipital epilepsy (n = 84), and photosensitivity (n = 123). Following interviews and review of medical records, individuals' seizure and epilepsy syndromes were classified. Three novel missense variants and one exon 3 deletion were predicted to be pathogenic by in silico tools, not found in population databases, and located in key evolutionary conserved domains. Median age at seizure onset was 3.5 years (0.5-10 years). Generalized, predominantly absence and myoclonic, and occipital seizures were seen in all families, often within the same individual (6/11). All individuals with epilepsy were photosensitive, and seven of 11 had cognitive abnormalities. Electroencephalograms showed generalized spike and wave and/or polyspike and wave. Here we show a striking RORB phenotype of overlap of photosensitive generalized and occipital epilepsy in both individuals and families. This is the first report of a gene associated with this overlap of epilepsy syndromes.
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Epilepsias Parciales/genética , Epilepsia Generalizada/genética , Epilepsia Refleja/genética , Predisposición Genética a la Enfermedad/genética , Miembro 2 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Mutación , LinajeRESUMEN
In vivo delivery of large RNA molecules has significant implications for novel gene therapy, biologics delivery, and vaccine applications. We have developed cationic nanolipoprotein particles (NLPs) to enhance the complexation and delivery of large self-amplifying mRNAs (replicons) in vivo. NLPs are high-density lipoprotein (HDL) mimetics, comprised of a discoidal lipid bilayer stabilized by apolipoproteins that are readily functionalized to provide a versatile delivery platform. Herein, we systematically screened NLP assembly with a wide range of lipidic and apolipoprotein constituents, using biophysical metrics to identify lead candidates for in vivo RNA delivery. NLPs formulated with cationic lipids successfully complexed with RNA replicons encoding luciferase, provided measurable protection from RNase degradation, and promoted replicon in vivo expression. The NLP complexation of the replicon and in vivo transfection efficiency were further enhanced by modulating the type and percentage of cationic lipid, the ratio of cationic NLP to replicon, and by incorporating additive molecules.
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Lipoproteínas HDL/metabolismo , ARN Mensajero/metabolismo , Apolipoproteínas/química , Apolipoproteínas/metabolismo , Biomimética , Membrana Dobles de Lípidos/química , Lipoproteínas HDL/química , ARN Mensajero/química , Replicón/genéticaRESUMEN
The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.
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Adenosina Trifosfato/metabolismo , Membrana Celular/química , Imagen Individual de Molécula/métodos , Dominio Catalítico , Sistema Libre de Células , Receptores ErbB/análisis , Receptores ErbB/genética , Receptores ErbB/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Lipoproteínas/química , Microscopía Confocal/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
PURPOSE: The purpose of this study was to expand the genetic architecture of neurodevelopmental disorders, and to characterize the clinical features of a novel cohort of affected individuals with variants in ZNF142, a C2H2 domain-containing transcription factor. METHODS: Four independent research centers used exome sequencing to elucidate the genetic basis of neurodevelopmental phenotypes in four unrelated families. Following bioinformatic filtering, query of control data sets, and secondary variant confirmation, we aggregated findings using an online data sharing platform. We performed in-depth clinical phenotyping in all affected individuals. RESULTS: We identified seven affected females in four pedigrees with likely pathogenic variants in ZNF142 that segregate with recessive disease. Affected cases in three families harbor either nonsense or frameshifting likely pathogenic variants predicted to undergo nonsense mediated decay. One additional trio bears ultrarare missense variants in conserved regions of ZNF142 that are predicted to be damaging to protein function. We performed clinical comparisons across our cohort and noted consistent presence of intellectual disability and speech impairment, with variable manifestation of seizures, tremor, and dystonia. CONCLUSION: Our aggregate data support a role for ZNF142 in nervous system development and add to the emergent list of zinc finger proteins that contribute to neurocognitive disorders.
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Discapacidades del Desarrollo/genética , Trastornos del Neurodesarrollo/genética , Transactivadores/genética , Adolescente , Adulto , Niño , Estudios de Cohortes , Biología Computacional/métodos , Distonía/genética , Familia , Femenino , Humanos , Discapacidad Intelectual/genética , Mutación , Mutación Missense , Linaje , Fenotipo , Convulsiones/genética , Trastornos del Habla/genética , Transactivadores/metabolismo , Secuenciación del ExomaRESUMEN
A nanolipoprotein particle (NLP) is a lipid bilayer disc stabilized by two amphipathic "scaffold" apolipoproteins. It has been most notably utilized as a tool for solubilizing a variety of membrane proteins while preserving structural and functional properties. Transfer of functional proteins from NLPs into model membrane systems such as supported lipid bilayers (SLBs) would enable new opportunities, for example, two-dimensional protein crystallization and studies on protein-protein interactions. This work used fluorescence microscopy and atomic force microscopy to investigate the interaction between NLPs and SLBs. When incubated with SLBs, NLPs were found to spontaneously deliver lipid and protein cargo. The impact of membrane composition on lipid exchange was explored, revealing a positive correlation between the magnitude of lipid transfer and concentration of defects in the target SLB. Incorporation of lipids capable of binding specifically to polyhistidine tags encoded into the apolipoproteins also boosted transfer of NLP cargo. Optimal conditions for lipid and protein delivery from NLPs to SLBs are proposed based on interaction mechanisms.
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Membrana Dobles de Lípidos/química , Lipoproteínas/química , Nanopartículas/química , Resinas Acrílicas/químicaRESUMEN
Chlamydia is a prevalent sexually transmitted disease that infects more than 100 million people worldwide. Although most individuals infected with Chlamydia trachomatis are initially asymptomatic, symptoms can arise if left undiagnosed. Long-term infection can result in debilitating conditions such as pelvic inflammatory disease, infertility, and blindness. Chlamydia infection, therefore, constitutes a significant public health threat, underscoring the need for a Chlamydia-specific vaccine. Chlamydia strains express a major outer-membrane protein (MOMP) that has been shown to be an effective vaccine antigen. However, approaches to produce a functional recombinant MOMP protein for vaccine development are limited by poor solubility, low yield, and protein misfolding. Here, we used an Escherichia coli-based cell-free system to express a MOMP protein from the mouse-specific species Chlamydia muridarum (MoPn-MOMP or mMOMP). The codon-optimized mMOMP gene was co-translated with Δ49apolipoprotein A1 (Δ49ApoA1), a truncated version of mouse ApoA1 in which the N-terminal 49 amino acids were removed. This co-translation process produced mMOMP supported within a telodendrimer nanolipoprotein particle (mMOMP-tNLP). The cell-free expressed mMOMP-tNLPs contain mMOMP multimers similar to the native MOMP protein. This cell-free process produced on average 1.5 mg of purified, water-soluble mMOMP-tNLP complex in a 1-ml cell-free reaction. The mMOMP-tNLP particle also accommodated the co-localization of CpG oligodeoxynucleotide 1826, a single-stranded synthetic DNA adjuvant, eliciting an enhanced humoral immune response in vaccinated mice. Using our mMOMP-tNLP formulation, we demonstrate a unique approach to solubilizing and administering membrane-bound proteins for future vaccine development. This method can be applied to other previously difficult-to-obtain antigens while maintaining full functionality and immunogenicity.
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Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/química , Vacunas Bacterianas/inmunología , Infecciones por Chlamydia/inmunología , Chlamydia muridarum/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Bases , Sistema Libre de Células , Infecciones por Chlamydia/microbiología , Femenino , Ratones , Ratones Endogámicos BALB CRESUMEN
BACKGROUND: Value-based health care aims to optimize the balance of patient outcomes and health care costs. To improve value in perinatal care using this strategy, standard outcomes must first be defined. The objective of this work was to define a minimum, internationally appropriate set of outcome measures for evaluating and improving perinatal care with a focus on outcomes that matter to women and their families. METHODS: An interdisciplinary and international Working Group was assembled. Existing literature and current measurement initiatives were reviewed. Serial guided discussions and validation surveys provided consumer input. A series of nine teleconferences, incorporating a modified Delphi process, were held to reach consensus on the proposed Standard Set. RESULTS: The Working Group selected 24 outcome measures to evaluate care during pregnancy and up to 6 months postpartum. These include clinical outcomes such as maternal and neonatal mortality and morbidity, stillbirth, preterm birth, birth injury and patient-reported outcome measures (PROMs) that assess health-related quality of life (HRQoL), mental health, mother-infant bonding, confidence and success with breastfeeding, incontinence, and satisfaction with care and birth experience. To support analysis of these outcome measures, pertinent baseline characteristics and risk factor metrics were also defined. CONCLUSIONS: We propose a set of outcome measures for evaluating the care that women and infants receive during pregnancy and the postpartum period. While validation and refinement via pilot implementation projects are needed, we view this as an important initial step towards value-based improvements in care.
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Evaluación de Resultado en la Atención de Salud/normas , Atención Perinatal/normas , Consenso , Atención a la Salud/normas , Parto Obstétrico/normas , Femenino , Humanos , Lactante , Recién Nacido , Relaciones Madre-Hijo , Medición de Resultados Informados por el Paciente , Embarazo , Resultado del Embarazo , Nacimiento Prematuro/etiología , Nacimiento Prematuro/prevención & control , Calidad de Vida , Factores de RiesgoRESUMEN
Nanoparticles (NPs) are often functionalized with reactive groups such as amines and thiols for the subsequent conjugation of further molecules, e.g., stabilizing polymers, drugs, and proteins for targeting cells or specific diseases. In addition to the quantitative estimation of the reactive conjugation sites, their molecular positioning and nanoscale arrangement on single nanoparticles become more and more important for the tailored engineering and design of functional nanomaterials. Here, we use maleimide or sulfo-succinimidyl ester-modified 1.4 nm gold nanoclusters (AuNCs) to specifically label reactive thiol and amine groups with sub-2-nm precision on metal oxide and polymeric nanostructures. We confirm the binding of AuNCs by measuring and modeling sedimentation properties using analytical centrifugation, imaging their surface distribution and surface distances by transmission electron microscopy (TEM), and comparing the results to ensemble measurements of numbers of reactive surface groups obtained by common photometric assays. We map thiol and amine groups introduced on silica NPs (SiNPs), titania stars (Ti), silica inverse opals (SiOps), and polystyrene NPs (PS NPs). We show that the method is suitable for mapping local, clustered inhomogeneities of the reactive sites on single SiNPs introduced by masking certain areas during surface functionalization. Mapping precise positions of reactive surface groups is essential to the design and tailored ligation of multifunctional nanomaterials.
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BACKGROUND: Recombinant expression of toxic proteins remains a challenging problem. One potential method to shield toxicity and thus improve expression of these proteins is to encapsulate them within protein compartments to sequester them away from their targets. Many bacteria naturally produce so-called bacterial microcompartments (BMCs) in which enzymes comprising a biosynthetic pathway are encapsulated in a proteinaeous shell, which is in part thought to shield the cells from the toxicity of reaction intermediates. As a proof-of-concept, we attempted to encapsulate toxic, lysis protein E (E) from bacteriophage ÏX174 inside recombinant BMCs to enhance its expression and achieve higher yields during downstream purification. RESULTS: E was fused with various N-terminal BMC targeting tags (PduP-, PduD-, and EutC-tags, 18-20 amino acids) and co-expressed with appropriate BMC shell proteins that associate with the tags and are required to form BMCs. Only BMC targeted E fusions, but not non-tagged E, could be successfully cloned, suggesting that the BMC tags reduce the toxicity of E. A PduP-tagged E system appeared to achieve the highest expression of E. Co-expression of Pdu BMC shell proteins with PduP-E increased its expression by 20-50%. Affinity purification of PduP-E via Ni-NTA in the presence of Empigen BB detergent yielded 270 µg of PduP-E per L of induced culture. Removal of the PduP-tag via proteolysis resulted in a final yield of 200 µg of E per L of induced culture, a nearly order of magnitude (~sevenfold) improvement compared to prior reports. CONCLUSIONS: These results demonstrate improved expression of ÏX174 lysis protein E via re-directed BMC systems and ultimately higher E purification yields. Similar strategies can be used to enhance expression of other toxic proteins in recombinant Escherichia coli systems.
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Escherichia coli/genética , Expresión Génica , Proteínas Virales/biosíntesis , Proteínas Virales/genética , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Compartimento Celular , Medios de Cultivo/química , Escherichia coli/citología , Escherichia coli/metabolismo , Proteolisis , Proteínas Recombinantes/biosíntesis , Proteínas Virales/aislamiento & purificaciónRESUMEN
There are 160,000 cancer patients worldwide treated with particle radiotherapy (RT). With the advent of proton, and high (H) charge (Z) and energy (E) HZE ionizing particle RT, the cardiovascular diseases risk estimates are uncertain. In addition, future deep space exploratory-type missions will expose humans to unknown but low doses of particle irradiation (IR). We examined molecular responses using transcriptome profiling in left ventricular murine cardiomyocytes isolated from mice that were exposed to 90 cGy, 1 GeV proton ((1)H) and 15 cGy, 1 GeV/nucleon iron ((56)Fe) over 28 days after exposure. Unsupervised clustering analysis of gene expression segregated samples according to the IR response and time after exposure, with (56)Fe-IR showing the greatest level of gene modulation. (1)H-IR showed little differential transcript modulation. Network analysis categorized the major differentially expressed genes into cell cycle, oxidative responses, and transcriptional regulation functional groups. Transcriptional networks identified key nodes regulating expression. Validation of the signal transduction network by protein analysis and gel shift assay showed that particle IR clearly regulates a long-lived signaling mechanism for ERK1/2, p38 MAPK signaling and identified NFATc4, GATA4, STAT3, and NF-κB as regulators of the response at specific time points. These data suggest that the molecular responses and gene expression to (56)Fe-IR in cardiomyocytes are unique and long-lasting. Our study may have significant implications for the efforts of National Aeronautics and Space Administration to develop heart disease risk estimates for astronauts and for patients receiving conventional and particle RT via identification of specific HZE-IR molecular markers.
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Redes Reguladoras de Genes/efectos de la radiación , Radioisótopos de Hierro/toxicidad , Miocitos Cardíacos/efectos de la radiación , Radioterapia de Alta Energía/efectos adversos , Transducción de Señal/efectos de la radiación , Animales , Células Cultivadas , Análisis por Conglomerados , Activación Enzimática , Fibrosis , Factor de Transcripción GATA4/genética , Factor de Transcripción GATA4/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de la radiación , Masculino , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/genética , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena en Tiempo Real de la Polimerasa , Medición de Riesgo , Factor de Transcripción STAT3/genética , Factor de Transcripción STAT3/metabolismo , Factores de Tiempo , Transcripción Genética/efectos de la radiación , Transcriptoma/efectos de la radiación , Irradiación Corporal TotalRESUMEN
Marine benthic communities face multiple anthropogenic pressures that compromise the future of some of the most biodiverse and functionally important ecosystems in the world. Yet one of the pressures these ecosystems face, night-time lighting, remains unstudied. Light is an important cue in guiding the settlement of invertebrate larvae, and altering natural regimes of nocturnal illumination could modify patterns of recruitment among sessile epifauna. We present the first evidence of night-time lighting changing the composition of temperate epifaunal marine invertebrate communities. Illuminating settlement surfaces with white light-emitting diode lighting at night, to levels experienced by these communities locally, both inhibited and encouraged the colonization of 39% of the taxa analysed, including three sessile and two mobile species. Our results indicate that ecological light pollution from coastal development, shipping and offshore infrastructure could be changing the composition of marine epifaunal communities.
Asunto(s)
Invertebrados/fisiología , Iluminación/efectos adversos , Animales , Organismos Acuáticos , Ecosistema , Contaminación Ambiental , Larva/fisiologíaRESUMEN
INTRODUCTION: Persistent foetal tachyarrythmias complicated by hydrops fetalis carry a poor prognosis, with foetal death reported in excess of a quarter despite treatment. We present our experience with direct intraperitoneal amiodarone administration in eight hydropic foetuses with resistant supraventricular tachycardia. METHODS: Amiodarone was injected slowly into foetal peritoneal cavity under ultrasound guidance. All mothers were loaded with oral amiodarone before the procedure and maintained on it. The procedure was repeated guided by foetal rhythm. RESULT: All eight cases had severe hydrops with a median foetal heart rate of 255 bpm (range 240-300 bpm), and the median gestational age was 27+1 weeks (range 21-33+3 weeks) at presentation. In six cases, the average time for supraventricular tachycardia to revert to sinus rhythm from the first procedure was 11.5 days. In one case, intravascular injection of amiodarone into the umbilical vein was performed before intraperitoneal injection, which resulted in conversion to sinus rhythm sustained until delivery. In the last case, supraventricular tachycardia and severe hydrops persisted and the baby was delivered 5 days later at 34 weeks' gestation. Hydrops resolved in five foetuses with a mean resolution time of 28.4 days. The mean gestational age at delivery was 34+5 days and seven of eight cases survived beyond the neonatal period with good postnatal outcomes. CONCLUSION: Intraperitoneal administration of amiodarone is a relatively simple and effective strategy in refractory supraventricular tachycardia complicated by severe hydrops. The intraperitoneal route assures delivery of the drug to the severely hydropic foetus and enables a bolus dose to be delivered for sustained absorption.