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1.
PLoS Comput Biol ; 16(9): e1008193, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32925919

RESUMO

Segmenting cell nuclei within microscopy images is a ubiquitous task in biological research and clinical applications. Unfortunately, segmenting low-contrast overlapping objects that may be tightly packed is a major bottleneck in standard deep learning-based models. We report a Nuclear Segmentation Tool (NuSeT) based on deep learning that accurately segments nuclei across multiple types of fluorescence imaging data. Using a hybrid network consisting of U-Net and Region Proposal Networks (RPN), followed by a watershed step, we have achieved superior performance in detecting and delineating nuclear boundaries in 2D and 3D images of varying complexities. By using foreground normalization and additional training on synthetic images containing non-cellular artifacts, NuSeT improves nuclear detection and reduces false positives. NuSeT addresses common challenges in nuclear segmentation such as variability in nuclear signal and shape, limited training sample size, and sample preparation artifacts. Compared to other segmentation models, NuSeT consistently fares better in generating accurate segmentation masks and assigning boundaries for touching nuclei.


Assuntos
Núcleo Celular/fisiologia , Aprendizado Profundo , Processamento de Imagem Assistida por Computador/métodos , Microscopia/métodos , Algoritmos , Artefatos , Biologia Computacional , Células HeLa , Humanos , Software
2.
Nat Commun ; 11(1): 4581, 2020 09 11.
Artigo em Inglês | MEDLINE | ID: mdl-32917893

RESUMO

Yes-associated protein 1 (YAP) is a transcriptional regulator with critical roles in mechanotransduction, organ size control, and regeneration. Here, using advanced tools for real-time visualization of native YAP and target gene transcription dynamics, we show that a cycle of fast exodus of nuclear YAP to the cytoplasm followed by fast reentry to the nucleus ("localization-resets") activates YAP target genes. These "resets" are induced by calcium signaling, modulation of actomyosin contractility, or mitosis. Using nascent-transcription reporter knock-ins of YAP target genes, we show a strict association between these resets and downstream transcription. Oncogenically-transformed cell lines lack localization-resets and instead show dramatically elevated rates of nucleocytoplasmic shuttling of YAP, suggesting an escape from compartmentalization-based control. The single-cell localization and transcription traces suggest that YAP activity is not a simple linear function of nuclear enrichment and point to a model of transcriptional activation based on nucleocytoplasmic exchange properties of YAP.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Sistemas CRISPR-Cas , Cálcio/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica , Técnicas de Introdução de Genes , Células HEK293 , Humanos , Mecanotransdução Celular/fisiologia , Oncogenes/genética , Fatores de Transcrição/genética
3.
Int J Pediatr Otorhinolaryngol ; 137: 110218, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32658803

RESUMO

OBJECTIVES: The purpose of this paper is to consider the anatomical basis and surgical technique along with the utility of buccal fat pad (BFP) for the reconstruction in cleft lip and palate patients. METHODS: We reviewed 27 cases of CLAP treated with BFP over three year period in our institution which included 2 cases of primary palatoplasty & 25 cases of secondary palatoplasty. Inclusion criteria consisted of patients operated by a single surgeon with a minimum follow up of 2yrs. Exclusion criteria included all syndromic cleft lip and palate patients. Predictor variables recorded were demographic characters, follow up period, type of cleft, type of surgical procedure, site & dimension of the fistula. Outcome variables of the study were post-operative fistula formation, post-operative hemorrhage & speech assessment. RESULTS: The study included 8 female & 19 male patients with mean age group 3.75 ± 1.75yrs. Cleft of soft palate & Lt. CLAP were the most common type of cleft. The surgical technique used was: BFP with V-Y pushback palatoplasty for primary palate repair, BFP with Furlow's technique for VPI correction, and BFP in conjugation with rotation flap, straight-line closure or redohardpalatoplasty for fistula closure. All cases showed satisfactory healing with favorable speech assessment outcomes for 18 patients (67%). CONCLUSION: BFP along with other types of flap is the choice of treatment in cases of moderate defect owing to its favorable anatomic location & high vascularity. The size limitation of the BFP must be known to permit a successful outcome.

4.
Artigo em Inglês | MEDLINE | ID: mdl-32181591

RESUMO

The majority of patients with spinal muscular atrophy (SMA) identified to date harbor a biallelic exonic deletion of SMN1. However, there have been reports of SMA-like disorders that are independent of SMN1, including those due to pathogenic variants in the glycyl-tRNA synthetase gene (GARS1). We report three unrelated patients with de novo variants in GARS1 that are associated with infantile-onset SMA (iSMA). Patients were ascertained during inpatient hospital evaluations for complications of neuropathy. Evaluations were completed as indicated for clinical care and management and informed consent for publication was obtained. One newly identified, disease-associated GARS1 variant, identified in two out of three patients, was analyzed by functional studies in yeast complementation assays. Genomic analyses by exome and/or gene panel and SMN1 copy number analysis of three patients identified two previously undescribed de novo missense variants in GARS1 and excluded SMN1 as the causative gene. Functional studies in yeast revealed that one of the de novo GARS1 variants results in a loss-of-function effect, consistent with other pathogenic GARS1 alleles. In sum, the patients' clinical presentation, assessments of previously identified GARS1 variants and functional assays in yeast suggest that the GARS1 variants described here cause iSMA. GARS1 variants have been previously associated with Charcot-Marie-Tooth disease (CMT2D) and distal SMA type V (dSMAV). Our findings expand the allelic heterogeneity of GARS-associated disease and support that severe early-onset SMA can be caused by variants in this gene. Distinguishing the SMA phenotype caused by SMN1 variants from that due to pathogenic variants in other genes such as GARS1 significantly alters approaches to treatment.

5.
J Craniofac Surg ; 31(4): e394-e397, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32176021

RESUMO

AIM: The aim of this study is to determine the frequency of pediatric pathologies and their distribution according to age, gender, site and types of lesions reported over a period of 2004 - 2019 in two prominent Dental Colleges at Western UP, India. MATERIAL AND METHODS: All the cases of pediatric pathology were retrospectively analyzed and reviewed that reported in the Department of Oral And Maxillofacial Surgery of 2 prominent dental colleges of Western UP, India. All consecutive patients (< 18 years), between 2004 to 2019, histologically diagnosed as having an intraosseous tumor or tumor-like lesions and cystic lesion, vascular lesions, patients with space infections and hardware infection formed the study population. Patients fulfilling the inclusion criteria were only considered for further study. The study was granted an exemption by the institutes. The entire study material was analyzed and grouped into prominent categories for logical conclusions: The accumulated data was grouped, entered, and analyzed. RESULTS: One hundred two cases fulfilled the criteria. Gender distribution was equal, with mandible predominance and a predominance of non-odontogenic lesions. There were 4 malignant and 57 benign conditions. There were 23 lesions in the anterior jaw and 38 lesions were present in the posterior jaw. CONCLUSION: The pattern of pediatric pathology presentation from this part of India has been documented. In this study, the pediatric jaw tumors are less common compared to those in adult jaw tumors with non-odontogenic tumors being more common.


Assuntos
Cabeça/patologia , Pescoço/patologia , Adolescente , Criança , Humanos , Incidência , Índia , Neoplasias Maxilomandibulares/patologia , Prevalência , Estudos Retrospectivos , Cirurgia Bucal
6.
Biochim Biophys Acta Gen Subj ; 1864(3): 129503, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31816347

RESUMO

BACKGROUND: In recent times, coordination complexes of iron in various oxidation states along with variety of ligand systems have been designed and developed for effective treatment of cancer cells without adversely affecting the normal cell and tissues of various organs. METHODS: In this study, we have evaluated the mechanism of action of a Fe(II) Schiff base complex in the crop plant Trigonella foenum-graecum L. (Fenugreek) as the screening system by using morphological, cytological, biochemical and molecular approaches. Further functional characterization was performed using MCF-7 cell line and solid tumour model for the assessment of anti-tumour activity of the complex. RESULTS: Our results indicate efficiency of the Fe(II) Schiff base complex in the induction of double strand breaks in DNA. Complex treatment clearly induced cytotoxic and genotoxic damage in Trigonella seedlings. The Fe-complex treatment caused cell cycle arrest via the activation of ATM-ATR kinase mediated DNA damage response pathway with the compromised expression of CDK1, CDK2 and CyclinB1 protein in Trigonella seedlings. In cultured MCF-7 cells, the complex induces cytotoxicity and DNA fragmentation through intracellular ROS generation. Fe-complex treatment inhibited tumour growth in solid tumour model with no additional side effects. CONCLUSION: The growth inhibitory and cytotoxic effects of the complex result from activation of DNA damage response along with oxidative stress and cell cycle arrest. GENERAL SIGNIFICANCE: Overall, our results have provided comprehensive information on the mechanism of action and efficacy of a Fe(II) Schiff base complex in higher eukaryotic genomes and indicated its future implications as potential therapeutic agent.

7.
Cancer Res ; 79(24): 6190-6203, 2019 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-31672843

RESUMO

Master regulators of the unfolded protein response (UPR), IRE1α and PERK, promote adaptation or apoptosis depending on the level of endoplasmic reticulum (ER) stress. Although the UPR is activated in many cancers, its effects on tumor growth remain unclear. Derived from endocrine cells, pancreatic neuroendocrine tumors (PanNET) universally hypersecrete one or more peptide hormones, likely sensitizing these cells to high ER protein-folding stress. To assess whether targeting the UPR is a viable therapeutic strategy, we analyzed human PanNET samples and found evidence of elevated ER stress and UPR activation. Genetic and pharmacologic modulation of IRE1α and PERK in cultured cells, xenograft, and spontaneous genetic (RIP-Tag2) mouse models of PanNETs revealed that UPR signaling was optimized for adaptation and that inhibiting either IRE1α or PERK led to hyperactivation and apoptotic signaling through the reciprocal arm, thereby halting tumor growth and survival. These results provide a strong rationale for therapeutically targeting the UPR in PanNETs and other cancers with elevated ER stress. SIGNIFICANCE: The UPR is upregulated in pancreatic neuroendocrine tumors and its inhibition significantly reduces tumor growth in preclinical models, providing strong rationale for targeting the UPR in these cancers.

8.
Mol Metab ; 27S: S60-S68, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31500832

RESUMO

BACKGROUND: Myriad challenges to the proper folding and structural maturation of secretory pathway client proteins in the endoplasmic reticulum (ER) - a condition referred to as "ER stress" - activate intracellular signaling pathways termed the unfolded protein response (UPR). SCOPE OF REVIEW: Through executing transcriptional and translational programs the UPR restores homeostasis in those cells experiencing manageable levels of ER stress. But the UPR also actively triggers cell degeneration and apoptosis in those cells that are encountering ER stress levels that exceed irremediable thresholds. Thus, UPR outputs are "double-edged". In pancreatic islet ß-cells, numerous genetic mutations affecting the balance between these opposing UPR functions cause diabetes mellitus in both rodents and humans, amply demonstrating the principle that the UPR is critical for the proper functioning and survival of the cell. MAJOR CONCLUSIONS: Specifically, we have found that the UPR master regulator IRE1α kinase/endoribonuclease (RNase) triggers apoptosis, ß-cell degeneration, and diabetes, when ER stress reaches critical levels. Based on these mechanistic findings, we find that novel small molecule compounds that inhibit IRE1α during such "terminal" UPR signaling can spare ER stressed ß-cells from death, perhaps affording future opportunities to test new drug candidates for disease modification in patients suffering from diabetes.

9.
J Craniofac Surg ; 30(7): 2088-2090, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31503110

RESUMO

PURPOSE: The study was conducted to assess the efficacy of orbital chart in detecting postoperative complications of orbital fractures. MATERIALS AND METHODS: A retrospective study was conducted in the Department of OMFS, SDM College of Dental Sciences, Dharwad from January 2011 to December 2016. It included all the patients with orbital fractures who underwent surgical intervention for reduction of the fracture in the study. We recorded data for the type of fracture, type of intervention, and orbital and ocular changes. Orbital changes measured and charted for 5 parameters which were: pain, proptosis, visual acuity, size of the pupil, and pupillary reaction to direct light reflex. RESULTS: Two hundred thirty-six patients with orbital fractures underwent surgical intervention during these 5 years. The prevailing type of fracture for which they required orbital intervention remains zygomatic complex fractures (69%). The treatment protocol depended on the pattern and displacement of fracture and age of the patient. Pain was the most common symptom among these parameters (15.7%). CONCLUSION: Orbital chart monitoring represents a straightforward and effective method to detect any complications after surgical management of orbital fractures.


Assuntos
Fraturas Orbitárias/cirurgia , Protocolos Clínicos , Humanos , Fraturas Orbitárias/fisiopatologia , Complicações Pós-Operatórias , Estudos Retrospectivos , Acuidade Visual , Fraturas Zigomáticas/cirurgia
10.
Am J Med Genet A ; 179(12): 2459-2468, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31520464

RESUMO

Hartnup disease is an autosomal recessive condition characterized by neutral aminoaciduria and behavioral problems. It is caused by a loss of B0 AT1, a neutral amino acid transporter in the kidney and intestine. CLTRN encodes the protein collectrin that functions in the transportation and activation of B0 AT1 in the renal apical brush bordered epithelium. Collectrin deficient mice have severe aminoaciduria. However, the phenotype associated with collectrin deficiency in humans has not been reported. Here we report two patients, an 11-year-old male who is hemizygous for a small, interstitial deletion on Xp22.2 that encompasses CLTRN and a 22-year-old male with a deletion spanning exons 1 to 3 of CLTRN. Both of them present with neuropsychiatric phenotypes including autistic features, anxiety, depression, compulsions, and motor tics, as well as neutral aminoaciduria leading to a clinical diagnosis of Hartnup disease and treatment with niacin supplementation. Plasma amino acids were normal in both patients. One patient had low 5-hydroxyindoleacetic acid levels, a serotoninergic metabolite. We explored the expression of collectrin in the murine brain and found it to be particularly abundant in the hippocampus, brainstem, and cerebellum. We propose that collectrin deficiency in humans can be associated with aminoaciduria and a clinical picture similar to that seen in Hartnup disease. Further studies are needed to explore the role of collectrin deficiency in the neurological phenotypes.

11.
Int J Biol Macromol ; 139: 221-232, 2019 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-31376448

RESUMO

In this study, we propose the use of a plant tissue culture-based system for the production of polysaccharides with consistent chemical characteristics and reduced endotoxin content. Polysaccharides were isolated from suspension cultures of Panax quinquefolius (American ginseng), a widely used medicinal herb. A neutral fraction, AGC1, purified by anion exchange and size exclusion chromatography, displayed immunostimulatory activity in vitro and ex vivo. AGC1 (average molecular weight: 5.2kDa) was predominantly composed of galactose (>60%) along with the presence of several other neutral sugars such as arabinose, xylose, glucose, mannose and rhamnose in minor amounts. The major glycosidic linkages were found to be 3-Galp (48.5%), 3,6-Galp (10.2%), t-Galp (5.2%), 6-Galp (4.4%), 4-Glcp (5.7%), 4-Arap/5-Araf (4.0%) and t-Araf (4.5%). AGC1 significantly (p<0.05) stimulated the expression of a range of proinflammatory mediators in RAW 264.7 murine macrophages such as IL-6, TNF-α, MCP-1 and GM-CSF. Additionally, AGC1 treatment of RAW 264.7 cells stimulated NOS2 gene expression, leading to increased levels of iNOS and downstream NO. Consistent with this, AGC1 was able to act as an immunostimulant in primary murine splenocytes, enhancing cell proliferation, as well as NO and TNF-α production. Our results also indicate the partial role of NF-κB pathway in the immunostimulatory response.


Assuntos
Meios de Cultivo Condicionados/química , Meios de Cultivo Condicionados/farmacologia , Panax/química , Compostos Fitoquímicos/química , Compostos Fitoquímicos/farmacologia , Polissacarídeos/química , Polissacarídeos/farmacologia , Animais , Células Cultivadas , Citocinas/metabolismo , Fatores Imunológicos/química , Fatores Imunológicos/farmacologia , Imunomodulação/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/metabolismo , Panax/citologia , Panax/metabolismo , Compostos Fitoquímicos/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Polissacarídeos/isolamento & purificação , Células RAW 264.7
12.
Cancer Discov ; 9(10): 1438-1451, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31337617

RESUMO

By examination of the cancer genomics database, we identified a new set of mutations in core histones that frequently recur in cancer patient samples and are predicted to disrupt nucleosome stability. In support of this idea, we characterized a glutamate to lysine mutation of histone H2B at amino acid 76 (H2B-E76K), found particularly in bladder and head and neck cancers, that disrupts the interaction between H2B and H4. Although H2B-E76K forms dimers with H2A, it does not form stable histone octamers with H3 and H4 in vitro, and when reconstituted with DNA forms unstable nucleosomes with increased sensitivity to nuclease. Expression of the equivalent H2B mutant in yeast restricted growth at high temperature and led to defective nucleosome-mediated gene repression. Significantly, H2B-E76K expression in the normal mammary epithelial cell line MCF10A increased cellular proliferation, cooperated with mutant PIK3CA to promote colony formation, and caused a significant drift in gene expression and fundamental changes in chromatin accessibility, particularly at gene regulatory elements. Taken together, these data demonstrate that mutations in the globular domains of core histones may give rise to an oncogenic program due to nucleosome dysfunction and deregulation of gene expression. SIGNIFICANCE: Mutations in the core histones frequently occur in cancer and represent a new mechanism of epigenetic dysfunction that involves destabilization of the nucleosome, deregulation of chromatin accessibility, and alteration of gene expression to drive cellular transformation.See related commentary by Sarthy and Henikoff, p. 1346.This article is highlighted in the In This Issue feature, p. 1325.

13.
Genome Med ; 11(1): 48, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31349857

RESUMO

BACKGROUND: Although mosaic variation has been known to cause disease for decades, high-throughput sequencing technologies with the analytical sensitivity to consistently detect variants at reduced allelic fractions have only recently emerged as routine clinical diagnostic tests. To date, few systematic analyses of mosaic variants detected by diagnostic exome sequencing for diverse clinical indications have been performed. METHODS: To investigate the frequency, type, allelic fraction, and phenotypic consequences of clinically relevant somatic mosaic single nucleotide variants (SNVs) and characteristics of the corresponding genes, we retrospectively queried reported mosaic variants from a cohort of ~ 12,000 samples submitted for clinical exome sequencing (ES) at Baylor Genetics. RESULTS: We found 120 mosaic variants involving 107 genes, including 80 mosaic SNVs in proband samples and 40 in parental/grandparental samples. Average mosaic alternate allele fraction (AAF) detected in autosomes and in X-linked disease genes in females was 18.2% compared with 34.8% in X-linked disease genes in males. Of these mosaic variants, 74 variants (61.7%) were classified as pathogenic or likely pathogenic and 46 (38.3%) as variants of uncertain significance. Mosaic variants occurred in disease genes associated with autosomal dominant (AD) or AD/autosomal recessive (AR) (67/120, 55.8%), X-linked (33/120, 27.5%), AD/somatic (10/120, 8.3%), and AR (8/120, 6.7%) inheritance. Of note, 1.7% (2/120) of variants were found in genes in which only somatic events have been described. Nine genes had recurrent mosaic events in unrelated individuals which accounted for 18.3% (22/120) of all detected mosaic variants in this study. The proband group was enriched for mosaicism affecting Ras signaling pathway genes. CONCLUSIONS: In sum, an estimated 1.5% of all molecular diagnoses made in this cohort could be attributed to a mosaic variant detected in the proband, while parental mosaicism was identified in 0.3% of families analyzed. As ES design favors breadth over depth of coverage, this estimate of the prevalence of mosaic variants likely represents an underestimate of the total number of clinically relevant mosaic variants in our cohort.


Assuntos
Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Mosaicismo , Polimorfismo de Nucleotídeo Único , Sequenciamento Completo do Exoma , Alelos , Feminino , Variação Genética , Estudo de Associação Genômica Ampla/métodos , Humanos , Masculino
14.
Am J Med Genet A ; 179(10): 2138-2143, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31290619

RESUMO

Autosomal recessive COX4I1 deficiency has been previously reported in a single individual with a homozygous pathogenic variant in COX4I1, who presented with short stature, poor weight gain, dysmorphic features, and features of Fanconi anemia. COX4I1 encodes subunit 4, isoform 1 of cytochrome c oxidase. Cytochrome c oxidase is a respiratory chain enzyme that plays an important role in mitochondrial electron transport and reduces molecular oxygen to water leading to the formation of ATP. Defective production of cytochrome c oxidase leads to a variable phenotypic spectrum ranging from isolated myopathy to Leigh syndrome. Here, we describe two siblings, born to consanguineous parents, who presented with encephalopathy, developmental regression, hypotonia, pathognomonic brain imaging findings resembling Leigh-syndrome, and a novel homozygous variant on COX4I1, expanding the known clinical phenotype associated with pathogenic variants in COX4I1.

15.
Nat Commun ; 10(1): 3221, 2019 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324780

RESUMO

The Satb1 genome organizer regulates multiple cellular and developmental processes. It is not yet clear how Satb1 selects different sets of targets throughout the genome. Here we have used live-cell single molecule imaging and deep sequencing to assess determinants of Satb1 binding-site selectivity. We have found that Satb1 preferentially targets nucleosome-dense regions and can directly bind consensus motifs within nucleosomes. Some genomic regions harbor multiple, regularly spaced Satb1 binding motifs (typical separation ~1 turn of the DNA helix) characterized by highly cooperative binding. The Satb1 homeodomain is dispensable for high affinity binding but is essential for specificity. Finally, we find that Satb1-DNA interactions are mechanosensitive. Increasing negative torsional stress in DNA enhances Satb1 binding and Satb1 stabilizes base unpairing regions against melting by molecular machines. The ability of Satb1 to control diverse biological programs may reflect its ability to combinatorially use multiple site selection criteria.


Assuntos
Sítios de Ligação , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Nucleossomos/metabolismo , Sequência de Bases , Linhagem Celular , Cromatina , Proteínas de Ligação a DNA/genética , Técnicas de Inativação de Genes , Genoma , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/genética , Ligação Proteica , Domínios Proteicos
16.
Genome Med ; 11(1): 30, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101064

RESUMO

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.


Assuntos
Variações do Número de Cópias de DNA , Testes Genéticos/métodos , Análise em Microsséries/métodos , Sequenciamento Completo do Exoma/métodos , Aberrações Cromossômicas , Feminino , Testes Genéticos/normas , Homozigoto , Humanos , Limite de Detecção , Masculino , Análise em Microsséries/normas , Sequenciamento Completo do Exoma/normas
17.
BMC Res Notes ; 12(1): 268, 2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31088532

RESUMO

OBJECTIVE: QC analysis of carbohydrates has been historically cumbersome due to lengthy and laborious derivatization techniques and the requirement of complimentary instrumentation. HILIC-CAD has emerged as an effective platform for direct monosaccharide composition analysis of complex carbohydrates without derivatization. Although, several neutral sugars have been separated and detected using HILIC-CAD, there has not been any report on acidic and amino sugar analysis using this method. In this study, we developed a gradient method for simultaneous analysis of acidic, amino and select neutral monosaccharides. As an application of the HILIC-CAD method, we performed composition analysis of commercially purchased hyaluronic acid products. Additionally, since CAD is suitable for SEC experiments, we tested the homogeneity of hyaluronic acids using a SEC-CAD method. RESULTS: We separated common uronic acids (GlcA, GalA, LIdoA and Neu5Ac), amino sugars (GlcN, GalN and GlcNAc) and select neutral sugars (LRha, LFuc, Man and Gal) using a gradient HILIC-CAD method. The optimized gradient method demonstrated good linearity (R2 > 0.99), precision (RSD < 8%), LOD (< 85 ng/mL) and LOQ (< 280 ng/mL). HILIC-CAD analysis of commercially purchased hyaluronic acid products indicated that samples were composed of GlcNAc and GlcA. Additionally, SEC-CAD chromatograms indicated the heterogeneous nature of the samples.


Assuntos
Aerossóis/análise , Carboidratos/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida de Alta Pressão/normas , Polímeros/análise , Ácido Hialurônico/análise , Interações Hidrofóbicas e Hidrofílicas , Monossacarídeos/isolamento & purificação , Controle de Qualidade , Padrões de Referência
18.
Int J Biol Macromol ; 133: 76-85, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-30981779

RESUMO

Frankincense has a long history in religious, cultural, and medicinal use. In this study polysaccharides were extracted from frankincense from Boswellia carterii. The polysaccharides were purified by anion exchange chromatography on a DEAE-Sepharose Fast Flow 16/10 FPLC column. Six fractions were obtained and the three most active immunomodulatory fractions were further purified by size exclusion chromatography on a Superdex-200 column. The composition showed the monosaccharides present were predominantly galactose, arabinose, and glucuronic acid along with small amounts of rhamnose and glucose. The monosaccharide composition and glycosyl linkage analysis revealed the polysaccharides belong to the type II arabinogalactans. Fourier-transform infrared spectroscopy and bicinchoninic acid assay showed that the amount of protein in the samples was <1 wt%. One-dimensional 1H NMR were consistent with high molecular weight compounds. The monosaccharides were primarily in the ß conformation. The three fractions exhibited an immunostimulatory effect on RAW 264.7 murine macrophage cells. The most active immunostimulatory fraction FA2, stimulated a range of pro-inflammatory mediators including iNOS, NO, TNF-α, and IL-6 in RAW 264.7 cells. The fractions were effective in proliferating primary murine splenocytes. The results indicate that the polysaccharides isolated from frankincense have the potential to be used as an immunological stimulant or nutraceutical.


Assuntos
Adjuvantes Imunológicos/química , Adjuvantes Imunológicos/farmacologia , Franquincenso/química , Polissacarídeos/química , Polissacarídeos/farmacologia , Adjuvantes Imunológicos/isolamento & purificação , Animais , Glicosilação , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Peso Molecular , Óxido Nítrico/metabolismo , Polissacarídeos/isolamento & purificação , Células RAW 264.7 , Fator de Necrose Tumoral alfa/metabolismo
19.
Nat Chem Biol ; 15(4): 401-409, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30858596

RESUMO

We describe three optical tags, ArrayG, ArrayD and ArrayG/N, for intracellular tracking of single molecules over milliseconds to hours. ArrayG is a fluorogenic tag composed of a green fluorescent protein-nanobody array and monomeric wild-type green fluorescent protein binders that are initially dim but brighten ~26-fold on binding with the array. By balancing the rates of binder production, photobleaching and stochastic binder exchange, we achieve temporally unlimited tracking of single molecules. High-speed tracking of ArrayG-tagged kinesins and integrins for thousands of frames reveals novel dynamical features. Tracking of single histones at 0.5 Hz for >1 hour with the import competent ArrayG/N tag shows that chromosomal loci behave as Rouse polymers with visco-elastic memory and exhibit a non-Gaussian displacement distribution. ArrayD, based on a dihydrofolate reductase nanobody array and dihydrofolate reductase-fluorophore binder, enables dual-color imaging. The arrays combine brightness, fluorogenicity, fluorescence replenishment and extended fluorophore choice, opening new avenues for tracking single molecules in living cells.


Assuntos
Corantes Fluorescentes/química , Microscopia de Fluorescência/métodos , Imagem Individual de Molécula/métodos , Linhagem Celular , Cor , Corantes Fluorescentes/síntese química , Proteínas de Fluorescência Verde , Células HeLa , Humanos , Anticorpos de Domínio Único
20.
PLoS One ; 14(1): e0209824, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30625178

RESUMO

Endoplasmic reticulum stress (ER stress) has been implicated in the pathogenesis of idiopathic pulmonary fibrosis (IPF), a disease of progressive fibrosis and respiratory failure. ER stress activates a signaling pathway called the unfolded protein response (UPR) that either restores homeostasis or promotes apoptosis. The bifunctional kinase/RNase IRE1α is a UPR sensor/effector that promotes apoptosis if ER stress remains high and irremediable (i.e., a "terminal" UPR). Using multiple small molecule inhibitors against IRE1α, we show that ER stress-induced apoptosis of murine alveolar epithelial cells can be mitigated in vitro. In vivo, we show that bleomycin exposure to murine lungs causes early ER stress to activate IRE1α and the terminal UPR prior to development of pulmonary fibrosis. Small-molecule IRE1α kinase-inhibiting RNase attenuators (KIRAs) that we developed were used to evaluate the contribution of IRE1α activation to bleomycin-induced pulmonary fibrosis. One such KIRA-KIRA7-provided systemically to mice at the time of bleomycin exposure decreases terminal UPR signaling and prevents lung fibrosis. Administration of KIRA7 14 days after bleomycin exposure even promoted the reversal of established fibrosis. Finally, we show that KIRA8, a nanomolar-potent, monoselective KIRA compound derived from a completely different scaffold than KIRA7, likewise promoted reversal of established fibrosis. These results demonstrate that IRE1α may be a promising target in pulmonary fibrosis and that kinase inhibitors of IRE1α may eventually be developed into efficacious anti-fibrotic drugs.


Assuntos
Células Epiteliais Alveolares/efeitos dos fármacos , Endorribonucleases/antagonistas & inibidores , Fibrose/tratamento farmacológico , Pulmão/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Células Epiteliais Alveolares/metabolismo , Células Epiteliais Alveolares/patologia , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Fibrose/metabolismo , Fibrose/patologia , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Inibidores de Proteínas Quinases/uso terapêutico , Resposta a Proteínas não Dobradas/efeitos dos fármacos
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