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1.
Nucleic Acids Res ; 46(18): 9637-9646, 2018 10 12.
Artigo em Inglês | MEDLINE | ID: mdl-30184164

RESUMO

Cut-and-paste transposons are important tools for mutagenesis, gene-delivery and DNA sequencing applications. At the molecular level, the most thoroughly understood are Tn5 and Tn10 in bacteria, and mariner and hAT elements in eukaryotes. All bacterial cut-and-paste transposases characterized to date are monomeric prior to interacting with the transposon end, while all eukaryotic transposases are multimers. Although there is a limited sample size, we proposed that this defines two pathways for transpososome assembly which distinguishes the mechanism of the bacterial and eukaryotic transposons. We predicted that the respective pathways would dictate how the rate of transposition is related to transposase concentration and genome size. Here, we have tested these predictions by creating a single-chain dimer version of the bacterial Tn5 transposase. We show that artificial dimerization switches the transpososome assembly pathway from the bacterial-style to the eukaryotic-style. Although this had no effect in vitro, where the transposase does not have to search far to locate the transposon ends, it increased the rate of transposition in bacterial and HeLa cell assays. However, in contrast to the mariner elements, the Tn5 single-chain dimer remained unaffected by over-production inhibition, which is an emergent property of the transposase subunit structure in the mariner elements.


Assuntos
Elementos de DNA Transponíveis/genética , Células Eucarióticas/metabolismo , Tamanho do Genoma , Células Procarióticas/metabolismo , Transposases/genética , Sequência de Bases , Clivagem do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Cinética , Mutagênese Insercional , Mutação , Multimerização Proteica , Transposases/química , Transposases/metabolismo
2.
Ann Surg Oncol ; 25(5): 1403-1409, 2018 May.
Artigo em Inglês | MEDLINE | ID: mdl-29484563

RESUMO

BACKGROUND: Recurrent primary hyperparathyroidism (PHPT) presents a diagnostic challenge in localizing a hyperfunctioning gland. Although several imaging modalities are available for preoperative localization, 4D-CT is increasingly utilized for its ability to locate both smaller and previously unlocalized lesions. Currently, there is a paucity of data evaluating the utility of 4D-CT in the reoperative setting compared with ultrasound (US) and sestamibi. We aimed to determine the sensitivity of 4D-CT in localizing parathyroid adenomas in recurrent or persistent PHPT. METHODS: We performed a retrospective review of prospectively collected data from a tertiary-care hospital, and identified 58 patients who received preoperative 4D-CT with US and/or sestamibi between May 2008 and March 2016. Data regarding the size, shape, and number of parathyroid lesions were collected for each patient. RESULTS: A total of 62 lesions were identified intraoperatively among the 58 patients (6 with multigland disease) included in this investigation. 4D-CT missed 13 lesions identified intraoperatively, compared with 32 and 22 lesions missed by US and sestamibi, respectively. Sensitivity for correct lateralization of culprit lesions was 77.4% for 4D-CT, 38.5% for US, and 46% for sestamibi. 4D-CT was superior in lateralizing adenomas (49/62) compared with US (20/52; p < 0.001) and sestamibi (18/47; p < 0.001). The overall cure rate (6-month postoperative calcium < 10.7 mg/dL) was 89.7%. All patients with lesions correctly lateralized by 4D-CT were cured at 6 months. CONCLUSION: 4D-CT localized parathyroid adenomas with higher sensitivity among patients with recurrent or persistent PHPT compared with sestamibi or US-based imaging.


Assuntos
Adenoma/diagnóstico por imagem , Tomografia Computadorizada Quadridimensional , Recidiva Local de Neoplasia/diagnóstico por imagem , Neoplasias das Paratireoides/diagnóstico por imagem , Adenoma/patologia , Adenoma/cirurgia , Adulto , Idoso , Reações Falso-Negativas , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/cirurgia , Neoplasias das Paratireoides/patologia , Neoplasias das Paratireoides/cirurgia , Período Pré-Operatório , Cintilografia , Reoperação , Estudos Retrospectivos , Sensibilidade e Especificidade , Tecnécio Tc 99m Sestamibi , Resultado do Tratamento , Ultrassonografia
3.
Plant J ; 84(3): 545-57, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26358508

RESUMO

Repair of DNA double-strand breaks (DSBs) by recombination pathways is essential for plant growth and fertility. The recombination endonuclease MRE11 plays important roles in sensing and repair of DNA DSBs. Here we demonstrate protein interaction between Arabidopsis MRE11 and the histone acetyltransferase TAF1, a TATA-binding protein Associated Factor (TAF) of the RNA polymerase II transcription initiation factor complex TFIID. Arabidopsis has two TAF1 homologues termed TAF1 and TAF1b and mutant taf1b lines are viable and fertile. In contrast, taf1 null mutations are lethal, demonstrating that TAF1 is an essential gene. Heterozygous taf1+/- plants display abnormal segregation of the mutant allele resulting from defects in pollen tube development, indicating that TAF1 is important for gamete viability. Characterization of an allelic series of taf1 lines revealed that hypomorphic mutants are viable but display developmental defects and reduced plant fertility. Hypersensitivity of taf1 mutants lacking the C-terminal bromodomain to X-rays and mitomycin C, but not to other forms of abiotic stress, established a specific role for TAF1 in plant DNA repair processes. Collectively these studies reveal a function for TAF1 in plant resistance to genotoxic stress, providing further insight into the molecular mechanisms of the DNA damage response in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Pólen/fisiologia , Fatores de Transcrição/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica de Plantas , Proteína Homóloga a MRE11 , Mitomicina/farmacologia , Mutação , Tubo Polínico/genética , Tubo Polínico/metabolismo , Estrutura Terciária de Proteína , Fatores de Transcrição/genética
5.
Radiology ; 270(1): 168-75, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24009349

RESUMO

PURPOSE: To identify a set of parameters, which are based on tissue enhancement and native iodine content obtained from a standardized triple-phase four-dimensional (4D) computed tomographic (CT) scan, that define a multinomial logistic regression model that discriminates between parathyroid adenoma (PTA) and thyroid nodules or lymph nodes. MATERIALS AND METHODS: Informed consent was waived by the institutional review board for this retrospective HIPAA-compliant study. Electronic medical records were reviewed for 102 patients with hyperparathyroidism who underwent triple-phase 4D CT and parathyroid surgery resulting in pathologically proved removal of adenoma from July 2010 through December 2011. Hounsfield units were measured in PTA, thyroid, lymph nodes, and aorta and were used to determine seven parameters characterizing tissue contrast enhancement. These were used as covariates in 10 multinomial logistic regression models. Three models with one covariate, four models with two covariates, and three models with three covariates were investigated. Receiver operating characteristic (ROC) analysis was performed to determine how well each model discriminated between adenoma and nonadenomatous tissues. Statistical differences between the areas under the ROC curves (AUCs) for each model pair were calculated, as well as sensitivity, specificity, accuracy, negative predictive value, and positive predictive value. RESULTS: A total of 120 lesions were found; 112 (93.3%) lesions were weighed, and mean and median weights were 589 and 335 mg, respectively. The three-covariate models were significantly identical (P > .65), with largest AUC of 0.9913 ± 0.0037 (standard error), accuracy of 96.9%, and sensitivity, specificity, negative predictive value, and positive predictive value of 94.3%, 98.3%, 97.1%, and 96.7%, respectively. The one- and two-covariate models were significantly less accurate (P < .043). CONCLUSION: A three-covariate multinomial logistic model derived from a triple-phase 4D CT scan can accurately provide the probability that tissue is PTA and performs significantly better than models using one or two covariates.


Assuntos
Tomografia Computadorizada Quadridimensional/métodos , Neoplasias das Paratireoides/diagnóstico por imagem , Nódulo da Glândula Tireoide/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Meios de Contraste , Diagnóstico Diferencial , Feminino , Humanos , Hiperparatireoidismo/diagnóstico por imagem , Modelos Logísticos , Linfonodos/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Interpretação de Imagem Radiográfica Assistida por Computador , Estudos Retrospectivos
6.
PLoS One ; 18(3): e0281900, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-36913348

RESUMO

Machine learning (ML) algorithms to detect critical findings on head CTs may expedite patient management. Most ML algorithms for diagnostic imaging analysis utilize dichotomous classifications to determine whether a specific abnormality is present. However, imaging findings may be indeterminate, and algorithmic inferences may have substantial uncertainty. We incorporated awareness of uncertainty into an ML algorithm that detects intracranial hemorrhage or other urgent intracranial abnormalities and evaluated prospectively identified, 1000 consecutive noncontrast head CTs assigned to Emergency Department Neuroradiology for interpretation. The algorithm classified the scans into high (IC+) and low (IC-) probabilities for intracranial hemorrhage or other urgent abnormalities. All other cases were designated as No Prediction (NP) by the algorithm. The positive predictive value for IC+ cases (N = 103) was 0.91 (CI: 0.84-0.96), and the negative predictive value for IC- cases (N = 729) was 0.94 (0.91-0.96). Admission, neurosurgical intervention, and 30-day mortality rates for IC+ was 75% (63-84), 35% (24-47), and 10% (4-20), compared to 43% (40-47), 4% (3-6), and 3% (2-5) for IC-. There were 168 NP cases, of which 32% had intracranial hemorrhage or other urgent abnormalities, 31% had artifacts and postoperative changes, and 29% had no abnormalities. An ML algorithm incorporating uncertainty classified most head CTs into clinically relevant groups with high predictive values and may help accelerate the management of patients with intracranial hemorrhage or other urgent intracranial abnormalities.


Assuntos
Aprendizado Profundo , Humanos , Incerteza , Tomografia Computadorizada por Raios X/métodos , Hemorragias Intracranianas/diagnóstico por imagem , Algoritmos , Estudos Retrospectivos
7.
Transl Vis Sci Technol ; 12(1): 27, 2023 01 03.
Artigo em Inglês | MEDLINE | ID: mdl-36705928

RESUMO

Purpose: The purpose of this study was to develop a protocol to prepare buffered chlorhexidine (CHX) eye drops (0.2% w/v) in the United Kingdom that can be reproduced at a production facility in Uganda. Buffered CHX eye drops can prevent CHX degradation and improve ocular tolerability during the treatment of fungal keratitis. Methods: Buffered CHX eye drops in amber glass containers were prepared using sodium acetate buffer at pH 5.90 to 6.75. Two commercial CHX solutions and CHX in water were used as controls. Eye drops were stored at 40°C (70% humidity, 21 months) in the United Kingdom and at ambient temperature in Uganda (30 months). High-performance liquid chromatography was used to determine CHX stability over time, and pH was monitored. Sterility was achieved using an autoclave (121°C, 15 minutes) and water bath (100°C, 30 minutes). Results: The pH of acetate-buffered CHX eye drops did not change over 21 months at 40°C or at ambient temperature (30 months), whereas the pH of the unbuffered aqueous CHX displayed significant fluctuations, with an increase in acidity. The CHX concentration remained the same in both buffered and unbuffered eye-drop solutions. Eye drops sterilization was successful using an autoclave and a water bath. Conclusions: Stable, sterile, buffered CHX eye drops (pH 6.75) were successfully prepared first in the United Kingdom and then reproducibly in Uganda. This eye drops can be prepared in a hospital or pharmacy setting with limited resources, thus providing a cost-effective treatment for fungal keratitis. Translational Relevance: A protocol has been developed to prepare buffered CHX eye drops in low- and middle-income countries to treat fungal keratitis.


Assuntos
Clorexidina , Ceratite , Humanos , Uganda , Soluções Oftálmicas/química
8.
Nat Commun ; 14(1): 3294, 2023 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-37322051

RESUMO

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.


Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Humanos , Escherichia coli , Infecções por Escherichia coli/microbiologia , Virulência/genética , Fatores de Virulência/genética , Proteínas de Escherichia coli/genética , Filogenia
9.
Radiology ; 264(3): 789-95, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22798226

RESUMO

PURPOSE: To investigate multiphase multidetector four-dimensional computed tomography (CT) as a technique to correctly localize abnormal parathyroid glands in patients with primary hyperparathyroidism. MATERIALS AND METHODS: Informed consent was waived by the institutional review body for this retrospective, chart review study. Radiology reports from four-dimensional CT and surgical notes were reviewed in 143 patients with primary hyperparathyroidism (35 men, 108 women; median ages, 58 and 60 years, respectively) who underwent parathyroid surgery between August 2004 and January 2007 and in whom four-dimensional CT predicted a single lesion. Accuracy of four-dimensional CT was stratified by patient and was determined separately for localization to the correct side and quadrant (upper and lower for each side), with surgical findings serving as standard of reference. RESULTS: In 143 patients, 148 abnormal parathyroid glands were found at surgery; 137 (93%) of these were weighed, with mean and median weights of 757 and 417 mg, respectively. Four-dimensional CT lateralized the abnormal glands with 93.7% accuracy (134 of 143). For localization according to quadrant, the accuracy was 86.6% (116 of 134). CONCLUSION: Four-dimensional CT has sufficiently high accuracy in presurgical localization to allow confident performance of unilateral parathyroidectomy in patients with sporadic primary hyperparathyroidism. The superior accuracy compared with that of ultrasonography and technetium 99m sestamibi scanning may be sufficient to allow four-dimensional CT to be used as the sole presurgical localization method.


Assuntos
Tomografia Computadorizada Quadridimensional , Hiperparatireoidismo Primário/diagnóstico por imagem , Glândulas Paratireoides/diagnóstico por imagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Hiperparatireoidismo Primário/patologia , Hiperparatireoidismo Primário/cirurgia , Masculino , Pessoa de Meia-Idade , Glândulas Paratireoides/patologia , Glândulas Paratireoides/cirurgia , Estudos Retrospectivos
10.
Pharmaceuticals (Basel) ; 15(4)2022 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-35455440

RESUMO

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (-20 to 60 °C) and pH (5.0-9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38-39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections.

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