Transposase subunit architecture and its relationship to genome size and the rate of transposition in prokaryotes and eukaryotes.

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.

4D-CT is Superior to Ultrasound and Sestamibi for Localizing Recurrent Parathyroid Disease.

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.

Arabidopsis TAF1 is an MRE11-interacting protein required for resistance to genotoxic stress and viability of the male gametophyte.

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.

Discriminating parathyroid adenoma from local mimics by using inherent tissue attenuation and vascular information obtained with four-dimensional CT: formulation of a multinomial logistic regression model.

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.

Incorporating algorithmic uncertainty into a clinical machine deep learning algorithm for urgent head CTs.

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.

Local Ugandan Production of Stable 0.2% Chlorhexidine Eye Drops.

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.

Evolutionary and functional history of the Escherichia coli K1 capsule.

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.

Accuracy of four-dimensional CT for the localization of abnormal parathyroid glands in patients with primary hyperparathyroidism.

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.

Isolation and Characterisation of Bacteriophage Selective for Key Acinetobacter baumannii Capsule Chemotypes.

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.

Imaging characteristics of hyperfunctioning parathyroid adenomas using multiphase multidectector computed tomography: a quantitative and qualitative approach.

OBJECTIVE: The objective of the study was to characterize the enhancement pattern of hyperfunctioning parathyroid adenomas on multiphase multidetector computed tomography (CT) or 4-dimensional CT. METHODS: We retrospectively studied the enhancement patterns of 48 pathologically confirmed parathyroid adenomas with 4-dimensional CT, compliant with institutional review and the Health Insurance Portability and Accountability Act. Region-of-interest analysis was done at baseline and at arterial (25 seconds), venous (55 seconds), and delayed (85 seconds) enhancement phases over the adenoma and adjacent normal thyroid tissue. Qualitative and quantitative analysis was done. Discriminant functions were calculated using a multivariate logistic regression model, and receiver operating characteristic curves were measured. RESULTS: Adenomas are lower than thyroid in density, demonstrate avid early contrast enhancement, and show rapid wash-out of contrast. Adenomas and thyroid had baseline Hounsfield unit attenuations of 35 ± 11 and 94 ± 21 and enhancement percentage change from baseline to arterial of 493% ± 328% and 132% ± 148%, respectively (P < 0.0001 both). Quantitative analysis showed that these 2 measures of baseline density and the percentage change from baseline to arterial were the most powerful discriminatory features, with contrast wash-out from arterial peak to venous phase being a less powerful discriminator. Several discriminant functions were derived, the best of which was: X = 13.74 - (0.207 × baseline Hounsfield unit) - (0.006 × percent density change from baseline to arterial). X > 0.2 classifies tissue as parathyroid with high certainty (area under the receiver operating characteristic curve = 0.98; specificity, 0.938; sensitivity, 0.999). CONCLUSIONS: Parathyroid adenomas have a characteristic enhancement pattern that can be distinguished from thyroid tissue: the key diagnostic discriminators are baseline density, percentage change in density from baseline to arterial enhancement, and percentage decrease in density from arterial to venous phases.

The EXCEL Trial: The Interventionalists' Perspective.

Left main stem (LMS) disease is identified in up to 5% of diagnostic angiography cases, and is associated with significant morbidity and mortality due to the proportion of myocardium it subtends. In the past 10 years, there has been a significant change in the way we contemplate treating lesions in the LMS due to evolving experience and evidence in percutaneous coronary intervention (PCI) strategies and technologies. This has been reflected in recent changes in European and International guidance on managing patients with this lesion subset. Here, the authors provide an overview of the current literature regarding the management of LMS disease using PCI in light of new developments and emerging concepts in this field, specifically looking at the recent EXCEL trial.

Insertion Specificity of the hATx-6 Transposase of Hydra magnipapillata.

Transposable elements (TE) are mobile genetic elements, present in all domains of life. They commonly encode a single transposase enzyme, that performs the excision and reintegration reactions, and these enzymes have been used in mutagenesis and creation of next-generation sequencing libraries. All transposases have some bias in the DNA sequence they bind to when reintegrating the TE DNA. We sought to identify a transposase that showed minimal sequence bias and could be produced recombinantly, using information from the literature and a novel bioinformatic analysis, resulting in the selection of the hATx-6 transposase from Hydra vulgaris (aka Hydra magnipapillata) for further study. This transposase was tested and shown to be active both in vitro and in vivo, and we were able to demonstrate very low sequence bias in its integration preference. This transposase could be an excellent candidate for use in biotechnology, such as the creation of next-generation sequencing libraries.

Characterisation of Bacteriophage-Encoded Depolymerases Selective for Key Klebsiella pneumoniae Capsular Exopolysaccharides.

Capsular polysaccharides enable clinically important clones of Klebsiella pneumoniae to cause severe systemic infections in susceptible hosts. Phage-encoded capsule depolymerases have the potential to provide an alternative treatment paradigm in patients when multiple drug resistance has eroded the efficacy of conventional antibiotic chemotherapy. An investigation of 164 K. pneumoniae from intensive care patients in Thailand revealed a large number of distinct K types in low abundance but four (K2, K51, K1, K10) with a frequency of at least 5%. To identify depolymerases with the capacity to degrade capsules associated with these common K-types, 62 lytic phage were isolated from Thai hospital sewage water using K1, K2 and K51 isolates as hosts; phage plaques, without exception, displayed halos indicative of the presence of capsule-degrading enzymes. Phage genomes ranged in size from 41-348 kb with between 50 and 535 predicted coding sequences (CDSs). Using a custom phage protein database we were successful in applying annotation to 30 - 70% (mean = 58%) of these CDSs. The largest genomes, of so-called jumbo phage, carried multiple tRNAs as well as CRISPR repeat and spacer sequences. One of the smaller phage genomes was found to contain a putative Cas type 1E gene, indicating a history of host DNA acquisition in these obligate lytic phage. Whole-genome sequencing (WGS) indicated that some phage displayed an extended host range due to the presence of multiple depolymerase genes; in total, 42 candidate depolymerase genes were identified with up to eight in a single genome. Seven distinct virions were selected for further investigation on the basis of host range, phage morphology and WGS. Candidate genes for K1, K2 and K51 depolymerases were expressed and purified as his6-tagged soluble protein and enzymatic activity demonstrated against K. pneumoniae capsular polysaccharides by gel electrophoresis and Anton-Paar rolling ball viscometry. Depolymerases completely removed the capsule in K-type-specific fashion from K. pneumoniae cells. We conclude that broad-host range phage carry multiple enzymes, each with the capacity to degrade a single K-type, and any future use of these enzymes as therapeutic agents will require enzyme cocktails for utility against a range of K. pneumoniae infections.

Acute respiratory distress secondary to a huge chronic left ventricular pseudo-aneurysm.

Pseudo-aneurysms are a rare, potentially life-threatening complication of a myocardial infarction. We present the case of a 45-year-old male who was brought to the emergency department in extremis and had a previous history of a late presentation inferior ST-elevation myocardial infarction treated percutaneously. Clinical examination revealed evidence of cardiogenic shock, pulmonary edema and a pulsatile epigastric mass. Chest X-ray demonstrated marked cardiomegaly and pulmonary edema. Urgent echocardiography confirmed the presence of a huge basal inferior wall pseudo-aneurysm with bi-directional flow. This was also associated with severe mitral regurgitation, due to posterior mitral annular involvement. The patient was transferred to the local cardiothoracic surgical unit where he underwent emergency repair of the pseudo-aneurysm and mitral valve replacement. Despite the surgery being complex he made a full recovery.

Improved preoperative planning for directed parathyroidectomy with 4-dimensional computed tomography.

BACKGROUND: Four-dimensional computed tomography (4D-CT) provides both functional and highly detailed anatomic information about parathyroid tumors. The purpose of this study was to compare 4D-CT with sestamibi imaging and ultrasonography as methods for the accurate preoperative localization of hyperfunctioning parathyroid glands before parathyroidectomy. METHODS: A study of 75 patients with primary hyperparathyroidism was performed at a tertiary-care institution. Sestamibi imaging, ultrasonography, and 4D-CT were performed on each patient preoperatively. Results of the imaging studies were compared with operative findings, pathologic data, and biochemical measurements to assess the sensitivity and specificity of each of the imaging modalities. RESULTS: 4D-CT demonstrated improved sensitivity (88%) over sestamibi imaging (65%) and ultrasonography (57%), when the imaging studies were used to localize (lateralize) hyperfunctioning parathyroid glands to 1 side of the neck. Moreover, when used to localize parathyroid tumors to the correct quadrant of the neck (ie, right inferior, right superior, left inferior, or left superior), the sensitivity of 4D-CT (70%) was significantly higher than sestamibi imaging (33%) and ultrasonography (29%). CONCLUSION: 4D-CT provides significantly greater sensitivity than sestamibi imaging and ultrasonography for precise (quadrant) localization of hyperfunctioning parathyroid glands. This allows improved preoperative planning, particularly for the case of reoperation. In addition to the data that are provided, we present a novel classification scheme for use in parathyroid localization.

Acute studies of a new primate model of reversible middle cerebral artery occlusion.

The recent failure of many clinical trials of neuroprotective compounds may be due in part to poor animal models of human stroke. We have developed an endovascular stroke model in nonhuman primates that is compatible with serial magnetic resonance imaging (MRI) monitoring. Using cynomologous macaques (n = 4), a microcatheter was navigated transarterially (under fluoroscopic guidance) from the femoral artery to the middle cerebral artery (MCA). The microcatheter was wedged in a branch of the MCA for 3 hours to cause focal cerebral ischemia, as verified angiographically. During occlusion and/or reperfusion, animals were scanned with MRI, and imaging findings were compared with the stained brain sections. All animals demonstrated small stroke lesions in the expected vascular territory, as seen on diffusion-weighted MRI and confirmed by postmortem examination. Reperfusion after 3 hours was confirmed angiographically (n = 2) and also by MRI (n = 4). The mean initial lesion volume, measured on the postreperfusion MRI scans, was 2.3 +/- 1.3 mL (n = 4). There was good agreement between anatomic location of the lesion on MRI and postmortem histological staining (n = 3). A "minimally invasive" primate model of focal cerebral ischemia was developed that is ideally suited to MRI studies of both acute and chronic stroke. By using serial MRI scans to measure changes in lesion size over time, we will be able to control for variability in lesion size/location. This model should prove useful as a test bed for new stroke therapies, in which noninvasive imaging findings are readily comparable to human stroke.

Functional CT perfusion imaging in predicting the extent of cerebral infarction from a 3-hour middle cerebral arterial occlusion in a primate stroke model.

BACKGROUND AND PURPOSE: Our purpose was to determine whether cerebral perfusion functional CT (fCT), performed after endovascular middle cerebral artery (MCA) occlusion, can be used to predict final cerebral infarction extent in a primate model. METHODS: fCT with bolus tracking was performed before and 30 and 150 minutes after 3-hour digital subtraction angiography (DSA)-guided endovascular MCA occlusion in five baboons. Parametric cerebral blood flow (CBF), cerebral blood volume (CBV) and mean transit time (MTT) maps were constructed by voxel-by-voxel gamma variate fitting and used to determine lesion sizes. Animals were sacrificed 48 hours after the occlusion, and ex vivo MR imaging was performed. Lesion sizes on fCT and MR images were compared. RESULTS: Hypoperfusion was clearly identified on all images obtained after MCA occlusion. Thirty and 150 minutes after occlusion onset, respectively, mean lesion sizes were 737 mm(2) +/- 33 and 737 mm(2) +/- 44 for CBF, 722 mm(2) +/- 32 and 730 mm(2) +/- 43 for CBV, and 819 mm(2) +/- 14 and 847 mm(2) +/- 11 for MTT. Mean outcome infarct size on MR images was 733 mm(2) +/- 30. Measurements based on CBV and CBF (R(2) = 0.97 and 0.96, P <.001), but not MTT (R(2) = 0.40, P >.5), were highly correlated with final lesion size. CONCLUSION: An endovascular approach to MCA occlusion provides a minimally invasive, reproducible animal model for controlled studies of cerebral ischemia and infarction. Derived cerebral perfusion maps closely predict the 48-hour infarct size after 3-hour MCA occlusion.