We have developed technologies for creating saturating libraries of sequence-defined transposon insertion mutants in which each strain is maintained. Phenotypic analysis of such libraries should provide a virtually complete identification of nonessential genes required for any process for which a suitable screen can be devised. The approach was applied to Pseudomonas aeruginosa, an opportunistic pathogen with a 6.3-Mbp genome. The library that was generated consists of 30,100 sequence-defined mutants, corresponding to an average of five insertions per gene. About 12% of the predicted genes of this organism lacked insertions; many of these genes are likely to be essential for growth on rich media. Based on statistical analyses and bioinformatic comparison to known essential genes in E. coli, we estimate that the actual number of essential genes is 300 -400. Screening the collection for strains defective in two defined multigenic processes (twitching motility and prototrophic growth) identified mutants corresponding to nearly all genes expected from earlier studies. Thus, phenotypic analysis of the collection may produce essentially complete lists of genes required for diverse biological activities. The transposons used to generate the mutant collection have added features that should facilitate downstream studies of gene expression, protein localization, epistasis, and chromosome engineering.hole-genome sequences provide the foundation for the creation of relatively complete collections of strains carrying defined mutations in individual genes. Such libraries should facilitate the comprehensive identification of genes required for a wide range of biological processes. A nearly complete library of single-gene deletions of Saccharomyces cerevisiae has been assembled by an international consortium using a PCR-based mutagenesis approach (1). Other projects, also following a strategy of gene-by-gene disruption, are underway for Escherichia coli (E. coli genome project, www. genome.wisc.edu͞functional͞tnmutagenesis.htm), and have recently been completed for Bacillus subtilis (2).An alternative strategy for generating mutant libraries consists of ''random'' whole-genome transposon-insertion mutagenesis followed by sequence-based identification of insertion sites. The approach is cost-effective and applicable to a wide variety of microbes (3, 4). Studies with yeast, in which a collection of mutants corresponding to about one-third of the genes were represented, have illustrated that the generation of large, arrayed collections of insertion mutants is feasible (5). Other studies with bacteria have analyzed large numbers of transposon insertion mutants to identify genes essential for growth, although the mutants were analyzed within populations rather than being archived in a format allowing additional phenotypes to be examined (6)(7)(8). In this report, we describe the generation and initial phenotypic analysis of a near-saturation library of transposon insertion mutants of the opportunistic pathogen Pseudomonas aeruginos...

In this paper, we extend a previously reported intensity-based nonrigid registration algorithm by using a novel regularization term to constrain the deformation. Global motion is modeled by a rigid transformation while local motion is described by a free-form deformation based on B-splines. An information theoretic measure, normalized mutual information, is used as an intensity-based image similarity measure. Registration is performed by searching for the deformation that minimizes a cost function consisting of a weighted combination of the image similarity measure and a regularization term. The novel regularization term is a local volume-preservation (incompressibility) constraint, which is motivated by the assumption that soft tissue is incompressible for small deformations and short time periods. The incompressibility constraint is implemented by penalizing deviations of the Jacobian determinant of the deformation from unity. We apply the nonrigid registration algorithm with and without the incompressibility constraint to precontrast and post-contrast magnetic resonance (MR) breast images from 17 patients. Without using a constraint, the volume of contrast-enhancing lesions decreases by 1%-78% (mean 26%). Image improvement (motion artifact reduction) obtained using the new constraint is compared with that obtained using a smoothness constraint based on the bending energy of the coordinate grid by blinded visual assessment of maximum intensity projections of subtraction images. For both constraints, volume preservation improves, and motion artifact correction worsens, as the weight of the constraint penalty term increases. For a given volume change of the contrast-enhancing lesions (2% of the original volume), the incompressibility constraint reduces motion artifacts better than or equal to the smoothness constraint in 13 out of 17 cases (better in 9, equal in 4, worse in 4). The preliminary results suggest that incorporation of the incompressibility regularization term improves intensity-based free-form nonrigid registration of contrast-enhanced MR breast images by greatly reducing the problem of shrinkage of contrast-enhancing structures while simultaneously allowing motion artifacts to be substantially reduced.

It is generally reasoned that lethal infections caused by opportunistic pathogens develop permissively by invading a host that is both physiologically stressed and immunologically compromised. However, an alternative hypothesis might be that opportunistic pathogens actively sense alterations in host immune function and respond by enhancing their virulence phenotype. We demonstrate that interferon-gamma binds to an outer membrane protein in Pseudomonas aeruginosa, OprF, resulting in the expression of a quorum-sensing dependent virulence determinant, the PA-I lectin. These observations provide details of the mechanisms by which prokaryotic organisms are directly signaled by immune activation in their eukaryotic host.

Francisella tularensis, the causative agent of tularemia, is one of the most infectious bacterial pathogens known and is a category A select agent. We created a sequence-defined, near-saturation transposon mutant library of F. tularensis novicida, a subspecies that causes a tularemia-like disease in rodents. The library consists of 16,508 unique insertions, an average of >9 insertions per gene, which is a coverage nearly twice that of the greatest previously achieved for any bacterial species. Insertions were recovered in 84% (1,490) of the predicted genes. To achieve high coverage, it was necessary to construct transposons carrying an endogenous Francisella promoter to drive expression of antibiotic resistance. An analysis of genes lacking (or with few) insertions identified nearly 400 candidate essential genes, most of which are likely to be required for growth on rich medium and which represent potential therapeutic targets. To facilitate genome-scale screening using the mutant collection, we assembled a sublibrary made up of two purified mutants per gene. The library provides a resource for virtually complete identification of genes involved in virulence and other nonessential processes.essential genes ͉ promoter ͉ tularemia ͉ U112

A traditional method of localizing brain functions has been to identify shared areas of brain damage in individuals who have a particular deficit. The rationale of this 'lesion overlap' approach is straightforward: if the individuals can no longer perform the function, the area of brain damaged in most of these individuals must have been responsible for that function. However, the reciprocal association, i.e. the probability of the lesion causing the deficit, is often not evaluated. In this study, we illustrate potential weaknesses of this approach, by re-examining regions of the brain essential for orchestrating speech articulation. A particularly elegant and widely cited lesion overlap study identified the superior part of the precentral gyrus of the insula (in the anterior insula) as the shared area of damage in chronic stroke patients with 'apraxia of speech', a disorder of motor planning and programming of speech. Others have confirmed that patients with apraxia of speech commonly have damage to the anterior insula. However, this reliable association might reflect the vulnerability of the insula to damage following occlusion or narrowing of the middle cerebral artery (which can independently cause apraxia of speech and many other deficits). To evaluate this possibility, we examined the relationship between apraxia of speech and the insula in three unique ways: (i) we determined the probability of the lesion causing the deficit, as well as the deficit being associated with the lesion, by examining speech articulation and advanced MRIs in two consecutive series of patients with acute left hemisphere, non-lacunar stroke, 40 with and 40 without insular damage; (ii) we studied patients at stroke onset to identify the deficit before it resolved in cases of small stroke; and (iii) we identified regions of dysfunctional brain tissue, as well as structural damage. Using this approach, we found no association between apraxia of speech and lesions of the left insula, anterior insula or superior tip of the precentral gyrus of the insula. Instead, in patients with and without insular lesions, apraxia of speech was associated with structural damage or low blood flow in left posterior inferior frontal gyrus. These results illustrate a potential limitation of lesion overlap studies, and illustrate an alternative method for identifying brain-behaviour relationships.

Pathogenicity in Francisella tularensis subspecies

.Sequencing of the non-pathogenic Francisella tularensis sub-species novicida U112, and comparison with two pathogenic sub-species, provides insights into the evolution of pathogenicity in these species.

Purpose:To evaluate the incremental value of diffusion-weighted (DW) imaging and apparent diffusion coeffi cient (ADC) mapping in relation to conventional breast magnetic resonance (MR) imaging in the characterization of benign versus malignant breast lesions at 3.0 T. Materials and Methods:This retrospective HIPAA-compliant study was approved by the institutional review board, with the requirement for informed patient consent waived. Of 550 consecutive patients who underwent bilateral breast MR imaging over a 10-month period, 93 women with 101 lesions met the following study inclusion criteria: They had undergone three-dimensional (3D) high -spatial-resolution T1-weighted contrast material-enhanced MR imaging, dynamic contrastenhanced MR imaging, and DW imaging examinations at 3.0 T and either had received a pathologic analysis-proven diagnosis (96 lesions) or had lesion stability confi rmed at more than 2 years of follow-up (fi ve lesions). DW images were acquired with b values of 0 and 600 sec/mm 2 . Regions of interest were drawn on ADC maps of breast lesions and normal glandular tissue. Morphologic features (margin, enhancement pattern), dynamic contrastenhanced MR results (semiquantitative kinetic curve data), absolute ADCs, and glandular tissue-normalized ADCs were included in multivariate models to predict a diagnosis of benign versus malignant lesion. Results:Forty-one (44%) of the 93 patients were premenopausal, and 52 (56%) were postmenopausal. Thirty-three (32.7%) of the 101 lesions were benign, and 68 (67.3%) were malignant. Normalized ADCs were signifi cantly different between the benign (mean ADC, 1.1 3 10 2 3 mm 2 /sec 6 0.4 [standard deviation]) and malignant (mean ADC, 0.55 3 10 2 3 mm 2 /sec 6 0.16) lesions ( P , .001). Adding normalized ADCs to the 3D T1-weighted and dynamic contrast-enhanced MR data improved the diagnostic performance of MR imaging: The area under the receiver operating characteristic curve improved from 0 .89 to 0.98, and the false-positive rate decreased from 36% (nine of 25 lesions) to 24% (six of 25 lesions). Conclusion:DW imaging with glandular tissue-normalized ADC assessment improves the characterization of breast lesions beyond the characterization achieved with conventional 3D T1-weighted and dynamic contrast-enhanced MR imaging at 3.0 T.q RSNA, 2010

The increasing use of biomarkers in cancer have led to the concept of personalized medicine for patients. Personalized medicine provides better diagnosis and treatment options available to clinicians. Radiological imaging techniques provide an opportunity to deliver unique data on different types of tissue. However, obtaining useful information from all radiological data is challenging in the era of “big data”. Recent advances in computational power and the use of genomics have generated a new area of research termed Radiomics. Radiomics is defined as the high throughput extraction of quantitative imaging features or texture (radiomics) from imaging to decode tissue pathology and creating a high dimensional data set for feature extraction. Radiomic features provide information about the gray-scale patterns, inter-pixel relationships. In addition, shape and spectral properties can be extracted within the same regions of interest on radiological images. Moreover, these features can be further used to develop computational models using advanced machine learning algorithms that may serve as a tool for personalized diagnosis and treatment guidance.