Remarkable geographical variations between India and Europe in carriage of the staphylococcal surface protein-encoding sasX/sesI and in the population structure of methicillin-resistant Staphylococcus aureus belonging to clonal complex 8.

OBJECTIVES: sasX is a colonization-virulence factor that potentially underlies the success of methicillin-resistant Staphylococcus aureus (MRSA) sequence type (ST) 239 in Asia. We aimed to study the spread of sasX and the population structure of MRSA in two geographically distinct regions, Europe and India. METHODS: MRSA (n = 128) from screening and clinical samples from tertiary care patients in 12 European countries (n = 119), and from India (n = 9) were multilocus-sequence-typed and screened for sasX and its carrier φSPß-like prophage by PCR. Whole genome sequencing was performed on sasX-harbouring strains from India (n = 5) and Europe (n = 2) and on a selection non-harbouring sasX (n = 36) (2 × 150 bp, Miseq, Illumina). Reads were mapped to the ST239 reference strain, TW20. RESULTS: sasX and sesI, a sasX homologue native to Staphylococcus epidermidis, were detected in five of the nine Indian MRSA belonging to ST239 and to other sequence types of CC8. In contrast, sasX was restricted to two ST239 strains in Europe. The intact sasX and sesI carrier φSPß-like prophages were ∼80 kb and ∼118 kb, and integrated in the yeeE gene. We identified 'novel' ST239 clades in India and Serbia that showed significant differences in base substitution frequencies (0.130 and 0.007, respectively, Tamura-Nei model) (p <0.05). CONCLUSIONS: Our data highlight dissemination of sasX to non-ST239 sequence types of CC8. Detection of the S. epidermidis-associated sesI in MRSA provided unquestionable evidence of transfer between the two species. Stark differences in evolutionary rates between the novel Indian and Serbian ST239 clades identified here might be due to inherent clade characteristics or influenced by other environmental differences such as antibiotic use.

Quantitative evaluation of single cell spread on collagen matrices.

Cells change their morphology as a response to environmental cues. The quantitative evaluation of single cell spread on extracellular matrices, such as type I collagen, is a key tool in cancer research. Inherent to the manual scoring of cellular spread is inter-observer but also intra-observer variation. To overcome these problems, we have developed the Morphology Analysis Software (MAS). MAS scores phase-contrast images of cells on native type I collagen gels and identifies whether a cell has a spread or round morphology using a combination of four unique parameters: the presence of a cellular extension, the cell area, the cell eccentricity and cell circularity. The MAS software scores are equivalent to the average score of five independent observers but MAS is faster, more objective and standardized. A functional screening assay using six cytokines identified TGFα as a stimulator of HCT8/E11 and SK-BR-3 single cell spreading on top of type I collagen gels. This change in morphology correlates with increased migration potential as evidenced by xCELLigence migration assays and are counteracted by EGFR signaling pathway inhibitors. This underscores the use of morphology classification on a population of unlabeled cells as read-out of an important cancer cell property and the potential for the MAS software in drug screening strategies.

Fatty acid kinase A is an important determinant of biofilm formation in Staphylococcus aureus USA300.

BACKGROUND: Methicillin-resistant Staphylococcus aureus (MRSA)-USA300 is notorious for its ability to cause community- and healthcare-acquired infections, which are even more difficult to treat when associated with a biofilm phenotype. We aimed to characterize the genetic determinants of biofilm formation in a USA300 skin abscess isolate (UAS391) that formed prolific biofilms. METHODS: USA300 S. aureus strains, TCH1516 and FPR3757, were found to be closely related based on whole genome mapping (Argus™ Optical Mapping System, Opgen Inc, Gaithersburg, USA) to UAS391 (96.3-99.1 % similarity, P=0.0151), however differed markedly in biofilm formation (P=0.0001) on a dynamic assay (BioFlux 200, Fluxion Biosciences, USA). Comparison of whole genome sequences of these strains identified differences in a total of 18 genes. Corresponding Tn (bursa aurealis-bearing) knockout mutants in these target genes were obtained from a publicly available mutant library of the same clonal lineage (USA300-JE2) and were characterized phenotypically for biofilm formation. Tn mutants showing significant differences in biofilm formation were utilized for transduction into a plasmid-cured erythromycin-sensitive derivative of UAS391 and for complementation experiments. All strains were tested on the dynamic assay, and 17h-biofilms were stained (SYTO9, Life Technologies) and fluorescence intensity quantified by microscopy (Zeiss, ImageJ). Gene expression levels in Tn and transduced mutants were studied by quantitative reverse transcriptase PCR (StepOnePlusTM, Applied Biosystems®). RESULTS: Comparison of the sequenced genomes of TCH1516, FPR3757 and UAS391 yielded a limited number of variant genes (n=18) that were hypothesized to account for the observed difference in biofilm-forming capacity. Screening of Tn mutants disrupted in these target genes identified one mutant (NE229) bearing a transposon insertion in SAUSA300_1119 (fakA), which exhibited increased biofilm formation similar to UAS391 (P=0.9320). Transduction experiments confirmed that fakA::Tn corresponded to 1.9- to 4.6-fold increase in biofilm formation depending on the USA300 strain background (P≤0.0007), while complementation of the TCH1516 wild-type fakA allele in UAS391 resulted in a 4.3-fold reduction in biofilm formation (P<0.0001). CONCLUSIONS: This sequential approach, consisting of strain typing, genome comparison and functional genomics, identified fakA, a recently described fatty acid kinase in S. aureus that is essential for phospholipid synthesis and also impacts the transcription of numerous virulence factors, as a negative regulator of biofilm formation in S. aureus USA300.

Assessment of the short-term functional outcome after urethroplasty: a prospective analysis

OBJECTIVES: To assess the short-term functional outcomes on urinary symptoms, erectile function, urinary continence and patient's satisfaction after urethroplasty. MATERIALS AND METHODS: A prospective analysis was done in 21 patients who underwent urethroplasty. An assessment of the urinary flow, urinary symptoms (International Prostate Symptome Score

Assessment of the short-term functional outcome after urethroplasty: a prospective analysis.

OBJECTIVES: To assess the short-term functional outcomes on urinary symptoms, erectile function, urinary continence and patient's satisfaction after urethroplasty. MATERIALS AND METHODS: A prospective analysis was done in 21 patients who underwent urethroplasty. An assessment of the urinary flow, urinary symptoms (International Prostate Symptoms Score ), erectile function (International Index of Erectile Function-5 ) and urinary continence International Consultation Committee on Incontinence Questionnaire male Short Form ) was done before urethroplasty and 6 weeks and 6 months after urethroplasty. Patients were also asked to score their satisfaction with the urethroplasty after 6 weeks and 6 months. RESULTS: Mean patient's age was 48 years range: 26-80 years). Mean stricture length was 4.2 cm (range: 1-12 cm). Three patients suffered a stricture recurrence. Mean maximum urinary flow increased from 5.83 mL/s to 24.92 mL/s (p < 0.001). Mean IPSS preoperative, 6 weeks and 6 months postoperative was respectively 15.86, 4.60 and 6.41(p < 0.001). The mean IIEF-5 score preoperative, 6 weeks and 6 months postoperative was respectively 15, 12.13 and 11.62 (not significant). The mean ICI-Q-SF score preoperative, 6 weeks and 6 months postoperative was respectively 10.47, 8.33 (p = 0.04) and 9.47 (p = 0.31). Patient's satisfaction 6 weeks and 6 months postoperative was respectively 17.14/20 and 17.12/20. CONCLUSIONS: Urethroplasty leads to a significant improvement in urinary flow and IPSS and urinary continence is tending to improve. Although not significant, erectile function was slightly diminished after urethroplasty. Functional outcome should be assessed when urethroplasty is performed.

Tomographic spectroscopic imaging; an experimental proof of concept.

Recording the electron energy loss spectroscopy data cube with a series of energy filtered images is a dose inefficient process because the energy slit blocks most of the electrons. When recording the data cube by scanning an electron probe over the sample, perfect dose efficiency is attained; but due to the low current in nanoprobes, this often is slower, with a smaller field of view. In W. Van den Broek et al. [Ultramicroscopy, 106 (2006) 269], we proposed a new method to record the data cube, which is more dose efficient than an energy filtered series. It produces a set of projections of the data cube and then tomographically reconstructs it. In this article, we demonstrate these projections in practice, we present a simple geometrical model that allows for quantification of the projection angles and we present the first successful experimental reconstruction, all on a standard post-column instrument.

Multiscale white matter fiber tract coregistration: a new feature-based approach to align diffusion tensor data.

In this paper an automatic multiscale feature-based rigid-body coregistration technique for diffusion tensor imaging (DTI) based on the local curvature kappa and torsion tau of the white matter (WM) fiber pathways is presented. As a similarity measure, the mean squared difference (MSD) of corresponding fiber pathways in (kappa, tau)-space is chosen. After the MSD is minimized along the arc length of the curve, principal component analysis is applied to calculate the transformation parameters. In addition, a scale-space representation of the space curves is incorporated, resulting in a multiscale robust coregistration technique. This fully automatic technique inherently allows one to apply region of interest (ROI) coregistration, and is adequate for performing both global and local transformations. Simulations were performed on synthetic DT data to evaluate the coregistration accuracy and precision. An in vivo coregistration example is presented and compared with a voxel-based coregistration approach, demonstrating the feasibility and advantages of the proposed technique to align DT data of the human brain.

X linked agammaglobulinaemia and rheumatoid arthritis.

BACKGROUND: Much interest has been shown recently in the pathogenic role of B cells in rheumatoid arthritis (RA) owing to the marked clinical responses to anti-CD20 treatment in RA. CASE REPORT: A patient with X linked agammaglobulinaemia (XLA) presented with an erosive symmetric polyarthritis with histological features of RA, including formation of a destructive pannus. Furthermore, the patient developed subcutaneous nodules that were histologically indistinguishable from rheumatoid nodules. Surprisingly, lymphocytic infiltrates in both the synovium and nodule consisted almost exclusively of CD8+ T cells. DISCUSSION: Although some peculiar B cell subsets have been described in patients with XLA, no B cell subsets could be demonstrated in synovial tissue or the subcutaneous nodule. This case illustrates that classical RA can develop in the absence of mature B cells.

Fusion and merging of multispectral images with use of multiscale fundamental forms.

A new multispectral image wavelet representation is introduced, based on multiscale fundamental forms. This representation describes gradient information of multispectral images in a multiresolution framework. The representation is, in particular, extremely suited for fusion and merging of multispectral images. For fusion as well as for merging, a strategy is described. Experiments are performed on multispectral images, where Landsat Thematic Mapper images are fused and merged with SPOT Panchromatic images. The proposed techniques are compared with wavelet-based techniques described in the literature.