Elucidating the Regulon of the Multidrug Resistance Regulator RarA in Klebsiella pneumoniae

RarA is an AraC-type regulator in Klebsiella pneumoniae, which, when overexpressed, confers a low-level multidrug-resistant (MDR) phenotype linked to the upregulation of both the acrAB and oqxAB efflux genes. Increased rarA expression has also been shown to be integral in the development of tigecycline resistance in the absence of ramA in K. pneumoniae. Given its phenotypic role in MDR, microarray analyses were performed to determine the RarA regulon. Transcriptome analysis was undertaken using strains Ecl8?rarA/pACrarA-2 (rarA-expressing construct) and Ecl8?rarA/pACYC184 (vector-only control) using bespoke microarray slides consisting of probes derived from the genomic sequences of K. pneumoniae MGH 78578 (NC_009648.1) and Kp342 (NC_011283.1). Our results show that rarA overexpression resulted in the differential expression of 66 genes (42 upregulated and 24 downregulated). Under the COG (clusters of orthologous groups) functional classification, the majority of affected genes belonged to the category of cell envelope biogenesis and posttranslational modification, along with genes encoding the previously uncharacterized transport proteins (e.g., KPN_03141, sdaCB, and leuE) and the porin OmpF. However, genes associated with energy production and conversion and amino acid transport/metabolism (e.g., nuoA, narJ, and proWX) were found to be downregulated. Biolog phenotype analyses demonstrated that rarA overexpression confers enhanced growth of the overexpresser in the presence of several antibiotic classes (i.e., beta-lactams and fluoroquinolones), the antifungal/antiprotozoal compound clioquinol, disinfectants (8-hydroxyquinoline), protein synthesis inhibitors (i.e., minocycline and puromycin), membrane biogenesis agents (polymyxin B and amitriptyline), DNA synthesis (furaltadone), and the cytokinesis inhibitor (sanguinarine). Both our transcriptome and phenotypic microarray data support and extend the role of RarA in the MDR phenotype of K. pneumoniae.

TMAX – a Database System Integrating Tissue Biomarker & Genomic Data

Background: Popular approaches in human tissue-based biomarker discovery include tissue microarrays (TMAs) and DNA Microarrays (DMAs) for protein and gene expression profiling respectively. The data generated by these analytic platforms, together with associated image, clinical and pathological data currently reside on widely different information platforms, making searching and cross-platform analysis difficult. Consequently, there is a strong need to develop a single coherent database capable of correlating all available data types.

Method: This study presents TMAX, a database system to facilitate biomarker discovery tasks. TMAX organises a variety of biomarker discovery-related data into the database. Both TMA and DMA experimental data are integrated in TMAX and connected through common DNA/protein biomarkers. Patient clinical data (including tissue pathological data), computer assisted tissue image and associated analytic data are also included in TMAX to enable the truly high throughput processing of ultra-large digital slides for both TMAs and whole slide tissue digital slides. A comprehensive web front-end was built with embedded XML parser software and predefined SQL queries to enable rapid data exchange in the form of standard XML files.

Results & Conclusion: TMAX represents one of the first attempts to integrate TMA data with public gene expression experiment data. Experiments suggest that TMAX is robust in managing large quantities of data from different sources (clinical, TMA, DMA and image analysis). Its web front-end is user friendly, easy to use, and most importantly allows the rapid and easy data exchange of biomarker discovery related data. In conclusion, TMAX is a robust biomarker discovery data repository and research tool, which opens up the opportunities for biomarker discovery and further integromics research.

Concordance between digital pathology and light microscopy in general surgical pathology: A pilot study of 100 cases

Aim (1)
A pilot study to determine the accuracy of interpretation of whole slide digital images in a broad range of general histopathology cases of graded complexity. (2) To survey the participating histopathologists with regard to acceptability of digital pathology.

Materials and methods
Glass slides of 100 biopsies and minor resections were digitally scanned in their entirety, producing digital slides. These cases had been diagnosed by light microscopy at least 1 year previously and were subsequently reassessed by the original reporting pathologist (who was blinded to their original diagnosis) using digital pathology. The digital pathology-based diagnosis was compared with the original glass slide diagnosis and classified as concordant, slightly discordant (without clinical consequence) or discordant. The participants were surveyed at the end of the study.

Results
There was concordance between the original light microscopy diagnosis and digital pathology-based diagnosis in 95 of the 100 cases while the remaining 5 cases showed only slight discordance (with no clinical consequence). None of the cases were categorised as discordant. Participants had mixed experiences using digital pathology technology.

Conclusions
In the broad range of cases we examined, digital pathology is a safe and viable method of making a primary histopathological diagnosis.

The lip:A histologic and analytical approach of relevance to esthetic plastic surgery

This is a study done for the first time to understand the histologic features of the lip deeply, quantitatively, and analytically and identify the differences between the outer, intermediate, and inner parts of the lips. Previous researchers did not tackle the histologic features of the lip from this point of view. Half lip was obtained from different well-preserved cadavers where the upper lip, lower lip, and lip angle were evaluated by coronal histologic sections. A total of 43 slides were studied and photographed using light and digital microscopes (Nikon COOLSCOPE, Nikon Corporation, Tokyo, Japan), respectively. The cadavers (26 men and 17 women) were in the age group of 45 to 65 years old, and older than 65 years. Data were entered on a study pro forma and statistically analyzed. Normal histologic features of the upper lip, intermediate, and the inner lip sections were observed. Fibrous septations that sometimes had muscular components inserting into them could be identified. In between these septations, there were loose areas (chambers). The mean numbers of chambers identified in the upper and lower lips were higher in the red areas, and more septations and chambers were identified in the lower lip. New histologic observations were classified into types 1 to 7. Significant relations were identified between the total number of septations and chambers in relation to age group and sex. Higher means of septations and chambers were detected in the age group older than 65 years, and in women as a whole. In conclusion, the findings detected in this work could explain the congenital lip pits that are familial or syndromic and many post-lip augmentation complications. © 2009 Lippincott Williams & Wilkins, Inc.

Automated tumour annotation and analysis for molecular pathology using TissueMark (R)

Objective: Molecular pathology relies on identifying anomalies using PCR or analysis of DNA/RNA. This is important in solid tumours where molecular stratification of patients define targeted treatment. These molecular biomarkers rely on examination of tumour, annotation for possible macro dissection/tumour cell enrichment and the estimation of % tumour. Manually marking up tumour is error prone. Method: We have developed a method for automated tumour mark-up and % cell calculations using image analysis called TissueMark® based on texture analysis for lung, colorectal and breast (cases=245, 100, 100 respectively). Pathologists marked slides for tumour and reviewed the automated analysis. A subset of slides was manually counted for tumour cells to provide a benchmark for automated image analysis. Results: There was a strong concordance between pathological and automated mark-up (100 % acceptance rate for macro-dissection). We also showed a strong concordance between manually/automatic drawn boundaries (median exclusion/inclusion error of 91.70 %/89 %). EGFR mutation analysis was precisely the same for manual and automated annotation-based macrodissection. The annotation accuracy rates in breast and colorectal cancer were 83 and 80 % respectively. Finally, region-based estimations of tumour percentage using image analysis showed significant correlation with actual cell counts. Conclusion: Image analysis can be used for macro-dissection to (i) annotate tissue for tumour and (ii) estimate the % tumour cells and represents an approach to standardising/improving molecular diagnostics.

Bacteria microarrays as sensitive tools for exploring pathogen surface epitopes and recognition by host receptors

We describe a protocol for the generation and validation of bacteria microarrays and their application to the study of specific features of the pathogen's surface and interactions with host receptors. Bacteria were directly printed on nitrocellulose-coated glass slides, using either manual or robotic arrayers, and printing quality, immobilization efficiency and stability of the arrays were rigorously controlled by incorporating a fluorescent dye into the bacteria. A panel of wild type and mutant strains of the human pathogen Klebsiella pneumoniae, responsible for nosocomial and community-acquired infections, was selected as model bacteria, and SYTO-13 was used as dye. Fluorescence signals of the printed bacteria were found to exhibit a linear concentration-dependence in the range of 1 x 10(8) to 1 x 10(9) bacteria per ml. Similar results were obtained with Pseudomonas aeruginosa and Acinetobacter baumannii, two other human pathogens. Successful validation of the quality and applicability of the established microarrays was accomplished by testing the capacity of the bacteria array to detect recognition by anti-Klebsiella antibodies and by the complement subcomponent C1q, which binds K. pneumoniae in an antibody-independent manner. The biotin/AlexaFluor-647-streptavidin system was used for monitoring binding, yielding strain-and dose-dependent signals, distinctive for each protein. Furthermore, the potential of the bacteria microarray for investigating specific features, e.g. glycosylation patterns, of the cell surface was confirmed by examining the binding behaviour of a panel of plant lectins with diverse carbohydrate-binding specificities. This and other possible applications of the newly developed arrays, as e.g. screening/evaluation of compounds to identify inhibitors of host-pathogen interactions, make bacteria microarrays a useful and sensitive tool for both basic and applied research in microbiology, biomedicine and biotechnology.

Novel titania sol-gel derived film for luminescence-based oxygen sensing.

A novel method for the preparation of titania sol–gel derived oxygen sensors based on the ruthenium(II) dye, [Ru(bpy)3]2+, is described. A titania sol–gel paste film was cast onto microscope slides, and the dye ion-paired to the deprotonated, hydroxylated groups on the film's surface from an aqueous solution of the dye at pH 11. The resulting sensor film is extremely oxygen sensitive, with a PO2 (S = 1/2) value (i.e. the partial pressure of oxygen required in order to reduce the original, oxygen free, luminescence intensity by 50%) of 0.011 atm. The sensor undergoes 95% response to oxygen in 4 s, and shows 95% recovery of its luminescence in argon within 7 s.

A sedimentologic and 14C dating study of five eastern Australian upper continental slope submarine landslides

Sedimentologic and AMS 14C age data are reported for calcareous hemipelagic mud samples taken from gravity cores collected at sites within, or adjacent to five submarine landslides identified with multibeam bathymetry data on the Nerrang Plateau segment and surrounding canyons of eastern Australia's continental slope (Bribie Bowl, Coolangatta-2, Coolangatta-1, Cudgen and Byron). Sediments are comprised of mixtures of calcareous and terrigenous clay (10-20%), silt (50-65%) and sand (15-40%) and are generally uniform in appearance. Their carbonate contents vary between and 17% and 22% by weight while organic carbon contents are less than 10% by weight. Dating of conformably deposited material identified in ten of the twelve cores indicates a range of sediment accumulation rates between 0.017mka-1 and 0.2 mka-1 which are consistent with previous estimates reported for this area. One slide-adjacent core, and four within-landslide cores present depositional hiatus surfaces located at depths of 0.8 to 2.2 meters below the present-day seafloor and identified by a sharp, colour-change boundary; discernable but small increases in sediment stiffness; and a slight increase in sediment bulk density of 0.1 gcm-3. Distinct gaps in AMS 14C age of at least 20ka are recorded across these boundary surfaces. Examination of sub-bottom profiler records of transects through three of the within-slide core-sites and their nearby landslide scarps available for the Coolangatta-1 and Cudgen slides indicate that: 1) the youngest identifiable sediment layer reflectors upslope of these slides, terminate on and are truncated by slide rupture surfaces; and 2) there is no obvious evidence in the sub-bottom profiles for a post-slide sediment layer draped over or otherwise burying slide ruptures or exposed slide detachment surfaces. This suggests that both these submarine landslides are geologically recent and suggests that the hiatus surfaces identified in Coolangatta-1's and Cudgen's within-slide cores are either: a) erosional features that developed after the occurrence of the landslide in which case the hiatus surface age provides a minimum age for landslide occurrence or b) detachment surfaces from which slabs of near-surface sediment were removed during landsliding in which case the post-hiatus sediment dates indicates approximately when landsliding occurred. In either case, it is reasonable to suggest that these two spatially adjacent slides occurred penecontemporaneously approximately 20,000 years ago.

Chimaerism following allogeneic bone marrow transplantation:detection of residual host cells using the polymerase chain reaction

Chimaerism was assessed in five recipients following sex mismatched allogeneic bone marrow transplantation. Techniques included karyotyping of bone marrow cells, dot blot DNA analysis of blood and bone marrow suspensions, and in vitro amplification of DNA by the polymerase chain reaction (PCR) using blood and bone marrow suspensions and stored bone marrow slides. Results of karyotypic analysis suggested complete chimaerism in four patients, while in one patient mixed chimaerism was detected. Mixed chimaerism was also detected, however, in a second patient using PCR and confirmed by dot blot analysis on all tissues examined. PCR is a sensitive tool for investigation of chimaerism following bone marrow transplantation. Since this technique does not require radioactivity, it is an attractive method for use in a clinical laboratory. This technique represents a further development in the use of DNA methodologies in the assessment of haematological disease.

Automated tumor analysis for molecular profiling in lung cancer

The discovery and clinical application of molecular biomarkers in solid tumors, increasingly relies on nucleic acid extraction from FFPE tissue sections and subsequent molecular profiling. This in turn requires the pathological review of haematoxylin & eosin (H&E) stained slides, to ensure sample quality, tumor DNA sufficiency by visually estimating the percentage tumor nuclei and tumor annotation for manual macrodissection. In this study on NSCLC, we demonstrate considerable variation in tumor nuclei percentage between pathologists, potentially undermining the precision of NSCLC molecular evaluation and emphasising the need for quantitative tumor evaluation. We subsequently describe the development and validation of a system called TissueMark for automated tumor annotation and percentage tumor nuclei measurement in NSCLC using computerized image analysis. Evaluation of 245 NSCLC slides showed precise automated tumor annotation of cases using Tissuemark, strong concordance with manually drawn boundaries and identical EGFR mutational status, following manual macrodissection from the image analysis generated tumor boundaries. Automated analysis of cell counts for % tumor measurements by Tissuemark showed reduced variability and significant correlation (p < 0.001) with benchmark tumor cell counts. This study demonstrates a robust image analysis technology that can facilitate the automated quantitative analysis of tissue samples for molecular profiling in discovery and diagnostics.

Disseminated tumor cells and their prognostic significance in nonmetastatic prostate cancer patients

Detection of pretreatment disseminated cells (pre-DTC) reflecting its homing to bone marrow (BM) in prostate cancer (PCa) might improve the current model to predict recurrence or survival in men with nonmetastatic disease despite of primary treatment. Thereby, pre-DTC may serve as an early prognostic biomarker. Post-treatment DTCs (post-DTC) finding may supply the clinician with additional predictive information about the possible course of PCa. To assess the prognostic impact of DTCs in BM aspirates sampled before initiation of primary therapy (pre-DTC) and at least 2 years after (post-DTC) to established prognostic factors and survival in patients with PCa. Available BM of 129 long-term follow-up patients with T1-3N0M0 PCa was assessed in addition to 100 BM of those in whom a pretreatment BM was sampled. Patients received either combined therapy [n = 81 (63%)], radiotherapy (RT) with different duration of hormone treatment (HT) or monotherapy with RT or HT alone [n = 48 (37%)] adapted to the criteria of the SPCG-7 trial. Mononuclear cells were deposited on slides according to the cytospin methodology and DTCs were identified by immunocytochemistry using the pancytokeratin antibodies AE1/AE3. The median age of men at diagnosis was 64.5 years (range 49.5-73.4 years). The median long-term follow-up from first BM sampling to last observation was 11 years. Categorized clinically relevant factors in PCa showed only pre-DTC status as the statistically independent parameter for survival in the multivariate analysis. Pre-DTCs homing to BM are significantly associated with clinically relevant outcome independent to the patient's treatment at diagnosis with nonmetastatic PCa.

Investigation of tanpura string vibrations using a two-dimensional time-domain model incorporating coupling and bridge friction

Tanpura string vibrations have been investigated previously using numerical models based on energy conserving schemes derived from a Hamiltonian description in one-dimensional form. Such time-domain models have the property that, for the lossless case, the numerical Hamiltonian (representing total energy of the system) can be proven to be constant from one time step
to the next, irrespective of any of the system parameters; in practice the Hamiltonian can be shown to be conserved within machine precision. Models of this kind can reproduce a jvari effect, which results from the bridge-string interaction. However the one-dimensional formulation has recently been shown to fail to replicate the jvaris strong dependence on the thread placement. As a first step towards simulations which accurately emulate this sensitivity to the thread placement, a twodimensional model is proposed, incorporating coupling of controllable level between the two string polarisations at the string termination opposite from the barrier. In addition, a friction force acting when the string slides across the bridge in horizontal direction is introduced, thus effecting a further damping mechanism. In this preliminary study, the string is terminated at the position of the thread. As in the one-dimensional model, an implicit scheme has to be used to solve the system, employing Newton's method to calculate the updated positions and momentums of each string segment. The two-dimensional model is proven to be energy conserving when the loss parameters are set to zero, irrespective of the coupling constant. Both frequency-dependent and independent losses are then added to the string, so that the model can be compared to analogous instruments. The influence of coupling and the bridge friction are investigated.

Immunohistochemistry should undergo robust validation equivalent to that of molecular diagnostics

Immunohistochemistry (IHC) is a widely available and highly utilised tool in diagnostic histopathology and is used to guide treatment options as well as provide prognostic information. IHC is subjected to qualitative and subjective assessment, which has been criticised for a lack of stringency, while PCR-based molecular diagnostic validations by comparison are regarded as very rigorous. It is essential that IHC tests are validated through evidence-based procedures. With the move to ISO15189 (2012), not just of the accuracy, specificity and reproducibility of each test need to be determined and managed, but also the degree of uncertainty and the delivery of such tests. The recent update to ISO 15189 (2012) states that it is appropriate to consider the potential uncertainty of measurement of the value obtained in the laboratory and how that may impact on prognostic or predictive thresholds. In order to highlight the problems surrounding IHC validity, we reviewed the measurement of Ki67and p53 in the literature. Both of these biomarkers have been incorporated into clinical care by pathology laboratories worldwide. The variation seen appears excessive even when measuring centrally stained slides from the same cases. We therefore propose in this paper to establish the basis on which IHC laboratories can bring the same level of robust validation seen in the molecular pathology laboratories and the principles applied to all routine IHC tests.