Salmonella Typhi Colonization Provokes Extensive Transcriptional Changes Aimed at Evading Host Mucosal Immune Defense During Early Infection of Human Intestinal Tissue.

Commensal microorganisms influence a variety of host functions in the gut, including immune response, glucose homeostasis, metabolic pathways and oxidative stress, among others. This study describes how Salmonella Typhi, the pathogen responsible for typhoid fever, uses similar strategies to escape immune defense responses and survive within its human host. To elucidate the early mechanisms of typhoid fever, we performed studies using healthy human intestinal tissue samples and "mini-guts," organoids grown from intestinal tissue taken from biopsy specimens. We analyzed gene expression changes in human intestinal specimens and bacterial cells both separately and after colonization. Our results showed mechanistic strategies that S. Typhi uses to rearrange the cellular machinery of the host cytoskeleton to successfully invade the intestinal epithelium, promote polarized cytokine release and evade immune system activation by downregulating genes involved in antigen sampling and presentation during infection. This work adds novel information regarding S. Typhi infection pathogenesis in humans, by replicating work shown in traditional cell models, and providing new data that can be applied to future vaccine development strategies.

Open PHACTS computational protocols for in silico target validation of cellular phenotypic screens: knowing the knowns.

Phenotypic screening is in a renaissance phase and is expected by many academic and industry leaders to accelerate the discovery of new drugs for new biology. Given that phenotypic screening is per definition target agnostic, the emphasis of in silico and in vitro follow-up work is on the exploration of possible molecular mechanisms and efficacy targets underlying the biological processes interrogated by the phenotypic screening experiments. Herein, we present six exemplar computational protocols for the interpretation of cellular phenotypic screens based on the integration of compound, target, pathway, and disease data established by the IMI Open PHACTS project. The protocols annotate phenotypic hit lists and allow follow-up experiments and mechanistic conclusions. The annotations included are from ChEMBL, ChEBI, GO, WikiPathways and DisGeNET. Also provided are protocols which select from the IUPHAR/BPS Guide to PHARMACOLOGY interaction file selective compounds to probe potential targets and a correlation robot which systematically aims to identify an overlap of active compounds in both the phenotypic as well as any kinase assay. The protocols are applied to a phenotypic pre-lamin A/C splicing assay selected from the ChEMBL database to illustrate the process. The computational protocols make use of the Open PHACTS API and data and are built within the Pipeline Pilot and KNIME workflow tools.

Evaluation of EGFR gene copy number as a predictive biomarker for the efficacy of cetuximab in combination with chemotherapy in the first-line treatment of recurrent and/or metastatic squamous cell carcinoma of the head and neck: EXTREME study.

BACKGROUND: The phase III EXTREME study demonstrated that combining cetuximab with platinum/5-fluorouracil (5-FU) significantly improved overall survival in the first-line treatment of patients with recurrent and/or metastatic squamous cell carcinoma of the head and neck (R/M SCCHN) compared with platinum/5-FU alone. The aim of this investigation was to evaluate elevated tumor EGFR gene copy number as a predictive biomarker in EXTREME study patients. PATIENTS AND METHODS: Dual-color FISH was used to determine absolute and relative EGFR copy number. Models of differing stringencies were used to score and investigate whether increased copy number was predictive for the activity of cetuximab plus platinum/5-FU. RESULTS: Tumors from 312 of 442 patients (71%) were evaluable by FISH and met the criteria for statistical analysis. A moderate increase in EGFR copy number was common, with high-level amplification of the gene occurring in a small fraction of tumors (∼11%). Considering each of the models tested, no association of EGFR copy number with overall survival, progression-free survival or best overall response was found for patients treated with cetuximab plus platinum/5-FU. CONCLUSION: Tumor EGFR copy number is not a predictive biomarker for the efficacy of cetuximab plus platinum/5-FU as first-line therapy for patients with R/M SCCHN.

Risk factors for extended-spectrum beta-lactamase positivity in uropathogenic Escherichia coli isolated from community-acquired urinary tract infections.

The aim of this prospective cohort study was to determine the risk factors for community-acquired urinary tract infections (UTIs) caused by extended-spectrum beta-lactamase (ESBL)-positive Escherichia coli and the distribution of the ESBL enzyme types. Structured forms were filled in for patients diagnosed with community-acquired UTI in four different geographical locations in Turkey. The forms and the isolates were sent to the central laboratory at Baskent University Hospital, Ankara. Antimicrobial susceptibility was determined according to the CLSI criteria. PCR and DNA sequencing were used to characterize the bla(TEM), bla(CTX-M) and bla(SHV) genes. Multivariate analysis was performed using logistic regression. A total of 510 patients with UTI caused by Gram-negative bacteria were included in this study. ESBLs were detected in 17 of 269 (6.3%) uropathogenic E. coli isolates from uncomplicated UTIs and 34 of 195 (17.4%) E. coli isolates from complicated UTIs (p <0.001). According to multivariate analysis, more than three urinary tract infection episodes in the preceding year (OR 3.8, 95% CI 1.8-8.1, p <0.001), use of a beta-lactam antibiotic in the preceding 3 months (OR 4.6, 95% CI 2.0-0.7, p <0.001) and prostatic disease (OR 9.6, 95% CI 2.1-44.8, p 0.004) were found to be associated with ESBL positivity. The percentages of isolates with simultaneous resistance to trimethoprim-sulphamethoxazole, ciprofloxacin and gentamicin were found to be 4.6% in the ESBL-negative group and 39.2% in the ESBL-positive group (p <0.001). Forty-six of 51 ESBL-positive isolates (90.2%) were found to harbour CTX-M-15. Therapeutic alternatives for UTI, particularly in outpatients, are limited. Further clinical studies are needed to guide the clinicians in the management of community-acquired UTIs.

Influence of habitat structure and resource availability on the movements of Rhagoletis indifferens (Diptera: Tephritidae).

Habitat structure and resources availability may differentially influence movement between habitat patches. We examined fly movement decisions (stay or leave) at the scale of individual trees by measuring the response of marked Rhagoletis indifferens (Diptera: Tephritidae) to sweet cherry trees (Prunus avium) that were manipulated by changing the shape of the tree (structure treatment = normal or reduced) and the fruit load (fruit treatment = augmented, normal, or reduced). More than 600 observations were made at two field sites that differed in the average inter-tree distance: Senger site, 10.1 +/- 4.5 m; Tuemp site, 29.0 +/- 19.3 m. At the Senger site, flies were resighted most often in the normal structure-augmented fruit trees. At the Tuemp site, however, there were fewer transfers between trees, unusual tree preferences, and significant treatment interaction terms. Using a first principles diffusion model of attraction and by varying fly perceptual range to limit tree choice, we generated unusual tree preferences based on differential attraction to individual trees. Our results suggest that manipulating tree attractiveness may be a viable pest management strategy for closely spaced trees but not for dispersed trees. Further study into the relationship between the spatial arrangement of trees and the flies' ability to detect specific tree characteristics is warranted.

Identification of regions of the P2X(7) receptor that contribute to human and rat species differences in antagonist effects.

BACKGROUND AND PURPOSE: Several P2X(7) receptor antagonists are allosteric inhibitors and exhibit species difference in potency. Furthermore, N(2)-(3,4-difluorophenyl)-N(1)-(2-methyl-5-(1-piperazinylmethyl)phenyl)glycinamide dihydrochloride (GW791343) exhibits negative allosteric effects at the human P2X(7) receptor but is a positive allosteric modulator of the rat P2X(7) receptor. In this study we have identified several regions of the P2X(7) receptor that contribute to the species differences in antagonist effects. EXPERIMENTAL APPROACH: Chimeric human-rat P2X(7) receptors were constructed with regions of the rat receptor being inserted into the human receptor. Antagonist effects at these receptors were measured in ethidium accumulation and radioligand binding studies. KEY RESULTS: Exchanging regions of the P2X(7) receptor close to transmembrane domain 1 modified the effects of KN62, 4-(4-fluorophenyl)-2-(4-methylsulphinylphenyl)-5-(4-pyridyl)1H-imidazole (SB203580) and GW791343. Further studies, in which single amino acids were exchanged, identified amino acid 95 as being primarily responsible for the differential allosteric effects of GW791343 and, to varying degrees, the species differences in potency of SB203580 and KN62. The species selectivity of pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid was affected by multiple regions of the receptor, with potency being particularly affected by the amino acid 126 but not by amino acid 95. A further region of the rat receptor (amino acids 154-183) was identified that, when inserted into the corresponding position in the human receptor, increased ATP potency 10-fold. CONCLUSIONS: This study has identified several key residues responsible for the species differences in antagonist effects at the P2X(7) receptor and also identified a further region of the P2X(7) receptor that can significantly affect agonist potency at the P2X(7) receptor.

In vitro activity of tigecycline against resistant micro-organisms isolated from burn patients.

Infections in burn patients are usually caused by multidrug-resistant micro-organisms. Tigecycline, a derivative of glycylcyclines, is an effective antibiotic against the resistant strains. The aim of this study is to determine the in vitro activity of tigecycline against the multidrug-resistant bacteria isolated from burn patients. Fourty-seven bacteria isolated from 118 patients hospitalized in the burn unit during 2003-2006 were included in the study. Gram-negative bacteria that were resistant to at least six broad-spectrum antibiotics, methicillin-resistant staphylococci and ampicillin-resistant enterococci were studied. Minimal inhibitory concentration values of tigecycline against these bacteria were tested by E-test strips. Susceptibility breakpoints were determined according to the previous studies;

Urinary tract infections in renal transplant recipients.

Urinary tract infection (UTI) is the most common infectious complication following renal transplantation. The purposes of this study were to determine the causative agents of UTIs among renal transplant recipients and to compare the antibiotic susceptibilities of Escherichia coli strains isolated from renal transplant recipients and complicated community-acquired UTIs. We evaluated 75 episodes of 63 recipients with confirmed UTI who underwent transplantation during the period 1981 to 2006 at our center. Medical records of the patients were reviewed retrospectively. To compare the susceptibility rates of E coli, 226 isolates from nontransplant patients with complicated community-acquired UTIs were also evaluated. Ten episodes (13.3%) occurred in the first month following the transplantation, 11 (14.7%) in the period of the second month to the sixth month, and 54 (72%) after the sixth month of transplantation. Forty-six (61.3%) isolates were E coli. Among these isolates, ciprofloxacin resistance rates were 50% (2/4) in the first month after transplantation, 75% (6/8) in the period of the second month to the sixth month, and 32.4% (11/34) beyond 6 months after transplantation. The resistance rates of trimethoprim/sulfamethoxazole (TMP-SMX) in the same time periods were 100% (4/4), 87.5% (7/8), and 70.6% (24/34), respectively. The rates of resistance to TMP-SMX among E coli isolated from renal recipients were significantly higher than those in community-acquired complicated UTIs. The increased resistance of urinary pathogens to this agent is a major concern. Although high resistance rates of ciprofloxacin against E coli strains were determined in this group, it was not found to be statistically significant.

The quest for Factor VIIa exosite inhibitors.

Coagulation proteases are involved in a highly orchestrated proteolytic cascade which is essential for haemostasis and blood clotting. In particular, the initiator of the coagulation cascade, Factor VIIa, binds to its cofactor, tissue factor, and its substrate, Factor X, via exosite interactions to form a ternary catalytic complex named extrinsic Xase. These exosite interactions have also been shown to allosterically induce the active conformation of the catalytic site of Factor VIIa. We have developed a direct continuous fluorescence polarization-based extrinsic Xase assay, which has been used to screen in excess of 1 million structurally diverse low-molecular-mass compounds as a potential starting point for the development of anticoagulants. The primary screen hits were categorized with deconvolution assays into either active-site or exosite inhibitors. The latter category of hits displayed both competitive and uncompetitive modalities of inhibition with respect to Factor X activation. An uncompetitive mechanism of action is of particular interest as it offers a hypothetical inhibitory advantage in the context of inhibiting a proteolytic cascade such as the blood coagulation pathway.

End-to-end performance measurement of Internet based medical applications.

We present a method to obtain an end-to-end characterization of the performance of an application over a network. This method is not dependent on any specific application or type of network. The method requires characterization of network parameters, such as latency and packet loss, between the expected server or client endpoints, as well as characterization of the application's constraints on these parameters. A subjective metric is presented that integrates these characterizations and that operates over a wide range of applications and networks. We believe that this method may be of wide applicability as research and educational applications increasingly make use of computation and data servers that are distributed over the Internet.

Simulated learning environments in anatomy and surgery delivered via the next generation internet.

The Next Generation Internet (NGI) will provide high bandwidth, guaranteed Quality of Service, collaboration and security, features that are not available in today's Internet. Applications that take advantage of these features will need to build them into their pedagogic requirements. We present the Anatomy Workbench and the Surgery Workbench, two applications that require most of these features of the NGI. We used pedagogic need and NGI features to define a set of applications that would be difficult to operate on the current Internet, and that would require the features of the NGI. These applications require rich graphics and visualization, and extensive haptic interaction with biomechanical models that represent bony and soft tissue. We are in the process of implementing these applications, and some examples are presented here. An additional feature that we required was that the applications be scalable such that they could run on either on a low-end desktop device with minimal manipulation tools or on a fully outfitted high-end graphic computer with a realistic set of surgical tools. The Anatomy and Surgery Workbenches will be used to test the features of the NGI, and to show the importance of these new features for innovative educational applications.

Immunomodulation of ABA function affects early events in somatic embryo development.

Immunomodulation of abscisic acid (ABA) function during somatic embryogenesis of Nicotiana plumbaginifolia has been used to demonstrate for the first time the effect of this phytohormone on early embryonic events. A homozygous transgenic line constitutively expressing an anti-abscisic acid (ABA) single chain fragment variable antibody in the endoplasmic reticulum was established. Development of somatic embryos from the transgenic line and the wild type was compared. The ABA biosynthesis mutants aba1 and aba2 and wild type cultures treated with the ABA biosynthesis inhibitor fluridone were also used for the comparative investigations. The development of embryonic structures was disturbed in the early stages of all cultures in which ABA function was blocked or which were ABA-deficient. After ABA complementation of the in vitro cell cultures normal somatic embryo development was restored.

Ring opening of 1-methylcyclopropene and cyclopropene: matrix infrared spectroscopic identification of 2-butene-1,3-diyl and propene-1,3-diyl

Triplet 2-butene-1,3-diyl (T-11) was generated on irradiation of 1-methylcyclopropene (10) in a bromine-doped xenon matrix and was characterized by means of IR spectroscopy for the first time. Experimental results suggest that triplet propene-1,3-diyl (T-3) is formed from cyclopropene (1) under similar conditions. In accordance with theoretical calculations, the experimental data indicate that the reactions 1-->3 and 10-->11 are the lowest energy ground-state pathways for the ring opening of 1 and 10, respectively.

New approaches to virtual environment surgery.

This research focused on two main problems: 1) low cost, high fidelity stereoscopic imaging of complex tissues and organs; and 2) virtual cutting of tissue. A further objective was to develop these images and virtual tissue cutting methods for use in a telemedicine project that would connect remote sites using the Next Generation Internet. For goal one we used a CT scan of a human heart, a desktop PC with an OpenGL graphics accelerator card, and LCD stereoscopic glasses. Use of multiresolution meshes ranging from approximately 1,000,000 to 20,000 polygons speeded interactive rendering rates enormously while retaining general topography of the dataset. For goal two, we used a CT scan of an infant skull with premature closure of the right coronal suture, a Silicon Graphics Onyx workstation, a Fakespace Immersive WorkBench and CrystalEyes LCD glasses. The high fidelity mesh of the skull was reduced from one million to 50,000 polygons. The cut path was automatically calculated as the shortest distance along the mesh between a small number of hand selected vertices. The region outlined by the cut path was then separated from the skull and translated/rotated to assume a new position. The results indicate that widespread high fidelity imaging in virtual environment is possible using ordinary PC capabilities if appropriate mesh reduction methods are employed. The software cutting tool is applicable to heart and other organs for surgery planning, for training surgeons in a virtual environment, and for telemedicine purposes.

minifly, a Drosophila gene required for ribosome biogenesis.

We report here the genetic, molecular, and functional characterization of the Drosophila melanogaster minifly (mfl) gene. Genetic analysis shows that mfl is essential for Drosophila viability and fertility. While P-element induced total loss-of-function mutations cause lethality, mfl partial loss-of-function mutations cause pleiotropic defects, such as extreme reduction of body size, developmental delay, hatched abdominal cuticle, and reduced female fertility. Morphological abnormalities characteristic of apoptosis are found in the ovaries, and a proportion of eggs laid by mfl mutant females degenerates during embryogenesis. We show that mfl encodes an ubiquitous nucleolar protein that plays a central role in ribosomal RNA processing and pseudouridylation, whose known eukaryotic homologues are yeast Cfb5p, rat NAP57 and human dyskerin, encoded by the gene responsible for the X-linked dyskeratosis congenita disease. mfl genetic analysis represents the first in vivo functional characterization of a member of this highly conserved gene family from higher eukaryotes. In addition, we report that mfl hosts an intron encoded box H/ACA snoRNA gene, the first member of this class of snoRNAs identified so far from Drosophila.

Effect of eugenol-containing temporary cements on bond strength of composite to enamel.

The purpose of this study was to find out the degree to which eugenol-containing temporary cements or temporary filling materials affected shear bond strength of a dual-cure resin luting cement to acid-etched enamel. For this purpose 56 human caries-free third molars were embedded in acrylic resin and cross sectioned mesiodistally. One of the corresponding halves was covered with Temp-Bond (eugenol-containing, Group 1), Provicol (eugenol-free, Group 2), ZOE (Group 3), and eugenol (Group 4). The other half of each sectioned tooth was kept clean and served as a control. After 1 week the cements were removed; the pure eugenol group (Group 4) was terminated after 24 hours. Plastic cylinders were filled with Dual Cement and placed on the etched enamel and light cured. Shear bond strength data were recorded using a Zwick Universal Testing Machine, and the mode of failure was diagnosed using a light microscope. Significant differences in shear bond strength could neither be found between the treated halves and the controls nor among the four groups pretreated with eugenol-containing or eugenol-free temporary cements. Based on the results of this study, no adverse effects of eugenol on shear bond strength of a resin luting cement to enamel could be found.

An immersive environment for the direct visualization and segmentation of volumetric data sets.

This paper describes a software environment for visualizing and segmenting volumetric data sets such as CT, MRI and the Visible Human data set. The goal is to produce an intuitive environment where the expert knowledge of the end user can be employed to directly guide visualization and segmentation of the data. The environment is built around the Fakespace Immersive Workbench (TM), which provides the user with the illusion that the data set volume resides in the space directly above the workbench surface. Using a position/orientation-tracked probe the user is able to interact with the visualization algorithm and segment the data set to expose features of interest. Segmentation can be performed in either the ray space of the volume rendering algorithm or the coordinate space of the data volume itself. The segmentation results can be saved and used for other purposes including the construction of polygonal models.

Incorporating visible human project data into the undergraduate anatomy and physiology curriculum.

The University of Wisconsin-La Crosse is developing a software environment which will allow undergraduates in anatomy and physiology to directly manipulate the Visible Human Data Set. The software environment provides students with a "personal digital cadaver" for study. The system incorporates a volume rendering daemon for imaging the digital cadaver. Central to the system is the concept of an anatomical notebook in which students record and annotate the studies.