Quantitative discrimination between oil and water in drilled bore cores via Fast-Neutron Resonance Transmission Radiography.

A novel method utilizing the Fast Neutron Resonance Transmission Radiography is proposed for non-destructive, quantitative determination of the weight percentages of oil and water in cores taken from subterranean or underwater geological formations. The ability of the method to distinguish water from oil stems from the unambiguously-specific energy dependence of the neutron cross-sections for the principal elemental constituents. Monte-Carlo simulations and initial results of experimental investigations indicate that the technique may provide a rapid, accurate and non-destructive method for quantitative evaluation of core fluids in thick intact cores, including those of tight shales for which the use of conventional core analytical approaches appears to be questionable.

Abnormal cortical activity in patients with temporomandibular disorder evoked by cognitive and emotional tasks.

Patients with temporomandibular disorder (TMD) perform poorly in neuropsychological tests of cognitive function. These deficits might be related to dysfunction in brain networks that support pain and cognition, due to the impact of chronic pain and its related emotional processes on cognitive ability. We therefore tested whether patients with TMD perform poorly in cognitive and emotion tasks and whether they had abnormal task-evoked brain activity. Seventeen female subjects with nontraumatic TMD and 17 age-matched healthy female subjects underwent functional magnetic resonance imaging while performing counting Stroop tasks comprising neutral words, incongruent numbers, or emotional words, including TMD-specific words. Group differences in task-related brain responses were assessed. Connectivity between 2 pairs of coupled brain regions during the cognitive and emotional tasks (prefrontal-cingulate and amygdala-cingulate) was also examined. The patients had sluggish Stroop reaction times for all Stroop tasks. Furthermore, compared to controls, patients showed increased task-evoked responses in brain areas implicated in attention (eg, lateral prefrontal, inferior parietal), emotional processes (eg, amygdala, pregenual anterior cingulate), motor planning and performance (eg, supplementary and primary motor areas), and activation of the default-mode network (medial prefrontal and posterior cingulate). The patients also exhibited decoupling of the normally correlated activity between the prefrontal and cingulate cortices and between the amygdala and cingulate cortex. These findings suggest that the slow behavioral responses in idiopathic TMD may be due to attenuated, slower, and/or unsynchronized recruitment of attention/cognition processing areas. These abnormalities may be due to the salience of chronic pain, which inherently requires attention. Sluggish performance in cognitive and emotional interference tasks in patients with nontraumatic temporomandibular disorder is associated with pronounced and unsynchronized task-evoked fMRI brain responses.

Complete genome sequence and comparative genomics of Shigella flexneri serotype 2a strain 2457T.

We determined the complete genome sequence of Shigella flexneri serotype 2a strain 2457T (4,599,354 bp). Shigella species cause >1 million deaths per year from dysentery and diarrhea and have a lifestyle that is markedly different from those of closely related bacteria, including Escherichia coli. The genome exhibits the backbone and island mosaic structure of E. coli pathogens, albeit with much less horizontally transferred DNA and lacking 357 genes present in E. coli. The strain is distinctive in its large complement of insertion sequences, with several genomic rearrangements mediated by insertion sequences, 12 cryptic prophages, 372 pseudogenes, and 195 S. flexneri-specific genes. The 2457T genome was also compared with that of a recently sequenced S. flexneri 2a strain, 301. Our data are consistent with Shigella being phylogenetically indistinguishable from E. coli. The S. flexneri-specific regions contain many genes that could encode proteins with roles in virulence. Analysis of these will reveal the genetic basis for aspects of this pathogenic organism's distinctive lifestyle that have yet to be explained.

Actin-based motility of intracellular microbial pathogens.

A diverse group of intracellular microorganisms, including Listeria monocytogenes, Shigella spp., Rickettsia spp., and vaccinia virus, utilize actin-based motility to move within and spread between mammalian host cells. These organisms have in common a pathogenic life cycle that involves a stage within the cytoplasm of mammalian host cells. Within the cytoplasm of host cells, these organisms activate components of the cellular actin assembly machinery to induce the formation of actin tails on the microbial surface. The assembly of these actin tails provides force that propels the organisms through the cell cytoplasm to the cell periphery or into adjacent cells. Each of these organisms utilizes preexisting mammalian pathways of actin rearrangement to induce its own actin-based motility. Particularly remarkable is that while all of these microbes use the same or overlapping pathways, each intercepts the pathway at a different step. In addition, the microbial molecules involved are each distinctly different from the others. Taken together, these observations suggest that each of these microbes separately and convergently evolved a mechanism to utilize the cellular actin assembly machinery. The current understanding of the molecular mechanisms of microbial actin-based motility is the subject of this review.

N-WASP deficiency reveals distinct pathways for cell surface projections and microbial actin-based motility.

The Wiskott-Aldrich syndrome protein (WASP) family of molecules integrates upstream signalling events with changes in the actin cytoskeleton. N-WASP has been implicated both in the formation of cell-surface projections (filopodia) required for cell movement and in the actin-based motility of intracellular pathogens. To examine N-WASP function we have used homologous recombination to inactivate the gene encoding murine N-WASP. Whereas N-WASP-deficient embryos survive beyond gastrulation and initiate organogenesis, they have marked developmental delay and die before embryonic day 12. N-WASP is not required for the actin-based movement of the intracellular pathogen Listeria but is absolutely required for the motility of Shigella and vaccinia virus. Despite these distinct defects in bacterial and viral motility, N-WASP-deficient fibroblasts spread by using lamellipodia and can protrude filopodia. These results imply a crucial and non-redundant role for N-WASP in murine embryogenesis and in the actin-based motility of certain pathogens but not in the general formation of actin-containing structures.

The making of a gradient: IcsA (VirG) polarity in Shigella flexneri.

The generation and maintenance of subcellular organization in bacteria is critical for many cell processes and properties, including growth, structural integrity and, in pathogens, virulence. Here, we investigate the mechanisms by which the virulence protein IcsA (VirG) is distributed on the bacterial surface to promote efficient transmission of the bacterium Shigella flexneri from one host cell to another. The outer membrane protein IcsA recruits host factors that result in actin filament nucleation and, when concentrated at one bacterial pole, promote unidirectional actin-based motility of the pathogen. We show here that the focused polar gradient of IcsA is generated by its delivery exclusively to one pole followed by lateral diffusion through the outer membrane. The resulting gradient can be modified by altering the composition of the outer membrane either genetically or pharmacologically. The gradient can be reshaped further by the action of the protease IcsP (SopA), whose activity we show to be near uniform on the bacterial surface. Further, we report polar delivery of IcsA in Escherichia coli and Yersinia pseudotuberculosis, suggesting that the mechanism for polar delivery of some outer membrane proteins is conserved across species and that the virulence function of IcsA capitalizes on a more global mechanism for subcellular organization.

Polar targeting of Shigella virulence factor IcsA in Enterobacteriacae and Vibrio.

Asymmetric localization is key to the proper function of certain prokaryotic proteins important to virulence, chemotaxis, cell division, development, motility, and adhesion. Shigella IcsA is localized to the old pole of the bacterium, where it mediates assembly of an actin tail inside infected mammalian cells. IcsA (VirG) is essential to Shigella intracellular motility and virulence. We used translational fusions between portions of IcsA and the green fluorescent protein (GFP) to determine the regions of IcsA that are necessary and sufficient for its targeting to the bacterial old pole. An IcsA-GFP fusion that lacks a signal peptide localized to the old pole, indicating that signal peptide-mediated secretion is not required for polar localization. Two regions within IcsA were required for localization of an IcsA-GFP fusion to the old pole. Further characterization of these regions indicated that amino acids 1-104 and 507-620 were each independently sufficient for polar localization. Finally, when expressed in Escherichia coli, Salmonella typhimurium, Yersinia pseudotuberculosis, and Vibrio cholerae, each of the two targeting regions localized to the pole, indicating that the mechanism of polar targeting used by IcsA is present generally among Enterobacteriacae and Vibrio.

Complete DNA sequence and analysis of the large virulence plasmid of Shigella flexneri.

The complete sequence analysis of the 210-kb Shigella flexneri 5a virulence plasmid was determined. Shigella spp. cause dysentery and diarrhea by invasion and spread through the colonic mucosa. Most of the known Shigella virulence determinants are encoded on a large plasmid that is unique to virulent strains of Shigella and enteroinvasive Escherichia coli; these known genes account for approximately 30 to 35% of the virulence plasmid. In the complete sequence of the virulence plasmid, 286 open reading frames (ORFs) were identified. An astonishing 153 (53%) of these were related to known and putative insertion sequence (IS) elements; no known bacterial plasmid has previously been described with such a high proportion of IS elements. Four new IS elements were identified. Fifty putative proteins show no significant homology to proteins of known function; of these, 18 have a G+C content of less than 40%, typical of known virulence genes on the plasmid. These 18 constitute potentially unknown virulence genes. Two alleles of shet2 and five alleles of ipaH were also identified on the plasmid. Thus, the plasmid sequence suggests a remarkable history of IS-mediated acquisition of DNA across bacterial species. The complete sequence will permit targeted characterization of potential new Shigella virulence determinants.

Periplasmic transit and disulfide bond formation of the autotransported Shigella protein IcsA.

The Shigella outer membrane protein IcsA belongs to the family of type V secreted (autotransported) virulence factors. Members of this family mediate their own translocation across the bacterial outer membrane: the carboxy-terminal beta domain forms a beta barrel channel in the outer membrane through which the amino-terminal alpha domain passes. IcsA, which is localized at one pole of the bacterium, mediates actin assembly by Shigella, which is essential for bacterial intracellular movement and intercellular dissemination. Here, we characterize the transit of IcsA across the periplasm during its secretion. We show that an insertion in the dsbB gene, whose gene product mediates disulfide bond formation of many periplasmic intermediates, does not affect the surface expression or unipolar targeting of IcsA. However, IcsA forms one disulfide bond in the periplasm in a DsbA/DsbB-dependent fashion. Furthermore, cellular fractionation studies reveal that IcsA has a transient soluble periplasmic intermediate. Our data also suggest that IcsA is folded in a proteinase K-resistant state in the periplasm. From these data, we propose a novel model for the secretion of IcsA that may be applicable to other autotransported proteins.

Reduced neuropsychologic measures as predictors of treatment outcome in patients with temporomandibular disorders.

AIMS: To determine via a prospective investigation whether the presence of neuropsychologic or cognitive deficiencies could be identified in patients with temporomandibular disorders (TMD) and used to predict treatment outcome. This was based on the theory that measurable reductions in neuropsychologic and cognitive function might have a negative impact on treatment outcome in patients with essentially nontraumatic TMD, as has been shown for patients with posttraumatic TMD. METHODS: Various neuropsychologic, psychosocial, and clinical parameters (including but not limited to the Peterson-Peterson Consonant Trigram Test and the California Verbal Learning Test) were used to pretest patients suffering from TMD prior to treatment. Patients were then entered into treatment, after which determination of treatment success was made both by the use of visual analog scales for pain and global transitional outcome measures (e.g., "better," responders versus "same/worse," nonresponders). After determination of treatment success was made, treatment response was correlated with the various clinical, cognitive, and neuropsychologic test scores. RESULTS: Overall, the nonresponders did worse in both the neuropsychologic and psychosocial assessments, with greater memory deficits, sleep disturbances, depression, and fatigue and lower energy levels as compared to responders. Among the best predictors of treatment outcome were the Peterson-Peterson Consonant Trigram Test scores, as well as the scores on the California Verbal Learning Test (i.e., poorer test outcomes predicted nonresponse). Neither responders nor nonresponders could be distinguished from one another based on clinical parameters of maximum interincisal opening or muscle tenderness. Three psychosocial variables were also found to be predictors of poor outcome: sleep disturbance, fatigue, and income. Pretreatment pain on chewing was also found to be a reliable predictor of poor treatment outcome. CONCLUSION: We conclude that various neuropsychologic, psychosocial, and some clinical parameters may provide pretreatment prediction of treatment outcome in an idiopathic TMD population.

Sensory and affective components of orofacial pain: is it all in your brain?

In this paper, we shall review several chronic orofacial pain conditions with emphasis on those that are essentially refractory to treatment. We shall present a review of current and past literature that describes the various pain phenomena as well as their underlying central mechanisms. New data concerning refractory pain will be used to underscore the importance of central processing of pain, with particular emphasis on neuropsychological and cognitive function and capacity that may play important roles in pain processing and maintenance of the pain state. Further, neurophysiological data showing that the anterior cingulate cortex (ACC) and other structures in the brain may play key roles in modulation of chronic pain will also be discussed. Although peripheral triggering events surely play an important role in initiating pain, the development of chronic and, in particular, refractory pain may depend on changes or malfunctions in the central nervous system. These changes may be quite subtle and require sophisticated approaches, such as functional MRI, to study them, as is now being done. New findings obtained therefore may lead to more rational and reliable treatment for orofacial pain.

Gamma ray nuclear resonance absorption: an alternative method for in vivo body composition studies.

We have evaluated gamma ray nuclear resonance absorption (gamma-NRA) on nitrogen, a mature technology proposed and developed by Soreq NRC for detecting explosives, as an alternative to neutron activation for in vivo assaying of body nitrogen. The principles of the gamma-NRA method are outlined, and a test facility constructed at McMaster University's Accelerator Laboratory is described. The results of a feasibility study recently performed there on phantoms and animal tissue are presented and discussed. gamma-NRA is a full imaging technique that essentially constitutes element-specific absorptiometry--i.e., it can generate projections of the mass distribution for a specific element, along with a conventional radiograph of the patient. From the transmission profile of an individual scanned by 9.17 MeV gamma rays, local or whole body nitrogen content can be determined via the resonant attenuation undergone when the beam encounters regions of nitrogen concentration. The advantages of gamma-NRA over neutron activation are (a) radiation doses delivered to the body are at least one order of magnitude lower, thus allowing repeated measurements on individual patients and also rendering the method ethically acceptable for application to children; (b) gamma-NRA is inherently free from uncertainties related to nonuniform distributions of the element in question within the body; (c) it is applicable to patients of varying size and shape; and (d) it yields both nitrogen images and conventional radiographic images of the body.

Thymic independence of adaptive immunity to the intracellular pathogen Shigella flexneri serotype 2a.

Shigella flexneri is a facultative intracellular pathogen. While immunity to several intracellular pathogens is mediated by T lymphocytes, it is unknown whether cellular immune responses are important to adaptive immunity to S. flexneri. We show that vaccination with S. flexneri serotype 2a confers protection to mice that lack T lymphocytes or gamma interferon (IFN-gamma), specific depletion of T lymphocytes does not alter the protection, and adoptive transfer of splenocytes from vaccinated mice does not confer protection to naive mice. In contrast, vaccination conferred no protection to mice that lack B lymphocytes and adoptive transfer of immune sera conferred partial protection to naive mice. These data demonstrate that in the mouse bronchopulmonary model, adaptive immunity to S. flexneri 2a is an antibody-mediated, B-lymphocyte-dependent process and can be generated in the absence of T lymphocytes or IFN-gamma.

The unipolar Shigella surface protein IcsA is targeted directly to the bacterial old pole: IcsP cleavage of IcsA occurs over the entire bacterial surface.

Shigella flexneri is an intracellular pathogen that is able to move within the cytoplasm of infected cells by the continual assembly of actin onto one pole of the bacterium. IcsA, an outer membrane protein, is localized to the old pole of the bacterium and is both necessary and sufficient for actin assembly. IcsA is slowly cleaved from the bacterial surface by the protease IcsP (SopA). Absence of IcsP leads to an alteration in the distribution of surface IcsA, such that the polar cap is maintained and some IcsA is distributed along the lateral walls of the bacillus. The mechanism of unipolar localization of IcsA and the role of IcsP in its unipolar localization are incompletely understood. Here, we demonstrate that cleavage of IcsA occurs exclusively in the outer membrane and that IcsP is localized to the outer membrane. In addition, we show that IcsA at the old pole is susceptible to cleavage by IcsP and that native IcsP is active at the pole. Taken together, these data indicate that IcsP cleaves IcsA over the entire bacterial surface. Finally, we show that, immediately after induction from a tightly regulated promoter, IcsA is expressed exclusively at the old pole in both the icsP- icsA- and the icsA- background. These data demonstrate that unipolar localization of IcsA results from its direct targeting to the pole, followed by its diffusion laterally in the outer membrane.

Adaptive immune response to Shigella flexneri 2a cydC in immunocompetent mice and mice lacking immunoglobulin A.

Shigella flexneri cydC, which is deficient in cytochrome bd, was rapidly cleared from the lungs of intranasally inoculated mice and was Sereny negative, yet it induced 93% protection against challenge with wild-type S. flexneri. Mice that lack immunoglobulin A (IgA) were fully protected, suggesting that IgA may not be required for adaptive immunity in this model system.

Impact of either elevated or decreased levels of cytochrome bd expression on Shigella flexneri virulence.

Shigella spp. are the major cause of bacillary dysentery worldwide. The pathogenic process involves bacterial invasion and lysis of the phagocytic vacuole, followed by replication and movement within the cell cytoplasm and, ultimately, spread directly into adjacent cells. This study demonstrates that S. flexneri cytochrome bd expression is necessary for normal intracellular survival and virulence. Cytochrome bd is one of two terminal oxidases in the bacterial respiratory chain that reduce molecular oxygen to water, utilizing intermediates shuttled through the electron transport chain. S. flexneri mutants that contain a disruption in the cydC locus, which leads to defective cytochrome bd expression, or in the riboflavin (ribE) or ubiquinol-8 (ubiH) biosynthetic pathway, which leads to elevated cytochrome bd expression, were evaluated in intracellular survival and virulence assays. The cydC mutant formed significantly smaller plaques, had significantly decreased intracellular survival, and had a 100-fold increase in lethal dose for mice compared with the wild type. The ribE and ubiH mutants each formed significantly larger plaques and had a 10-fold decrease in lethal dose for mice compared with the wild type. The data indicate that expression of cytochrome bd is required for S. flexneri intracellular survival and virulence.

Functional analysis of a rickettsial OmpA homology domain of Shigella flexneri icsA.

Shigella flexneri is a gram-negative bacterium that causes diarrhea and dysentery by invasion and spread through the colonic epithelium. Bacteria spread by assembling actin and other cytoskeletal proteins of the host into "actin tails" at the bacterial pole; actin tail assembly provides the force required to move bacteria through the cell cytoplasm and into adjacent cells. The 120-kDa S. flexneri outer membrane protein IcsA is essential for actin assembly. IcsA is anchored in the outer membrane by a carboxy-terminal domain (the beta domain), such that the amino-terminal 706 amino acid residues (the alpha domain) are exposed on the exterior of the bacillus. The alpha domain is therefore likely to contain the domains that are important to interactions with host factors. We identify and characterize a domain of IcsA within the alpha domain that bears significant sequence similarity to two repeated domains of rickettsial OmpA, which has been implicated in rickettsial actin tail formation. Strains of S. flexneri and Escherichia coli that carry derivatives of IcsA containing deletions within this domain display loss of actin recruitment and increased accessibility to IcsA-specific antibody on the surface of intracytoplasmic bacteria. However, site-directed mutagenesis of charged residues within this domain results in actin assembly that is indistinguishable from that of the wild type, and in vitro competition of a polypeptide of this domain fused to glutathione S-transferase did not alter the motility of the wild-type construct. Taken together, our data suggest that the rickettsial homology domain of IcsA is required for the proper conformation of IcsA and that its disruption leads to loss of interactions of other IcsA domains within the amino terminus with host cytoskeletal proteins.

Clearance of Shigella flexneri infection occurs through a nitric oxide-independent mechanism.

Nitric oxide (NO) generated by gamma interferon (IFN-gamma) activation of macrophages mediates the killing of many intracellular pathogens. IFN-gamma is essential to innate resistance to Shigella flexneri infection. We demonstrate that NO is produced following S. flexneri infection both in mice and in activated cells in vitro and that while it is able to kill S. flexneri in a cell-free system, it is not required for clearance of S. flexneri in either infected mice or in activated cells in vitro.

An essential role for gamma interferon in innate resistance to Shigella flexneri infection.

Shigella spp. are the major cause of bacillary dysentery worldwide. To identify immune effectors associated with protection of the naive host during infection, the susceptibility to pulmonary Shigella infection of each of various mouse strains that have a targeted deletion in a specific aspect of the immune system was evaluated. Our results demonstrate that mice deficient in gamma interferon are 5 orders of magnitude more susceptible to Shigella than are wild-type mice, whereas mice deficient in B and T lymphocytes or in T lymphocytes alone exhibit no difference in susceptibility. Significantly lower numbers of shigellae were recovered from immunocompetent compared with gamma-interferon-deficient mice after infection. While immunocompetent mice were able to clear a sublethal Shigella inoculum by day 5 postinfection, progressively increasing numbers of shigellae were cultured from the lungs of gamma interferon-deficient mice over the same period. Histopathology of the lungs from immunocompetent mice infected with a sublethal Shigella inoculum showed mild inflammatory changes, whereas the lungs from gamma interferon-deficient mice demonstrated progressively worsening acute bronchiolitis with ulceration. Further, the time to death in gamma interferon-deficient mice correlates inversely with the size of the Shigella inoculum. To identify the cellular source of gamma interferon, we infected SCID mice, T-cell-receptor-deficient mice, beige mice (a mouse strain deficient in natural killer [NK] cell activity), and mice depleted of NK cells using anti-asialo-GM1. Each NK cell-deficient mouse strain exhibited a 10-fold-greater susceptibility to Shigella infection than immunocompetent mice. To test the protective effects of gamma interferon in vitro, survival of intracellular Shigella was examined in primary macrophages from wild-type mice, primary macrophages from gamma interferon-deficient mice, a macrophage cell line, and a fibroblast cell line. Following activation with gamma interferon, each cell type eradicated intracellular Shigella, while nonactivated macrophages fostered Shigella replication and nonactivated fibroblast cells fostered both Shigella replication and intercellular spread. Taken together, these data establish that NK cell-mediated gamma interferon is essential to resistance following primary Shigella infection.