Improved specificity for detection of Mycobacterium bovis in fresh tissues using IS6110 real-time PCR

<p>Abstract</p> <p>Background</p> <p>Culture of <it>M. bovis </it>from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the USA. Detection of <it>M. bovis </it>by PCR in tissue homogenates may provide a simple rapid method to complement bacterial culture. A significant impediment to PCR based assays on tissue homogenates is specificity since mycobacteria other than <it>M. bovis </it>may be associated with the tissues.</p> <p>Results</p> <p>Previously published IS<it>6110 </it>based PCR diagnostic assays, along with one developed in house, were tested against environmental mycobacteria commonly isolated from diagnostic tissues submitted to the National Veterinary Services Laboratory. A real-time PCR assay was developed (IS6110_T) that had increased specificity over other IS<it>6110 </it>based assays. Of the 13 non-tuberculous mycobacteria tested with IS6110_T only <it>M. wolinskyi </it>was positive. Thirty <it>M. bovis </it>infected tissue homogenates and 18 control tissues were used to evaluate the potential for the assay as a diagnostic test. In this small sample, IS6110_T detected 20/30 samples from <it>M. bovis </it>infected animals and 0/18 control tissues.</p> <p>Conclusions</p> <p>The IS6110_T assay provides a PCR based assay system that is compatible with current diagnostic protocols for the detection of <it>M. bovis </it>in the USA and compliments current testing strategies.</p

3D discomfort from vertical and torsional disparities in natural images

The two major aspects of camera misalignment that cause visual discomfort when viewing images on a 3D display are vertical and torsional disparities. While vertical disparities are uniform throughout the image, torsional rotations introduce a range of disparities that depend on the location in the image. The goal of this study was to determine the discomfort ranges for the kinds of natural image that people are likely to take with 3D cameras rather than the artificial line and dot stimuli typically used for laboratory studies. We therefore assessed visual discomfort on a five-point scale from ‘none’ to ‘severe’ for artificial misalignment disparities applied to a set of full-resolution images of indoor scenes. For viewing times of 2 s, discomfort ratings for vertical disparity in both 2D and 3D images rose rapidly toward the discomfort level of 4 (‘severe’) by about 60 arcmin of vertical disparity. Discomfort ratings for torsional disparity in the same image rose only gradually, reaching only the discomfort level of 3 (‘strong’) by about 50 deg of torsional disparity. These data were modeled with a second-order hyperbolic compression function incorporating a term for the basic discomfort of the 3D display in the absence of any misalignments through a Minkowski norm. These fits showed that, at a criterion discomfort level of 2 (‘moderate’), acceptable levels of vertical disparity were about 15 arcmin. The corresponding values for the torsional disparity were about 30 deg of relative orientation

Shotgun ion mobility mass spectrometry sequencing of heparan sulfate saccharides

Despite evident regulatory roles of heparan sulfate (HS) saccharides in numerous biological processes, definitive information on the bioactive sequences of these polymers is lacking, with only a handful of natural structures sequenced to date. Here, we develop a “Shotgun” Ion Mobility Mass Spectrometry Sequencing (SIMMS2) method in which intact HS saccharides are dissociated in an ion mobility mass spectrometer and collision cross section values of fragments measured. Matching of data for intact and fragment ions against known values for 36 fully defined HS saccharide structures (from di- to decasaccharides) permits unambiguous sequence determination of validated standards and unknown natural saccharides, notably including variants with 3O-sulfate groups. SIMMS2 analysis of two fibroblast growth factor-inhibiting hexasaccharides identified from a HS oligosaccharide library screen demonstrates that the approach allows elucidation of structure-activity relationships. SIMMS2 thus overcomes the bottleneck for decoding the informational content of functional HS motifs which is crucial for their future biomedical exploitation

Temporal Response Properties of the Auditory Nerve in Implanted Children with Auditory Neuropathy Spectrum Disorder and Implanted Children with Sensorineural Hearing Loss

This study aimed to 1) characterize temporal response properties of the auditory nerve in implanted children with auditory neuropathy spectrum disorder (ANSD); and 2) compare results recorded in implanted children with ANSD with those measured in implanted children with sensorineural hearing loss (SNHL)

Mycobacterium bovis: A model pathogen at the interface of livestock, wildlife, and humans

Complex and dynamic interactions involving domestic animals, wildlife, and humans create environments favorable to the emergence of new diseases, or reemergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals, and sometimes humans, exist in a range of countries and wild animal populations. Free-ranging populations of white-tailed deer in the US, brushtail possum in New Zealand, badger in the Republic of Ireland and the United Kingdom, and wild boar in Spain exemplify established reservoirs of M. bovis. Establishment of these reservoirs is the result of factors such as spillover from livestock, translocation of wildlife, supplemental feeding of wildlife, and wildlife population densities beyond normal habitat carrying capacities. As many countries attempt to eradicate M. bovis from livestock, efforts are impeded by spillback from wildlife reservoirs. It will not be possible to eradicate this important zoonosis from livestock unless transmission between wildlife and domestic animals is halted. Such an endeavor will require a collaborative effort between agricultural, wildlife, environmental, and political interests.Peer Reviewe

PROSAC: A Submillimeter Array Survey of Low-Mass Protostars. I. Overview of Program: Envelopes, Disks, Outflows and Hot Cores

This paper presents a large spectral line and continuum survey of 8 deeply embedded, low-mass protostellar cores using the Submillimeter Array. Each source was observed in high excitation lines of some of the most common molecular species, CO, HCO+, CS, SO, H2CO, CH3OH and SiO. Line emission from 11 species originating from warm and dense gas have been imaged at high angular resolution (1-3"; typically 200-600 AU) together with continuum emission at 230 GHz (1.3 mm) and 345 GHz (0.8 mm). Compact continuum emission is observed for all sources which likely originates in marginally optically thick circumstellar disks, with typical lower limits to their masses of 0.1 M_sun (1-10% of the masses of their envelopes) and having a dust opacity law with beta approximately 1. Prominent outflows are present in CO 2-1 observations in all sources: the most diffuse outflows are found in the sources with the lowest ratios of disk-to-envelope mass, and it is suggested that these sources are in a phase where accretion of matter from the envelope has almost finished and the remainder of the envelope material is being dispersed by the outflows. Other characteristic dynamical signatures are found with inverse P Cygni profiles indicative of infalling motions seen in the 13CO 2-1 lines toward NGC1333-IRAS4A and -IRAS4B. Outflow-induced shocks are present on all scales in the protostellar environments and are most clearly traced by the emission of CH3OH in NGC1333-IRAS4A and -IRAS4B. These observations suggest that the emission of CH3OH and H2CO from these proposed "hot corinos" are related to the shocks caused by the protostellar outflows. Only one source, NGC1333-IRAS2A, has evidence for hot, compact CH3OH emission coincident with the embedded protostar.Comment: Accepted for publication in ApJ (52 pages; 9 figures). Abstract abridge

Characterization of effector and memory T cell subsets in the immune response to bovine tuberculosis in cattle

Cultured IFN-γ ELISPOT assays are primarily a measure of central memory T cell (Tcm) responses with humans; however, this important subset of lymphocytes is poorly characterized in cattle. Vaccine-elicited cultured IFN-γ ELISPOT responses correlate with protection against bovine tuberculosis in cattle. However, whether this assay measures cattle Tcm responses or not is uncertain. The objective of the present study was to characterize the relative contribution of Tcm (CCR7+, CD62Lhi, CD45RO+), T effector memory (Tem, defined as: CCR7-, CD62Llow/int, CD45RO+), and T effector cells (CCR7-, CD62L-/low, CD45RO-), in the immune response to Mycobacterium bovis. Peripheral blood mononuclear cells (PBMC) from infected cattle were stimulated with a cocktail of M. bovis purified protein derivative, rTb10.4 and rAg85A for 13 days with periodic addition of fresh media and rIL-2. On day 13, cultured PBMC were re-stimulated with medium alone, rESAT-6:CFP10 or PPDb with fresh autologous adherent cells for antigen presentation. Cultured cells (13 days) or fresh PBMCs (ex vivo response) from the same calves were analyzed for IFN-γ production, proliferation, and CD4, CD45RO, CD62L, CD44, and CCR7 expression via flow cytometry after overnight stimulation. In response to mycobacterial antigens, ~75% of CD4+ IFN-γ+ cells in long-term cultures expressed a Tcm phenotype while less than 10% of the ex vivo response consisted of Tcm cells. Upon re-exposure to antigen, long-term cultured cells were highly proliferative, a distinctive characteristic of Tcm, and the predominant phenotype within the long-term cultures switched from Tcm to Tem. These findings suggest that proliferative responses of Tcm cells to some extent occurs simultaneously with reversion to effector phenotypes (mostly Tem). The present study characterizes Tcm cells of cattle and their participation in the response to M. bovis infection

Cmah-dystrophin deficient mdx mice display an accelerated cardiac phenotype that is improved following peptide-PMO exon skipping treatment

Duchenne muscular dystrophy (DMD) is caused by loss of dystrophin protein, leading to progressive muscle weakness and premature death due to respiratory and/or cardiac complications. Cardiac involvement is characterized by progressive dilated cardiomyopathy, decreased fractional shortening and metabolic dysfunction involving reduced metabolism of fatty acids—the major cardiac metabolic substrate. Several mouse models have been developed to study molecular and pathological consequences of dystrophin deficiency, but do not recapitulate all aspects of human disease pathology and exhibit a mild cardiac phenotype. Here we demonstrate that Cmah (cytidine monophosphate-sialic acid hydroxylase)-deficient mdx mice (Cmah−/−;mdx) have an accelerated cardiac phenotype compared to the established mdx model. Cmah−/−;mdx mice display earlier functional deterioration, specifically a reduction in right ventricle (RV) ejection fraction and stroke volume (SV) at 12 weeks of age and decreased left ventricle diastolic volume with subsequent reduced SV compared to mdx mice by 24 weeks. They further show earlier elevation of cardiac damage markers for fibrosis (Ctgf), oxidative damage (Nox4) and haemodynamic load (Nppa). Cardiac metabolic substrate requirement was assessed using hyperpolarized magnetic resonance spectroscopy indicating increased in vivo glycolytic flux in Cmah−/−;mdx mice. Early upregulation of mitochondrial genes (Ucp3 and Cpt1) and downregulation of key glycolytic genes (Pdk1, Pdk4, Ppara), also denote disturbed cardiac metabolism and shift towards glucose utilization in Cmah−/−;mdx mice. Moreover, we show long-term treatment with peptide-conjugated exon skipping antisense oligonucleotides (20-week regimen), resulted in 20% cardiac dystrophin protein restoration and significantly improved RV cardiac function. Therefore, Cmah−/−;mdx mice represent an appropriate model for evaluating cardiac benefit of novel DMD therapeutics