Development and evaluation of a diagnostic cytokine-release assay for Mycobacterium suricattae infection in meerkats (Suricata suricatta)

CITATION: Clarke, C., et al. 2017. Development and evaluation of a diagnostic cytokine-release assay for mycobacterium suricattae infection in meerkats (Suricata suricatta). BMC Veterinary Research, 13:2, doi:10.1186/s12917-016-0927-x.The original publication is available at http://bmcvetres.biomedcentral.comBackground: Sensitive diagnostic tools are necessary for the detection of Mycobacterium suricattae infection in meerkats (Suricata suricatta) in order to more clearly understand the epidemiology of tuberculosis and the ecological consequences of the disease in this species. We therefore aimed to develop a cytokine release assay to measure antigen-specific cell-mediated immune responses of meerkats. Results: Enzyme-linked immunosorbent assays (ELISAs) were evaluated for the detection of interferon-gamma (IFN-γ) and IFN-γ inducible protein 10 (IP-10) in meerkat plasma. An IP-10 ELISA was selected to measure the release of this cytokine in whole blood in response to Bovigam® PC-HP Stimulating Antigen, a commercial peptide pool of M. bovis antigens. Using this protocol, captive meerkats with no known M. suricattae exposure (n = 10) were tested and results were used to define a diagnostic cut off value (mean plus 2 standard deviations). This IP-10 release assay (IPRA) was then evaluated in free-living meerkats with known M. suricattae exposure, categorized as having either a low, moderate or high risk of infection with this pathogen. In each category, respectively, 24.7%, 27.3% and 82.4% of animals tested IPRA-positive. The odds of an animal testing positive was 14.0 times greater for animals with a high risk of M. suricattae infection compared to animals with a low risk. Conclusion: These results support the use of this assay as a measure of M. suricattae exposure in meerkat populations. Ongoing longitudinal studies aim to evaluate the value of the IPRA as a diagnostic test of M. suricattae infection in individual animals.http://bmcvetres.biomedcentral.com/articles/10.1186/s12917-016-0927-xPublisher's versio

High-Throughput Single-Cell Manipulation in Brain Tissue

The complexity of neurons and neuronal circuits in brain tissue requires the genetic manipulation, labeling, and tracking of single cells. However, current methods for manipulating cells in brain tissue are limited to either bulk techniques, lacking single-cell accuracy, or manual methods that provide single-cell accuracy but at significantly lower throughputs and repeatability. Here, we demonstrate high-throughput, efficient, reliable, and combinatorial delivery of multiple genetic vectors and reagents into targeted cells within the same tissue sample with single-cell accuracy. Our system automatically loads nanoliter-scale volumes of reagents into a micropipette from multiwell plates, targets and transfects single cells in brain tissues using a robust electroporation technique, and finally preps the micropipette by automated cleaning for repeating the transfection cycle. We demonstrate multi-colored labeling of adjacent cells, both in organotypic and acute slices, and transfection of plasmids encoding different protein isoforms into neurons within the same brain tissue for analysis of their effects on linear dendritic spine density. Our platform could also be used to rapidly deliver, both ex vivo and in vivo, a variety of genetic vectors, including optogenetic and cell-type specific agents, as well as fast-acting reagents such as labeling dyes, calcium sensors, and voltage sensors to manipulate and track neuronal circuit activity at single-cell resolution

More GABA, less distraction: a neurochemical predictor of motor decision speed

People vary markedly in the efficiency with which they can resolve competitive action decisions, even simple ones such as shifting gaze to one stimulus rather than another. We found that an individual's ability to rapidly resolve such competition is predicted by the concentration of GABA, the main inhibitory neurotransmitter, in a region of frontal cortex that is relevant for eye movements, but not in a control region (occipital cortex)

Rescuing Loading Induced Bone Formation at Senescence

The increasing incidence of osteoporosis worldwide requires anabolic treatments that are safe, effective, and, critically, inexpensive given the prevailing overburdened health care systems. While vigorous skeletal loading is anabolic and holds promise, deficits in mechanotransduction accrued with age markedly diminish the efficacy of readily complied, exercise-based strategies to combat osteoporosis in the elderly. Our approach to explore and counteract these age-related deficits was guided by cellular signaling patterns across hierarchical scales and by the insight that cell responses initiated during transient, rare events hold potential to exert high-fidelity control over temporally and spatially distant tissue adaptation. Here, we present an agent-based model of real-time Ca2+/NFAT signaling amongst bone cells that fully described periosteal bone formation induced by a wide variety of loading stimuli in young and aged animals. The model predicted age-related pathway alterations underlying the diminished bone formation at senescence, and hence identified critical deficits that were promising targets for therapy. Based upon model predictions, we implemented an in vivo intervention and show for the first time that supplementing mechanical stimuli with low-dose Cyclosporin A can completely rescue loading induced bone formation in the senescent skeleton. These pre-clinical data provide the rationale to consider this approved pharmaceutical alongside mild physical exercise as an inexpensive, yet potent therapy to augment bone mass in the elderly. Our analyses suggested that real-time cellular signaling strongly influences downstream bone adaptation to mechanical stimuli, and quantification of these otherwise inaccessible, transient events in silico yielded a novel intervention with clinical potential

Computational subunits in thin dendrites of pyramidal cells

The thin basal and oblique dendrites of cortical pyramidal neurons receive most of the cells' synaptic input, but their integrative properties remain uncertain. Previous studies have most often reported global linear or sublinear summation. An alternative view,supported by biophysical modeling studies, holds that thin dendrites provide a layer of independent computational 'subunits' that sigmoidally modulate their inputs prior to global summation. To distinguish these possibilities, we combined confocal imaging and dual-site focal synaptic stimulation of identified thin dendrites in rat neocortical pyramidal neurons. We found that nearby inputs on the same branch summed sigmoidally, whereas widely separated inputs or inputs to different branches summed linearly. This strong spatial compartmentalization effect is incompatible with a global summation rule and provides the first experimental support for a two-layer "neural network" [The quotes are left in to refer to a standard architecture in the artificial neural network field] model of pyramidal neuron thin-branch integration. Our findings could have important implications for the computing and memory-related functions of cortical tissue

Pseudoproline-containing analogues of morphiceptin and endomorphin-2: Evidence for a cis Tyr-Pro amide bond in the bioactive conformation

Analogues of the opioid peptides [D-Phe(3)]morphiceptin (H-Tyr-Pro-D-Phe-Pro-NH2) and endomorphin-2 (H-Tyr-Pro-Phe-Phe-NH2) containing the pseudoproline (Psi Pro) (4R)-thiazolidine-4-carboxylic acid (CyS[Psi (R1,R2) pro]) or (4S)-oxazolidine-4-carboxylic acid (Ser[Psi (R1,R2)pro]) in place of Pro(2) were synthesized. The pseudoproline ring in these compounds was either unsubstituted (R-1, R-2 = H) or dimethylated (R-1, R-2 = CH3) at the 2-C position. 2-C-dimethylated pseudoprolines are known to be quantitative or nearly quantitative inducers of the Cis conformation around the Xaa(i-1)-Xaa(i)[Psi (CH3,CH3)pro] imide bond. All dihydropseudoproline-containing analogues (R1, R2 = H) showed goody opioid agonist potency in the guinea pig ileum (GPI) assay, high mu receptor binding affinity in the rat brain membrane binding assay, and, like their parent peptides, excellent mu receptor binding selectivity. H-1 NMR spectroscopic analysis of the Cys[Psi (H,H)pro](2)- and Ser[Psi (H,H)pro](2)-containing analogues in DMSO-d(6) revealed that they existed in a conformational equilibrium around the Tyr-Xaa[Psi (H,H)pro] peptide bond with cis/trans ratios of 40:60 and 45:55, respectively. The dimethylated thiazolidine- and oxazolidine-containing [D-Phe3]morphiceptin- and endomorphin-2 analogues (R-1, R-2 = CH3) all retained full mu agonist potency in the GPI assay and displayed mu receptor binding affinities in the nanomolar range and high mu receptor selectivity. As expected, no conformers of the latter analogues with a trans conformation around the Tyr-Xaa[Psi (CH3,CH3)pro] imide bond were detected by H-1 NMR spectral analysis, indicating that in these compounds the cis conformation is highly predominant (> 98%). These results represent the most direct evidence obtained so far to indicate that morphiceptin and endomorphin-2 have the cis conformation around the Tyr-Pro peptide bond in their bioactive conformations