First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank,Palestinian Territories

Background

Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area.

Methodology/principal findings

A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample.

Significance

Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.     

Expression of vascular antigens by bone cells during bone regeneration in a membranous bone distraction system

An in vivo system of membranous bone formation during distraction has been investigated in order to follow cells that express vascular markers with the objective of understanding the neovascularization process. Concomitantly, sustained proliferation of preskeletal cells was achieved through the application of mechanical force. New capillaries and leading edges that arose by angiogenesis from the periosteal and mucosal surfaces and invaded the central zone of the regenerating distraction tissue temporally preceded the growth of delicate woven bone trabeculae from both edges of the cut bone. Concentrically arranged 'onion-like' configurations were abundant in paracentral zones and in association with mesenchymal condensations, suggesting their de novo formation in situ. Vascular specific markers, the angiopoietin receptor Tie-2 and factor VIII-related antigen (FVIIIrAg), were localized immunohistochemically in order to follow cells of vascular origin. Endothelial cells of the new capillaries, centrally located cells of the concentric configurations, pericytes, and most of the adjacent polygonal mesenchymal cells stained positively with specific antibodies to both antigens. Moreover, preosteoblasts and osteoblasts that lie adjacent to or already embedded in the osteiod of the newly formed trabeculae were also FVIIIrAg and Tie-2 immunopositive. As the source of the bone-forming cells in regenerating tissue during distraction is not yet fully understood, this observation might support the possibility of their vascular origin.     

T cell derived HIV-1 is present in the CSF in the face of suppressive antiretroviral therapy

HIV cerebrospinal fluid (CSF) escape, where HIV is suppressed in blood but detectable in CSF, occurs when HIV persists in the CNS despite antiretroviral therapy (ART). To determine the virus producing cell type and whether lowered CSF ART levels are responsible for CSF escape, we collected blood and CSF from 156 neurosymptomatic participants from Durban, South Africa. We observed that 28% of participants with an undetectable HIV blood viral load showed CSF escape. We detected host cell surface markers on the HIV envelope to determine the cellular source of HIV in participants on the first line regimen of efavirenz, emtricitabine, and tenofovir. We confirmed CD26 as a marker which could differentiate between T cells and macrophages and microglia, and quantified CD26 levels on the virion surface, comparing the result to virus from in vitro infected T cells or macrophages. The measured CD26 level was consistent with the presence of T cell produced virus. We found no significant differences in ART concentrations between CSF escape and fully suppressed individuals in CSF or blood, and did not observe a clear association with drug resistance mutations in CSF virus which would allow HIV to replicate. Hence, CSF HIV in the face of ART may at least partly originate in CD4+ T cell populations.     

Identification Procedure in a model of single fibre action potential – Part I: Estimation of fibre diameter and radial distance

The Dimitrov–Dimitrova (D–D) model generates a single fibre action potential (SFAP) as the convolution of an excitation function and a filter impulse function. We propose a method to estimate the parameters involved in these functions from a SFAP waveform (inverse problem) and call it Identification Procedure. The Identification Procedure comprises two parts. The present paper, Part I, is centred on the estimation of the radial distance, r, and the fibre diameter, d, of a given SFAP. To this end we develop a technique which we call the candidate pair method (CP-method), and we test it on fibrillation potentials (FPs) as experimental data. We found that the D–D model cannot synthesize all the SFAP waveforms observed in the experimental recordings, but in the cases where it can, the CP-method then provides the values of r and d that are more likely to have synthesized the SFAP. Having a method that provides information about the fibre diameter straight from a SFAP waveform is very desirable as this parameter has clinical and physiological relevance. Moreover, the CP-method plays a major role in the Identification Procedure that is carried out in Part II.     

Generation of a fluorescently labeled endogenous protein library in living human cells

We present a protocol to tag proteins expressed from their endogenous chromosomal locations in individual mammalian cells using central dogma tagging. The protocol can be used to build libraries of cell clones, each expressing one endogenous protein tagged with a fluorophore such as the yellow fluorescent protein. Each round of library generation produces 100-200 cell clones and takes about 1 month. The protocol integrates procedures for high-throughput single-cell cloning using flow cytometry, high-throughput cDNA generation and 3' rapid amplification of cDNA ends, semi-automatic protein localization screening using fluorescent microscopy and freezing cells in 96-well format.     

Identification procedure in a model of single fibre action potential – Part II: Global approach and experimental results

The present paper describes a global procedure for estimating all the synthesis parameters that generate a single fibre action potential (SFAP) in the Dimitrov–Dimitrova (D–D) convolutional model. We call this inverse problem Identification Procedure, and it is presented in two parts, this paper being the second. The procedure incorporates the candidate pair (CP) method developed in Part I, which provides the values of radial distance r and fibre diameter d of the simulated SFAP that best matches a potential under study. The CP-method required prior knowledge of all the excitation parameters. However, since the Identification Procedure makes no assumption about the excitation, multiple combinations of the synthesis parameters result in very similar SFAPs whose shape is close the signal under study. Analysis of the possible combinations reveals that r and d can be modelled as two jointly Gaussian random variables. The interest of the Identification Procedure is that, for a certain SFAP, it provides estimates of r and d, along with estimates of different parameters that determine the IAP waveform. Moreover, the procedure is able to determine the degree of error that accompanies the estimation of r and d.     

Mitochondrial Phylogeography Illuminates the Origin of the Extinct Caspian Tiger and Its Relationship to the Amur Tiger

The Caspian tiger (Panthera tigris virgata) flourished in Central Asian riverine forest systems in a range disjunct from that of other tigers, but was driven to extinction in 1970 prior to a modern molecular evaluation. For over a century naturalists puzzled over the taxonomic validity, placement, and biogeographic origin of this enigmatic animal. Using ancient-DNA (aDNA) methodology, we generated composite mtDNA haplotypes from twenty wild Caspian tigers from throughout their historic range sampled from museum collections. We found that Caspian tigers carry a major mtDNA haplotype differing by only a single nucleotide from the monomorphic haplotype found across all contemporary Amur tigers (P. t. altaica). Phylogeographic analysis with extant tiger subspecies suggests that less than 10,000 years ago the Caspian/Amur tiger ancestor colonized Central Asia via the Gansu Corridor (Silk Road) from eastern China then subsequently traversed Siberia eastward to establish the Amur tiger in the Russian Far East. The conservation implications of these findings are far reaching, as the observed genetic depletion characteristic of modern Amur tigers likely reflects these founder migrations and therefore predates human influence. Also, due to their evolutionary propinquity, living Amur tigers offer an appropriate genetic source should reintroductions to the former range of the Caspian tiger be implemented.     

High yellow pigments production by root culture of <Emphasis Type=Italic>Carthamus tinctorius</Emphasis> and its release in medium under gas oil treatment

In this study, the ability of safflower-isolated root cultures to produce yellow pigments was tested. Initially, the growth of isolated roots in static liquid medium was evaluated with different volumes of culture medium. A volume of 6 ml of medium per flask of 250 ml gave the best growth performance and, in this condition of culture, production of pigments from isolated roots treated or not by light has been determined by spectrophotometry (321 and 400 nm). Under these conditions, the production of yellow pigments amounted to 13.18 mg g⁻¹ fresh weight and the light stimulated the synthesis of these pigments by isolated roots. Total yellow pigments of 24.12, 38.91 and 46.38 mg g⁻¹ fresh weight was produced by the roots treated with 9, 13.5 and 18% (v/v) gas oil, respectively, representing high values of production. The pigments were released in large quantities in the medium. The increased synthesis of pigments as a result of gas oil treatment was accompanied by a reduction of the peroxidase activity of roots. Given the high production of yellow pigments, systems of isolated root culture could be considered for the study of a larger scale production of safflower pigments widely used for various industrial purposes.     

Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved β-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the β-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.