Macrophage-specific responses to human- and animal-adapted tubercle bacilli reveal pathogen and host factors driving multinucleated cell formation

The Mycobacterium tuberculosis complex (MTBC) is a group of related pathogens that cause tuberculosis (TB) in mammals. MTBC species are distinguished by their ability to sustain in distinct host populations. While Mycobacterium bovis (Mbv) sustains transmission cycles in cattle and wild animals and causes zoonotic TB, M. tuberculosis (Mtb) affects human populations and seldom causes disease in cattle. The host and pathogen determinants underlying host tropism between MTBC species are still unknown. Macrophages are the main host cell that encounters mycobacteria upon initial infection, and we hypothesised that early interactions between the macrophage and mycobacteria influence species-specific disease outcome. To identify factors that contribute to host tropism, we analysed blood-derived primary human and bovine macrophages (hMϕ or bMϕ, respectively) infected with Mbv and Mtb. We show that Mbv and Mtb reside in different cellular compartments and differentially replicate in hMϕ whereas both Mbv and Mtb efficiently replicate in bMϕ. Specifically, we show that out of the four infection combinations, only the infection of bMϕ with Mbv promoted the formation of multinucleated giant cells (MNGCs), a hallmark of tuberculous granulomas. Mechanistically, we demonstrate that both MPB70 from Mbv and extracellular vesicles released by Mbv-infected bMϕ promote macrophage multinucleation. Importantly, we extended our in vitro studies to show that granulomas from Mbv-infected but not Mtb-infected cattle contained higher numbers of MNGCs. Our findings implicate MNGC formation in the contrasting pathology between Mtb and Mbv for the bovine host and identify MPB70 from Mbv and extracellular vesicles from bMϕ as mediators of this process.     

Measurement of the Infection and Dissemination of Bluetongue Virus in Culicoides Biting Midges Using a Semi-Quantitative RT-PCR Assay and Isolation of Infectious Virus

Background

Culicoides biting midges (Diptera: Ceratopogonidae) are the biological vectors of globally significant arboviruses of livestock including bluetongue virus (BTV), African horse sickness virus (AHSV) and the recently emerging Schmallenberg virus (SBV). From 2006–2009 outbreaks of BTV in northern Europe inflicted major disruption and economic losses to farmers and several attempts were made to implicate Palaearctic Culicoides species as vectors. Results from these studies were difficult to interpret as they used semi-quantitative RT-PCR (sqPCR) assays as the major diagnostic tool, a technique that had not been validated for use in this role. In this study we validate the use of these assays by carrying out time-series detection of BTV RNA in two colony species of Culicoides and compare the results with the more traditional isolation of infectious BTV on cell culture.

Methodology/Principal Findings

A BTV serotype 1 strain mixed with horse blood was fed to several hundred individuals of Culicoides sonorensis (Wirth & Jones) and C. nubeculosus (Mg.) using a membrane-based assay and replete individuals were then incubated at 25°C. At daily intervals 25 Culicoides of each species were removed from incubation, homogenised and BTV quantified in each individual using sqPCR (C_q values) and virus isolation on a KC-C. sonorensis embryonic cell line, followed by antigen enzyme-linked immunosorbent assay (ELISA). In addition, comparisons were also drawn between the results obtained with whole C. sonorensis and with individually dissected individuals to determine the level of BTV dissemination.

Conclusions/Significance

C_q values generated from time-series infection experiments in both C. sonorensis and C. nubeculosus confirmed previous studies that relied upon the isolation and detection of infectious BTV. Implications on the testing of field-collected Culicoides as potential virus vectors by PCR assays and the use of such assays as front-line tools for use in diagnostic laboratories in this role are discussed.     

Phytoextraction of high value elements and contaminants from mining and mineral wastes: opportunities and limitations

Plant and Soil - Phytoextraction is an in situ technique that can be applied to minerals and mining wastes using hyperaccumulator plants to purposely bio-concentrate high levels of metals or...     

Involvement of Schizosaccharomyces pombe rrp1+ and rrp2+ in the Srs2- and Swi5/Sfr1-dependent pathway in response to DNA damage and replication inhibition

Previously we identified Rrp1 and Rrp2 as two proteins required for the Sfr1/Swi5-dependent branch of homologous recombination (HR) in Schizosaccharomyces pombe. Here we use a yeast two-hybrid approach to demonstrate that Rrp1 and Rrp2 can interact with each other and with Swi5, an HR mediator protein. Rrp1 and Rrp2 form co-localizing methyl methanesulphonate–induced foci in nuclei, further suggesting they function as a complex. To place the Rrp1/2 proteins more accurately within HR sub-pathways, we carried out extensive epistasis analysis between mutants defining Rrp1/2, Rad51 (recombinase), Swi5 and Rad57 (HR-mediators) plus the anti-recombinogenic helicases Srs2 and Rqh1. We confirm that Rrp1 and Rrp2 act together with Srs2 and Swi5 and independently of Rad57 and show that Rqh1 also acts independently of Rrp1/2. Mutants devoid of Srs2 are characterized by elevated recombination frequency with a concomitant increase in the percentage of conversion-type recombinants. Strains devoid of Rrp1 or Rrp2 did not show a change in HR frequency, but the number of conversion-type recombinants was increased, suggesting a possible function for Rrp1/2 with Srs2 in counteracting Rad51 activity. Our data allow us to propose a model placing Rrp1 and Rrp2 functioning together with Swi5 and Srs2 in a synthesis-dependent strand annealing HR repair pathway.     

Electrotactile stimulation on the tongue: Intensity perception,discrimination, and cross-modality estimation

Due to its high sensitivity and conductivity, electrotactile stimulation (ETS) on the tongue has proven to be a useful and technically convenient tool to substitute and/or augment sensory capabilities. However, most of its applications have only provided spatial attributes and little is known about (a) the ability of the tongue's sensory system to process electrical stimuli of varying magnitudes and (b) how modulation of ETS intensity affects subjects’ ability to decode stimulus intensity. We addressed these questions by quantifying: (1) the magnitude of the dynamic range (DR; maximal comfortable intensity/perception threshold) and its sensitivity to prolonged exposure; (2) subjects’ ability to perceive intensity changes; and (3) subjects’ ability to associate intensity with angular excursions of a protractor's handle. We found that the average DR (17 dB) was generally large in comparison with other tactile loci and of a relatively constant magnitude among subjects, even after prolonged exposure, despite a slight but significant upward drift (p < 0.001). Additionally, our results showed that as stimulus intensity increased, subjects’ ability to discriminate ETS stimuli of different intensities improved (p < 0.05) while estimation accuracy, in general, slightly decreased (increasing underestimation). These results suggest that higher ETS intensity may increase recruitment of rapidly adapting mechanoreceptor fibers, as these are specialized for coding stimulus differences rather than absolute intensities. Furthermore, our study revealed that the tongue's sensory system can effectively convey electrical stimuli despite minimal practice and when information transfer is limited by memory and DR drift.     

Hyperaccumulators of metal and metalloid trace elements: Facts and fiction

Background

Plants that accumulate metal and metalloid trace elements to extraordinarily high concentrations in their living biomass have inspired much research worldwide during the last decades. Hyperaccumulators have been recorded and experimentally confirmed for elements such as nickel, zinc, cadmium, manganese, arsenic and selenium. However, to date, hyperaccumulation of lead, copper, cobalt, chromium and thallium remain largely unconfirmed. Recent uses of the term in relation to rare-earth elements require critical evaluation.

Scope

Since the mid-1970s the term ‘hyperaccumulator’ has been used millions of times by thousands of people, with varying degrees of precision, aptness and understanding that have not always corresponded with the views of the originators of the terminology and of the present authors. There is therefore a need to clarify the circumstances in which the term ‘hyperaccumulator’ is appropriate and to set out the conditions that should be met when the terms are used. We outline here the main considerations for establishing metal or metalloid hyperaccumulation status of plants, (re)define some of the terminology and note potential pitfalls.

Conclusions

Unambiguous communication will require the international scientific community to adopt standard terminology and methods for confirming the reliability of analytical data in relation to metal and metalloid hyperaccumulators.     

Detection of Proton Movement Directly across Viral Membranes To Identify Novel Influenza Virus M2 Inhibitors

The influenza virus M2 protein is a well-validated yet underexploited proton-selective ion channel essential for influenza virus infectivity. Because M2 is a toxic viral ion channel, existing M2 inhibitors have been discovered through live virus inhibition or medicinal chemistry rather than M2-targeted high-throughput screening (HTS), and direct measurement of its activity has been limited to live cells or reconstituted lipid bilayers. Here, we describe a cell-free ion channel assay in which M2 ion channels are incorporated into virus-like particles (VLPs) and proton conductance is measured directly across the viral lipid bilayer, detecting changes in membrane potential, ion permeability, and ion channel function. Using this approach in high-throughput screening of over 100,000 compounds, we identified 19 M2-specific inhibitors, including two novel chemical scaffolds that inhibit both M2 function and influenza virus infectivity. Counterscreening for nonspecific disruption of viral bilayer ion permeability also identified a broad-spectrum antiviral compound that acts by disrupting the integrity of the viral membrane. In addition to its application to M2 and potentially other ion channels, this technology enables direct measurement of the electrochemical and biophysical characteristics of viral membranes.     

Multiple C-Terminal Tails within a Single E. coli SSB Homotetramer Coordinate DNA Replication and Repair

Escherichia coli single-stranded DNA binding protein (SSB) plays essential roles in DNA replication, recombination and repair. SSB functions as a homotetramer with each subunit possessing a DNA binding domain (OB-fold) and an intrinsically disordered C-terminus, of which the last nine amino acids provide the site for interaction with at least a dozen other proteins that function in DNA metabolism. To examine how many C-termini are needed for SSB function, we engineered covalently linked forms of SSB that possess only one or two C-termini within a four-OB-fold “tetramer”. Whereas E. coli expressing SSB with only two tails can survive, expression of a single-tailed SSB is dominant lethal. E. coli expressing only the two-tailed SSB recovers faster from exposure to DNA damaging agents but accumulates more mutations. A single-tailed SSB shows defects in coupled leading and lagging strand DNA replication and does not support replication restart in vitro. These deficiencies in vitro provide a plausible explanation for the lethality observed in vivo. These results indicate that a single SSB tetramer must interact simultaneously with multiple protein partners during some essential roles in genome maintenance.