Vaccination with Recombinant Microneme Proteins Confers Protection against Experimental Toxoplasmosis in Mice

Toxoplasmosis, a zoonotic disease caused by Toxoplasma gondii, is an important public health problem and veterinary concern. Although there is no vaccine for human toxoplasmosis, many attempts have been made to develop one. Promising vaccine candidates utilize proteins, or their genes, from microneme organelle of T. gondii that are involved in the initial stages of host cell invasion by the parasite. In the present study, we used different recombinant microneme proteins (TgMIC1, TgMIC4, or TgMIC6) or combinations of these proteins (TgMIC1-4 and TgMIC1-4-6) to evaluate the immune response and protection against experimental toxoplasmosis in C57BL/6 mice. Vaccination with recombinant TgMIC1, TgMIC4, or TgMIC6 alone conferred partial protection, as demonstrated by reduced brain cyst burden and mortality rates after challenge. Immunization with TgMIC1-4 or TgMIC1-4-6 vaccines provided the most effective protection, since 70% and 80% of mice, respectively, survived to the acute phase of infection. In addition, these vaccinated mice, in comparison to non-vaccinated ones, showed reduced parasite burden by 59% and 68%, respectively. The protective effect was related to the cellular and humoral immune responses induced by vaccination and included the release of Th1 cytokines IFN-γ and IL-12, antigen-stimulated spleen cell proliferation, and production of antigen-specific serum antibodies. Our results demonstrate that microneme proteins are potential vaccines against T. gondii, since their inoculation prevents or decreases the deleterious effects of the infection

VgrG and PAAR Proteins Define Distinct Versions of a Functional Type VI Secretion System

The Type VI secretion system (T6SS) is widespread among bacterial pathogens and acts as an effective weapon against competitor bacteria and eukaryotic hosts by delivering toxic effector proteins directly into target cells. The T6SS utilises a bacteriophage-like contractile machinery to expel a puncturing device based on a tube of Hcp topped with a VgrG spike, which can be extended by a final tip from a PAAR domain-containing protein. Effector proteins are believed to be delivered by specifically associating with particular Hcp, VgrG or PAAR proteins, either covalently ('specialised') or non-covalently ('cargo' effectors). Here we used the T6SS of the opportunistic pathogen Serratia marcescens, together with integratecd genetic, proteomic and biochemical approaches, to elucidate the role of specific VgrG and PAAR homologues in T6SS function and effector specificity, revealing new aspects and unexpected subtleties in effector delivery by the T6SS. We identified effectors, both cargo and specialised, absolutely dependent on a particular VgrG for delivery to target cells, and discovered that other cargo effectors can show a preference for a particular VgrG. The presence of at least one PAAR protein was found to be essential for T6SS function, consistent with designation as a 'core' T6SS component. We showed that specific VgrG-PAAR combinations are required to assemble a functional T6SS and that the three distinct VgrG-PAAR assemblies in S. marcescens exhibit distinct effector specificity and efficiency. Unexpectedly, we discovered that two different PAAR-containing Rhs proteins can functionally pair with the same VgrG protein. Showing that accessory EagR proteins are involved in these interactions, native VgrG-Rhs-EagR complexes were isolated and specific interactions between EagR and cognate Rhs proteins identified. This study defines an essential yet flexible role for PAAR proteins in the T6SS and highlights the existence of distinct versions of the machinery with differential effector specificity and efficiency of target cell delivery

MicroRNAs hsa-miR-99b, hsa-miR-330, hsa-miR-126 and hsa-miR-30c: Potential Diagnostic Biomarkers in Natural Killer (NK) Cells of Patients with Chronic Fatigue Syndrome (CFS)/ Myalgic Encephalomyelitis (ME)

Chronic Fatigue Syndrome (CFS/ME) is a complex multisystem disease of unknown aetiology which causes debilitating symptoms in up to 1% of the global population. Although a large cohort of genes have been shown to exhibit altered expression in CFS/ME patients, it is currently unknown whether microRNA (miRNA) molecules which regulate gene translation contribute to disease pathogenesis. We hypothesized that changes in microRNA expression in patient leukocytes contribute to CFS/ME pathology, and may therefore represent useful diagnostic biomarkers that can be detected in the peripheral blood of CFS/ME patients.miRNA expression in peripheral blood mononuclear cells (PBMC) from CFS/ME patients and healthy controls was analysed using the Ambion Bioarray V1. miRNA demonstrating differential expression were validated by qRT-PCR and then replicated in fractionated blood leukocyte subsets from an independent patient cohort. The CFS/ME associated miRNA identified by these experiments were then transfected into primary NK cells and gene expression analyses conducted to identify their gene targets.Microarray analysis identified differential expression of 34 miRNA, all of which were up-regulated. Four of the 34 miRNA had confirmed expression changes by qRT-PCR. Fractionating PBMC samples by cell type from an independent patient cohort identified changes in miRNA expression in NK-cells, B-cells and monocytes with the most significant abnormalities occurring in NK cells. Transfecting primary NK cells with hsa-miR-99b or hsa-miR-330-3p, resulted in gene expression changes consistent with NK cell activation but diminished cytotoxicity, suggesting that defective NK cell function contributes to CFS/ME pathology.This study demonstrates altered microRNA expression in the peripheral blood mononuclear cells of CFS/ME patients, which are potential diagnostic biomarkers. The greatest degree of miRNA deregulation was identified in NK cells with targets consistent with cellular activation and altered effector function

Current and prospective pharmacological targets in relation to antimigraine action

Migraine is a recurrent incapacitating neurovascular disorder characterized by unilateral and throbbing headaches associated with photophobia, phonophobia, nausea, and vomiting. Current specific drugs used in the acute treatment of migraine interact with vascular receptors, a fact that has raised concerns about their cardiovascular safety. In the past, α-adrenoceptor agonists (ergotamine, dihydroergotamine, isometheptene) were used. The last two decades have witnessed the advent of 5-HT1B/1D receptor agonists (sumatriptan and second-generation triptans), which have a well-established efficacy in the acute treatment of migraine. Moreover, current prophylactic treatments of migraine include 5-HT2 receptor antagonists, Ca2+ channel blockers, and β-adrenoceptor antagonists. Despite the progress in migraine research and in view of its complex etiology, this disease still remains underdiagnosed, and available therapies are underused. In this review, we have discussed pharmacological targets in migraine, with special emphasis on compounds acting on 5-HT (5-HT1-7), adrenergic (α1, α2, and β), calcitonin gene-related peptide (CGRP 1 and CGRP2), adenosine (A1, A2, and A3), glutamate (NMDA, AMPA, kainate, and metabotropic), dopamine, endothelin, and female hormone (estrogen and progesterone) receptors. In addition, we have considered some other targets, including gamma-aminobutyric acid, angiotensin, bradykinin, histamine, and ionotropic receptors, in relation to antimigraine therapy. Finally, the cardiovascular safety of current and prospective antimigraine therapies is touched upon

Constrained pre-equalization accounting for multi-path fading emulated using large RC networks: applications to wireless and photonics communications

Multi-path propagation is modelled assuming a multi-layer RC network with randomly allocated resistors and capacitors to represent the transmission medium. Due to frequency-selective attenuation, the waveforms associated with each propagation path incur path-dependent distortion. A pre-equalization procedure that takes into account the capabilities of the transmission source as well as the transmission properties of the medium is developed. The problem is cast within a Mixed Integer Linear Programming optimization framework that uses the developed nominal RC network model, with the excitation waveform customized to optimize signal fidelity from the transmitter to the receiver. The objective is to match a Gaussian pulse input accounting for frequency regions where there would be pronounced fading. Simulations are carried out with different network realizations in order to evaluate the sensitivity of the solution with respect to changes in the transmission medium mimicking the multi-path propagation. The proposed approach is of relevance where equalization techniques are difficult to implement. Applications are discussed within the context of emergent communication modalities across the EM spectrum such as light percolation as well as emergent indoor communications assuming various modulation protocols or UWB schemes as well as within the context of space division multiplexing

Adenylyl Cyclase α and cAMP Signaling Mediate Plasmodium Sporozoite Apical Regulated Exocytosis and Hepatocyte Infection

Malaria starts with the infection of the liver of the host by Plasmodium sporozoites, the parasite form transmitted by infected mosquitoes. Sporozoites migrate through several hepatocytes by breaching their plasma membranes before finally infecting one with the formation of an internalization vacuole. Migration through host cells induces apical regulated exocytosis in sporozoites. Here we show that apical regulated exocytosis is induced by increases in cAMP in sporozoites of rodent (P. yoelii and P. berghei) and human (P. falciparum) Plasmodium species. We have generated P. berghei parasites deficient in adenylyl cyclase α (ACα), a gene containing regions with high homology to adenylyl cyclases. PbACα-deficient sporozoites do not exocytose in response to migration through host cells and present more than 50% impaired hepatocyte infectivity in vivo. These effects are specific to ACα, as re-introduction of ACα in deficient parasites resulted in complete recovery of exocytosis and infection. Our findings indicate that ACα and increases in cAMP levels are required for sporozoite apical regulated exocytosis, which is involved in sporozoite infection of hepatocytes

Understanding Actions of Others: The Electrodynamics of the Left and Right Hemispheres. A High-Density EEG Neuroimaging Study

Background: When we observe an individual performing a motor act (e.g. grasping a cup) we get two types of information on the basis of how the motor act is done and the context: what the agent is doing (i.e. grasping) and the intention underlying it (i.e. grasping for drinking). Here we examined the temporal dynamics of the brain activations that follow the observation of a motor act and underlie the observer’s capacity to understand what the agent is doing and why. Methodology/Principal Findings: Volunteers were presented with two-frame video-clips. The first frame (T0) showed an object with or without context; the second frame (T1) showed a hand interacting with the object. The volunteers were instructed to understand the intention of the observed actions while their brain activity was recorded with a high-density 128-channel EEG system. Visual event-related potentials (VEPs) were recorded time-locked with the frame showing the hand-object interaction (T1). The data were analyzed by using electrical neuroimaging, which combines a cluster analysis performed on the group-averaged VEPs with the localization of the cortical sources that give rise to different spatiotemporal states of the global electrical field. Electrical neuroimaging results revealed four major steps: 1) bilateral posterior cortical activations; 2) a strong activation of the left posterior temporal and inferior parietal cortices with almost a complete disappearance of activations in the right hemisphere; 3) a significant increase of the activations of the right temporo-parieta

Phylogenomics and the rise of the angiosperms

Angiosperms are the cornerstone of most terrestrial ecosystems and human livelihoods1,2. A robust understanding of angiosperm evolution is required to explain their rise to ecological dominance. So far, the angiosperm tree of life has been determined primarily by means of analyses of the plastid genome3,4. Many studies have drawn on this foundational work, such as classification and first insights into angiosperm diversification since their Mesozoic origins5–7. However, the limited and biased sampling of both taxa and genomes undermines confidence in the tree and its implications. Here, we build the tree of life for almost 8,000 (about 60%) angiosperm genera using a standardized set of 353 nuclear genes8. This 15-fold increase in genus-level sampling relative to comparable nuclear studies9 provides a critical test of earlier results and brings notable change to key groups, especially in rosids, while substantiating many previously predicted relationships. Scaling this tree to time using 200 fossils, we discovered that early angiosperm evolution was characterized by high gene tree conflict and explosive diversification, giving rise to more than 80% of extant angiosperm orders. Steady diversification ensued through the remaining Mesozoic Era until rates resurged in the Cenozoic Era, concurrent with decreasing global temperatures and tightly linked with gene tree conflict. Taken together, our extensive sampling combined with advanced phylogenomic methods shows the deep history and full complexity in the evolution of a megadiverse clade

Plasmodium Protease ROM1 Is Important for Proper Formation of the Parasitophorous Vacuole

Apicomplexans are obligate intracellular parasites that invade host cells by an active process leading to the formation of a non-fusogenic parasitophorous vacuole (PV) where the parasite replicates within the host cell. The rhomboid family of proteases cleaves substrates within their transmembrane domains and has been implicated in the invasion process. Although its exact function is unknown, Plasmodium ROM1 is hypothesized to play a role during invasion based on its microneme localization and its ability to cleave essential invasion adhesins. Using the rodent malaria model, Plasmodium yoelii, we carried out detailed quantitative analysis of pyrom1 deficient parasites during the Plasmodium lifecycle. Pyrom1(-) parasites are attenuated during erythrocytic and hepatic stages but progress normally through the mosquito vector with normal counts of oocyst and salivary gland sporozoites. Pyrom1 steady state mRNA levels are upregulated 20-fold in salivary gland sporozoites compared to blood stages. We show that pyrom1(-) sporozoites are capable of gliding motility and traversing host cells normally. Wildtype and pyrom1(-) sporozoites do not differ in the rate of entry into Hepa1–6 hepatocytes. Within the first twelve hours of hepatic development, however, only 50% pyrom1(-) parasites have developed into exoerythrocytic forms. Immunofluorescence microscopy using the PVM marker UIS4 and transmission electron microscopy reveal that the PV of a significant fraction of pyrom1(-) parasites are morphologically aberrant shortly after invasion. We propose a novel function for PyROM1 as a protease that promotes proper PV modification to allow parasite development and replication in a suitable environment within the mammalian host

The Transcription Factor AmrZ Utilizes Multiple DNA Binding Modes to Recognize Activator and Repressor Sequences of Pseudomonas aeruginosa Virulence Genes

AmrZ, a member of the Ribbon-Helix-Helix family of DNA binding proteins, functions as both a transcriptional activator and repressor of multiple genes encoding Pseudomonas aeruginosa virulence factors. The expression of these virulence factors leads to chronic and sustained infections associated with worsening prognosis. In this study, we present the X-ray crystal structure of AmrZ in complex with DNA containing the repressor site, amrZ1. Binding of AmrZ to this site leads to auto-repression. AmrZ binds this DNA sequence as a dimer-of-dimers, and makes specific base contacts to two half sites, separated by a five base pair linker region. Analysis of the linker region shows a narrowing of the minor groove, causing significant distortions. AmrZ binding assays utilizing sequences containing variations in this linker region reveals that secondary structure of the DNA, conferred by the sequence of this region, is an important determinant in binding affinity. The results from these experiments allow for the creation of a model where both intrinsic structure of the DNA and specific nucleotide recognition are absolutely necessary for binding of the protein. We also examined AmrZ binding to the algD promoter, which results in activation of the alginate exopolysaccharide biosynthetic operon, and found the protein utilizes different interactions with this site. Finally, we tested the in vivo effects of this differential binding by switching the AmrZ binding site at algD, where it acts as an activator, for a repressor binding sequence and show that differences in binding alone do not affect transcriptional regulation