Search

Background Damage to olfactory sensory neurons (OSNs), situated within the neuroepithelium of the olfactory cleft, may be associated with anosmia. Although their direct contact with the nasal airspace make OSNs vulnerable to injury and death, multiple mechanisms maintain epithelium integrity and olfactory function. We hypothesized that BMI1, a polycomb protein found to be enriched in OSNs, may function in neuroprotection. Here, we explored BMI1 function in a mouse model. Methods Utilizing a mouse genetic approach to delete Bmi1 selectively in mature OSNs, we investigated changes in OE homeostasis by performing immunohistochemical, biochemical, and functional assays. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR), immunostaining, and electro-olfactograms were used to compare gene expression, cell composition, and olfactory function in OSN-specific BMI1 knockout mice (n = 3 to 5) and controls. Chromatin studies were also performed to identify protein-DNA interactions between BMI1 and its target genes (n = 3). Results OSN-specific BMI1 knockout led to increased neuron death and basal cell activation. Chromatin studies suggested a mechanism of increased neurodegeneration due to de-repression of a pro-apoptosis gene, p19ARF. Despite the increased turnover, we found that olfactory neuroepithelium thickness and olfactory function remained intact. Our studies also revealed the presence of additional polycomb group proteins that may compensate for the loss of BMI1 in mature OSNs. Conclusion The olfactory neuroepithelium employs multiple mechanisms to maintain epithelial homeostasis. Our findings provide evidence that in a mouse model of BMI1 deletion, the overall integrity and function of the olfactory neuroepithelium are not compromised, despite increased neuronal turnover, reflecting a remarkable reparative capacity to sustain a critical sensory system.

Toxicity thresholds for dissolved oil applied in tropical ocean risk assessments are largely based on the sensitivities of temperate and/or freshwater species. To explore the suitability of these thresholds for tropical habitats we experimentally determined toxicity thresholds for eight tropical species for a partially weathered gas condensate, applied the target lipid model (TLM) to predict toxicity of fresh and weathered condensates and compared sensitivities of the tropical species with model predictions. The experimental condensate-specific hazard concentration (HC5) was 167 μg L−1 total aromatic hydrocarbons (TAH), with the TLM-modelled HC5 (78 μg L−1 TAH) being more conservative, supporting TLM-modelled thresholds for tropical application. Putative species-specific critical target lipid body burdens (CTLBBs) indicated that several of the species tested were among the more sensitive species in the TLM database ranging from 5.1 (coral larvae) to 97 (sponge larvae) μmol g−1 octanol and can be applied in modelling risk for tropical marine ecosystems.

Additional Sex Combs-Like 1 (ASXL1) is mutated at a high frequency in all forms of myeloid malignancies associated with poor prognosis. We generated a Vav1 promoter-driven Flag-Asxl1Y588X transgenic mouse model, Asxl1Y588X Tg, to express a truncated FLAG-ASXL1aa1-587 protein in the hematopoietic system. The Asxl1Y588X Tg mice had an enlarged hematopoietic stem cell (HSC) pool, shortened survival, and predisposition to a spectrum of myeloid malignancies, thereby recapitulating the characteristics of myeloid malignancy patients with ASXL1 mutations. ATAC- and RNA-sequencing analyses revealed that the ASXL1aa1-587 truncating protein expression results in more open chromatin in cKit+ cells compared with wild-type cells, accompanied by dysregulated expression of genes critical for HSC self-renewal and differentiation. Liquid chromatography-tandem mass spectrometry and coimmunoprecipitation experiments showed that ASXL1aa1-587 acquired an interaction with BRD4. An epigenetic drug screening demonstrated a hypersensitivity of Asxl1Y588X Tg bone marrow cells to BET bromodomain inhibitors. This study demonstrates that ASXL1aa1-587 plays a gain-of-function role in promoting myeloid malignancies. Our model provides a powerful platform to test therapeutic approaches of targeting the ASXL1 truncation mutations in myeloid malignancies.

Pannexins are membrane channel proteins that play a role in a number of critical biological processes (Panchin et al., 2000; Shestopalov, Panchin, 2008). Among other cellular functions, pannexin hemichannels serve as purine nucleoside conduits providing ATP efflux into the extracellular space (Dahl, 2015), where it is rapidly degraded to adenosine. Pannexin1 (Panx1) is abundantly expressed in the brain and has been shown to contribute to adenosine signaling in nervous system tissues (Prochnow et al., 2012). We hypothesized that pannexin1 may contribute to sleep-wake cycle regulation through extracellular adenosine, a well-established paracrine factor in slow wave sleep. To investigate this link, EEG and movement activity throughout the light/dark cycle were compared in Panx1-/- and Panx1+/+ mice. We found a significant increase in waking and a correspondent decrease in slow wave sleep percentages in the Panx1-/- animals. These changes were especially pronounced during the dark period. Furthermore, we found a significant increase in movement activity of Panx1-/- mice. These findings are consistent with the hypothesis that extracellular adenosine is relatively depleted in Panx1-/- animals due to the absence of the ATP-permeable hemichannels. At the same time, sleep rebound after a 6-h sleep deprivation remained unchanged in Panx1-/- mice as compared to the control animals. Behavioral tests revealed that Panx1-/- mice were significantly faster during their descent along the vertical pole but more sluggish during their run through the horizontal pole as compared to the control mice.

BAP1 regulates developmental switch in lineages commonly affected by BAP1-mutant cancers., The BAP1 tumor suppressor is mutated in many human cancers such as uveal melanoma, leading to poor patient outcome. It remains unclear how BAP1 functions in normal biology or how its loss promotes cancer progression. Here, we show that Bap1 is critical for commitment to ectoderm, mesoderm, and neural crest lineages during Xenopus laevis development. Bap1 loss causes transcriptional silencing and failure of H3K27ac to accumulate at promoters of key genes regulating pluripotency-to-commitment transition, similar to findings in uveal melanoma. The Bap1-deficient phenotype can be rescued with human BAP1, by pharmacologic inhibition of histone deacetylase (HDAC) activity or by specific knockdown of Hdac4. Similarly, BAP1-deficient uveal melanoma cells are preferentially vulnerable to HDAC4 depletion. These findings show that Bap1 regulates lineage commitment through H3K27ac-mediated transcriptional activation, at least in part, by modulation of Hdac4, and they provide insights into how BAP1 loss promotes cancer progression.

Lung cancer patients often suffer from severe airway stenosis, the symptoms of which can be relieved by the implantation of stents. Different respiratory stents are commercially available, but the impact of their mechanical performance on tissue responses is not well understood. Two novel laser-cut and hand-braided nitinol stents, partially covered with polycarbonate urethane, were bench tested and implanted in Rhön sheep for 6 weeks. Bench testing highlighted differences in mechanical behavior: the laser-cut stent showed little foreshortening when crimped to a target diameter of 7.5 mm, whereas the braided stent elongated by more than 50%. Testing also revealed that the laser-cut stent generally exerted higher radial resistive and chronic outward forces than the braided stent, but the latter produced significantly higher radial resistive forces at diameters below 9 mm. No migration was observed for either stent type in vivo. In terms of granulation, most stents exerted a low to medium tissue response with only minimal formation of granulation tissue. We have developed a mechanical and in vivo framework to compare the behavior of different stent designs in a large animal model, providing data, which may be employed to improve current stent designs and to achieve better treatment options for lung cancer patients.

Currently, there is no optimal treatment available for end stage tumour patients with airway stenosis. The PulmoStent concept aims on overcoming current hurdles in airway stenting by combining a nitinol stent with a nutrient-permeable membrane, which prevents tumour ingrowth. Respiratory epithelial cells can be seeded onto the cover to restore mucociliary clearance. In this study, a novel hand-braided dog bone stent was developed, covered with a polycarbonate urethane nonwoven and mechanically tested. Design and manufacturing of stent and cover were improved in an iterative process according to predefined requirements for permeability and mechanical properties and finally tested in a proof of concept animal study in sheep for up to 24 weeks. In each animal two stents were implanted, one of which was cell-seeded by endoscopic spraying in situ. We demonstrated the suitability of this membrane for our concept by glucose transport testing and in vitro culture of respiratory epithelial cells. In the animal study, no migration occurred in any of the twelve stents. There was only mild granulation tissue formation and tissue reaction; no severe mucus plugging was observed. Thus, the PulmoStent concept might be a step forward for palliative treatment of airway stenosis with a biohybrid stent device.

Autoantibodies against the M2 subtype of muscarinic acetylcholine receptors with functional activities have been found in the sera of patients with dilated cardiomyopathy (DCM), and the second extracellular loop has been established as the predominant epitope. However, it has been shown that the third intracellular loop is recognized by Chagas disease patients with severe cardiac dysfunction. In this work, BALB/c mice were immunized with plasmids encoding these two epitopes, and a control group received the empty plasmid (pcDNA3 vector). Serum from these DNA-immunized animals had elevated and persistent titres of antibodies against respective antigens. Heart echocardiography indicated diminished left ventricular wall thickness and reduced ejection fraction for both epitope-immunized groups, and ergospirometry tests showed a significant decrease in the exercise time and oxygen consumption. Transfer of serum from these immunized mice into naive recipients induced the same alterations in cardiac structure and function. Furthermore, electron microscopy analysis of donor-immunized animals revealed several ultrastructural alterations suggestive of autophagy and mitophagy, suggesting novel roles for these autoantibodies. Overall, greater functional and structural impairment was observed in the donor and recipient epitope groups, implicating the third intracellular loop epitope in the pathological effects for the first-time. Therefore, the corresponding peptides could be useful for autoimmune DCM diagnosis and targeted therapy.

Summary: Stem cell-based therapies have been proposed as a strategy to replace damaged tissues, especially in the nervous system. A primary sensory modality, olfaction, is impaired in 12% of the US population, but lacks treatment options. We report here the development of a novel mouse model of inducible hyposmia and demonstrate that purified tissue-specific stem cells delivered intranasally engraft to produce olfactory neurons, achieving recovery of function. Adult mice were rendered hyposmic by conditional deletion of the ciliopathy-related IFT88 gene in the olfactory sensory neuron lineage and following experimentally induced olfactory injury, received either vehicle or stem cell infusion intranasally. Engraftment-derived olfactory neurons were identified histologically, and functional improvements were measured via electrophysiology and behavioral assay. We further explored mechanisms in culture that promote expansion of engraftment-competent adult olfactory basal progenitor cells. These findings provide a basis for translational research on propagating adult tissue-specific sensory progenitor cells and testing their therapeutic potential. : Olfactory sensory losses lack treatment options. The corresponding author and colleagues report the testing of a cellular therapy for olfactory loss. Inducible hyposmia was produced by targeting ciliopathy to the olfactory lineage in mice, and wild-type adult stem cells engrafted to produce functional neurons, assessed using histology, electrophysiology, and behavioral assay. Keywords: olfaction, anosmia, stem cells, neuron, ciliopathy

Gouvea, AL, Martinez, CG, and Kurtenbach, E. Determining maximal muscle strength in mice: validity and reliability of an adapted swimming incremental overload test. J Strength Cond Res 34(8): 2360-2368, 2020-At present, there are no reliable methods to determine maximal muscle strength in small rodents. Here, we established an adapted swimming incremental overload test (SIOT) as an instrument for this purpose. First, to validate the test, BALB/c mice received 20 mg·kg·d of dexamethasone (DEXA group) or water (control group). After 14 days, with a cumulative dose of 120 mg·kg of dexamethasone, the SIOT could detect a decrease of approximately 7% in muscle strength. In addition, this decrease was consistent with a significant reduction in body (above 13.5%) and muscle (approximately 15%) weight in DEXA atrophic animals. To establish the SIOT reliability, another group of animals was evaluated for 5 consecutive days. In this second protocol, the SIOT was executed with an initial load corresponding to 12% of the mouse body weight (BW) fixed to the tail. Increments between 1 and 5% of the BW were added during each attempt to obtain the highest load that was tolerated for a time interval of 5-7 seconds. On the last day, the SIOT reliability test was performed by 2 different raters to obtain the inter-rater reproducibility. The adapted SIOT was shown to be reliable when measured by the same rater (intraclass correlation coefficient [ICC] = 0.939) and by 2 different raters (ICC = 0.830). The Bland-Altman graphical representation did not demonstrate heteroscedastic errors. Therefore, the SIOT proved to be a sensitive and reliable method to measure muscle strength, and it can be applied to small animals in different models of muscle atrophy.

Macromolecular bioscience : MBS 18(7), 1700292 (2018). doi:10.1002/mabi.201700292, Published by Wiley-VCH, Weinheim

Usher syndrome is a genetically and clinically heterogeneous disease in humans, characterized by sensorineural hearing loss, retinitis pigmentosa and vestibular dysfunction. This disease is caused by mutations in genes encoding proteins that form complex networks in different cellular compartments. Currently, it remains unclear whether the Usher proteins also form networks within the olfactory epithelium (OE). Here, we describe Usher gene expression at the mRNA and protein level in the OE of mice and showed interactions between these proteins and olfactory signaling proteins. Additionally, we analyzed the odor sensitivity of different Usher syndrome mouse models using electro-olfactogram recordings and monitored significant changes in the odor detection capabilities in mice expressing mutant Usher proteins. Furthermore, we observed changes in the expression of signaling proteins that might compensate for the Usher protein deficiency. In summary, this study provides novel insights into the presence and purpose of the Usher proteins in olfactory signal transduction.

Carnosine (β-alanyl-L-histidine) is an imidazole dipeptide synthesized in excitable tissues of many animals, whose biochemical properties include carbonyl scavenger, anti-oxidant, bivalent metal ion chelator, proton buffer, and immunomodulating agent, although its precise physiological role(s) in skeletal muscle and brain tissues in vivo remain unclear. The aim of the present study was to investigate the in vivo effects of acute carnosine administration on various aspects of brain bioenergetics of young Wistar rats. The activity of mitochondrial enzymes in cerebral cortex was assessed using a spectrophotometer, and it was found that there was an increase in the activities of complexes I-III and II-III and succinate dehydrogenase in carnosine-treated rats, as compared to vehicle-treated animals. However, quantitative real-time RT-PCR (RT-qPCR) data on mRNA levels of mitochondrial biogenesis-related proteins (nuclear respiratory factor 1 (Nrf1), peroxisome proliferator-activated receptor-γ coactivator 1-α (Ppargc1α), and mitochondrial transcription factor A (Tfam)) were not altered significantly and therefore suggest that short-term carnosine administration does not affect mitochondrial biogenesis. It was in agreement with the finding that immunocontent of respiratory chain complexes was not altered in animals receiving carnosine. These observations indicate that acute carnosine administration increases the respiratory chain and citric acid cycle enzyme activities in cerebral cortex of young rats, substantiating, at least in part, a neuroprotector effect assigned to carnosine against oxidative-driven disorders.

Background: Sepsis is associated with high mortality rates in intensive care units worldwide and represents a systemic inflammatory response to infection. P2X7 is an ionotropic purine receptor with known proinflammatory activity. Here, we investigated the role of the P2X7 receptor in sepsis induced by cecal ligation and puncture (CLP). Methods: Wild-type (WT) and P2X7KO (P2X7 null) mice were subjected to CLP and their survival was monitored for 7 days. Blood, peritoneal wash and lungs were collected 24 h after CLP and used to measure bacterial load, immune cell infiltration, nitric oxide (NO), cytokine levels, and peritoneal cell death and to assess lung injury. Results: P2X7KO mice showed significantly increased survival 7 days after CLP (30% compared to 60% in WT animals) accompanied by an overall attenuated inflammatory response, with decreased cell recruitment to the peritoneum, no or limited increases in the levels of NO and proinflammatory cytokines (IL-1β, IL-6, IL-12, IL-17, and IL-4), reduced peritoneal cell apoptosis, and less pronounced lung infiltration and morphological changes. Conclusions: Our data show the P2X7 receptor is required for the development of the inflammatory response associated with sepsis and support the notion that P2X7 receptor is a valid therapeutic target against inflammatory diseases.

BEACH domain proteins are involved in membrane protein traffic and human diseases, but their molecular mechanisms are not understood. The BEACH protein LRBA has been implicated in immune response and cell proliferation, and human LRBA mutations cause severe immune deficiency. Here, we report a first functional and molecular phenotype outside the immune system of LRBA-knockout mice: compromised olfaction, manifesting in reduced electro-olfactogram response amplitude, impaired food-finding efficiency, and smaller olfactory bulbs. LRBA is prominently expressed in olfactory and vomeronasal chemosensory neurons of wild-type mice. Olfactory impairment in the LRBA-KO is explained by markedly reduced concentrations (20–40% of wild-type levels) of all three subunits αolf, β1 and γ13 of the olfactory heterotrimeric G-protein, Golf, in the sensory cilia of olfactory neurons. In contrast, cilia morphology and the concentrations of many other proteins of olfactory cilia are not or only slightly affected. LRBA is also highly expressed in photoreceptor cells, another cell type with a specialized sensory cilium and heterotrimeric G-protein-based signalling; however, visual function appeared unimpaired by the LRBA-KO. To our knowledge, this is the first observation that a BEACH protein is required for the efficient subcellular localization of a lipid-anchored protein, and of a ciliary protein.

Pannexin 1 (Panx1) forms ATP-permeable membrane channels that play a key role in purinergic signaling in the nervous system in both normal and pathological conditions. In the retina, particularly high levels of Panx1 are found in retinal ganglion cells (RGCs), but the normal physiological function in these cells remains unclear. In this study, we used patch clamp recordings in the intact inner retina to show that evoked currents characteristic of Panx1 channel activity were detected only in RGCs, particularly in the OFF-type cells. The analysis of pattern electroretinogram (PERG) recordings indicated that Panx1 contributes to the electrical output of the retina. Consistently, PERG amplitudes were significantly impaired in the eyes with targeted ablation of the Panx1 gene in RGCs. Under ocular hypertension and ischemic conditions, however, high Panx1 activity permeated cell membranes and facilitated the selective loss of RGCs or stably transfected Neuro2A cells. Our results show that high expression of the Panx1 channel in RGCs is essential for visual function in the inner retina but makes these cells highly sensitive to mechanical and ischemic stresses. These findings are relevant to the pathophysiology of retinal disorders induced by increased intraocular pressure, such as glaucoma.

The olfactory epithelium contains basal cells with stem cell characteristics, which have the capacity to differentiate throughout life into olfactory receptor neurons (ORNs). Here we investigate the in vitro characteristics of stem cells taken from the olfactory bulb (OB) and the olfactory epithelium (OE) of neonatal TIS21 knock-in mice. The major aim of the study was the generation of olfactory neurospheres (ONS) derived from OB and OE of neonatal mice as a tool to further analyze the elementary processes of ORN development. Our data showed that the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) leads to a significant increase in number of ONS derived from OB but not from OE. The differentiation of ONSs led to the formation of different neuronal cell types, in particular to bipolar-shaped cells as well as putative pyramidal-neurons, astrocytes and oligodendrocytes. Immunohistochemical staining confirmed the presence of astrocytes and neurons in both types of ONSs. In order to investigate the functionality of the neurons we performed calcium imaging and patch-clamp experiments. Calcium imaging experiments revealed that the application of high potassium concentration provokes calcium transients. No excitable properties, neither sodium currents nor action potentials, were observed for the bipolar-shaped cells derived from OB and OE neurospheres, which means that these types of cells morphologically defined as putative neuronal cells, were not physiologically active. Interestingly, patch-clamp recordings performed in the pyramidal-shaped cells of OB neurospheres showed sodium and potassium currents as well as action potentials. Our study will help to establish further models in the field of olfactology.

The hard tick Ixodes ricinus is the principal vector of Lyme borreliosis (LB) group spirochaetes in Europe, but it also transmits a large number of other microbial pathogens that are of importance to animal and human health. Here, we characterise geographically distinct populations of this important ectoparasite based on multilocus sequence typing (MLST) of multiple mitochondrial (mt) genes (mtMLST). Internal fragments of approximately 500 bp were amplified and sequenced for 6 protein-encoding and ribosomal genes (atp6, coi, coii, coiii, cytB, and 12s). The samples analysed consisted of 506 questing nymphs collected in Britain and Latvia in 2006-2008 and in Latvia in 2002. Although little genetic structure has previously been observed in I. ricinus ticks among Europe, our data could clearly differentiate these 2 populations. Here, we argue that this novel scheme provides additional phylogenetic resolution which is important for understanding the genetic and geographic structure of I. ricinus populations. This in turn will benefit monitoring and management of tick-borne diseases.

The Lyme borreliosis (LB) group of spirochaetes currently comprises 18 named species that vary in their geographic distribution, host specificity and ability to cause disease in humans. In Europe three species are most abundant, Borrelia afzelii, Borrelia garinii and Borrelia valaisiana but only two of these (B. garinii and B. afzelii) are regularly found in Asia as well. A recently published study has shown that Borrelia species associated with birds, such as B. garinii, showed limited geographic structuring between European countries while, the rodent associated species, B. afzelii, showed extensive spatial structuring in Europe. Here, we use multilocus sequence analysis to show that when the wider, inter-continental, distribution is considered, there is evidence of spatial structuring even in the bird-associated species B. garinii. Furthermore, our investigations into historical LB populations provided evidence for range expansions of B. garinii and B. afzelii populations in Europe in the distant past. We propose that the expansion of B. afzelii in Europe may be linked to rodent population expansions after the last glacial maximum.

The abilities of the most common European genospecies of Borrelia burgdorferi sensu lato to survive blood meals taken by ticks feeding on birds were analyzed. A pattern of differential survival of the spirochetes in feeding ticks was observed. The result is consistent with the concept of selective transmission of Lyme borreliosis spirochetes.

In this work we characterized the degenerative process of ovarian follicles of the bug Rhodnius prolixus challenged with the non-entomopathogenic fungus Aspergillus niger. An injection of A. niger conidia directly into the hemocoel of adult R. prolixus females at the onset of vitellogenesis caused no effect on host lifespan but elicited a net reduction in egg batch size. Direct inspection of ovaries from the mycosed insects revealed that fungal challenge led to atresia of the vitellogenic follicles. Light microscopy and DAPI staining showed follicle shrinkage, ooplasm alteration and disorganization of the monolayer of follicle cells in the atretic follicles. Transmission electron microscopy of thin sections of follicle epithelium also showed nuclei with condensed chromatin, electron dense mitochondria and large autophagic vacuoles. Occurrence of apoptosis of follicle cells in these follicles was visualized by TUNEL labeling. Resorption of the yolk involved an increase in protease activities (aspartyl and cysteinyl proteases) which were associated with precocious acidification of yolk granules and degradation of yolk protein content. The role of follicle atresia in nonspecific host–pathogen associations and the origin of protease activity that led to yolk resorption are discussed.

BACKGROUND: Northward expansion of the tick Ixodes scapularis is driving Lyme disease (LD) emergence in Canada. Information on mechanisms involved is needed to enhance surveillance and identify where LD risk is emerging. OBJECTIVES: We used passive and active surveillance and phylogeographic analysis of Borrelia burgdorferi to investigate LD risk emergence in Quebec. METHODS: In active surveillance, we collected ticks from the environment and from captured rodents. B. burgdorferi transmission was detected by serological analysis of rodents and by polymerase chain reaction assays of ticks. Spatiotemporal trends in passive surveillance data assisted interpretation of active surveillance. Multilocus sequence typing (MLST) of B. burgdorferi in ticks identified likely source locations of B. burgdorferi. RESULTS: In active surveillance, we found I. scapularis at 55% of sites, and we were more likely to find them at sites with a warmer climate. B. burgdorferi was identified at 13 I. scapularis-positive sites, but infection prevalence in ticks and animal hosts was low. Low infection prevalence in ticks submitted in passive surveillance after 2004-from the tick-positive regions identified in active surveillance-coincided with an exponential increase in tick submissions during this time. MLST analysis suggested recent introduction of B. burgdorferi from the northeastern United States. CONCLUSIONS: These data are consistent with I. scapularis ticks dispersed from the United States by migratory birds, founding populations where the climate is warmest, and then establishment of B. burgdorferi from the United States several years after I. scapularis have established. These observations provide vital information for public health to minimize the impact of LD in Canada.

Analysis of Lyme borreliosis (LB) spirochetes, using a novel multilocus sequence analysis scheme, revealed that OspA serotype 4 strains (a rodent-associated ecotype) of Borrelia garinii were sufficiently genetically distinct from bird-associated B. garinii strains to deserve species status. We suggest that OspA serotype 4 strains be raised to species status and named Borrelia bavariensis sp. nov. The rooted phylogenetic trees provide novel insights into the evolutionary history of LB spirochetes.

Blackbirds ( Turdus merula ) and song thrushes ( Turdus philomelos ) were found to carry 95% of all spirochete-infected tick larvae among 40 bird species captured in Central Europe. More than 90% of the infections were typed as Borrelia garinii and Borrelia valaisiana . We conclude that thrushes are key players in the maintenance of these spirochete species in this region of Central Europe.

Two proteins from the eggshell of Rhodnius prolixus were isolated, characterized and named Rp30 and Rp45 according to their molecular masses. Purified proteins were used to obtain specific antiserum which was later used for immunolocalization. The antiserum against Rp30 and Rp45 detected their presence inside the follicle cells, their secretion and their association with oocyte microvilli. Both proteins are expressed during the final stage of vitellogenesis, preserved during embryogenesis and discarded together with the eggshell. The amino terminals were sequenced and both proteins were further cloned using degenerated primers. The amino acid sequences appear to have a tripartite arrangement with a highly conserved central domain which presents a repetitive motif of valine-proline-valine (VPV) at intervals of 15 amino acid residues. Their amino acid sequence showed no similarity to any known eggshell protein. The expression of these proteins was also investigated; the results demonstrated that this occurred strictly in choriogenic follicles. Antifungal activity against Aspergillus niger was found to be associated with Rp45 but not with Rp30. A. niger exposed to Rp45 protein induced growth inhibition and several morphological changes such as large vacuoles, swollen mitochondria, multi-lamellar structures and a disorganized cell wall as demonstrated by electron microscopy analysis.

Plant defensins, components of the plant innate immune system, are cationic cysteine-rich antifungal peptides. Evidence from the literature [Thevissen, K., et al. (2003) Peptides 24, 1705-1712] has demonstrated that patches of fungi membrane containing mannosyldiinositolphosphorylceramide and glucosylceramides are selective binding sites for the plant defensins isolated from Dahlia merckii and Raphanus sativus, respectively. Whether plant defensins interact directly or indirectly with fungus intracellular targets is unknown. To identify physical protein-protein interactions, a GAL4-based yeast two-hybrid system was performed using the antifungal plant peptide Pisum sativum defensin 1 (Psd1) as the bait. Target proteins were screened within a Neurospora crassa cDNA library. Nine out of 11 two-hybrid candidates were nuclear proteins. One clone, detected with high frequency per screening, presented sequence similarity to a cyclin-like protein, with F-box and WD-repeat domains, related to the cell cycle control. GST pull-down assay corroborated in vitro this two-hybrid interaction. Fluorescence microscopy analysis of FITC-conjugated Psd1 and DAPI-stained fungal nuclei showed in vivo the colocalization of the plant peptide Psd1 and the nucleus. Analysis of the DNA content of N. crassa conidia using flow cytometry suggested that Psd1 directed cell cycle impairment and caused conidia to undergo endoreduplication. The developing retina of neonatal rats was used as a model to observe the interkinetic nuclear migration during proliferation of an organized tissue from the S toward the M phase of the cell cycle in the presence of Psd1. The results demonstrated that the plant defensin Psd1 regulates interkinetic nuclear migration in retinal neuroblasts.

Autoantibodies against the M2 receptors (M2AChR) have been associated with Dilated Cardiomyopathy (DCM). In the heart, P2×7 receptors influence electrical conduction, coronary circulation and response to ischemia. They can also trigger pro-inflammatory responses and the development of neurological, cardiac and renal disorders. Here, P2×7−/− mice displayed an increased heart rate and ST segment depression, but similar exercise performance when compared to wild type (WT) animals. After immunization with plasmid containing M2AChR cDNA sequence, WT mice produced anti-M2AChR antibodies, while P2×7−/− mice showed an attenuated production. Despite this, WT and P2×7−/− showed left ventricle cavity enlargement and decreased exercise tolerance. Transfer of serum from M2AChR WT immunized mice to näive recipients led to an alteration in heart shape. P2×7−/− mice displayed a significant increase in the frequency of spleen regulatory T cells population, which is mainly composed by the FoxP3+CD25− subset. M2AChR WT immunized mice showed an increase in IL-1β, IFNγ and IL-17 levels in the heart, while P2×7−/− group produced lower amounts of IL-1β and IL-17 and higher amounts of IFNγ. These results pointed to previously unnoticed roles of P2×7 in cardiovascular and immune systems and underscored the participation of IL-17 and IFNγ in the progress of autoimmune DCM.

Fitness of tick-borne pathogens may be determined by the degree to which their infection dynamics in vertebrate hosts permits transmission cycles if infective and uninfected tick stages are active at different times of the year. To investigate this hypothesis we developed a simulation model that integrates the transmission pattern imposed by seasonally asynchronous nymphal and larvalIxodes scapularisticks in northeastern North America, with a model of infection in white-footed mice (Peromyscus leucopus) reservoir hosts, using the bacteriaBorrelia burgdorferiandAnaplasma phagocytophilumas examples. In simulations, survival of microparasites, their sensitivity to reduced rodent and tick abundance, and to ‘dilution’ by a reservoir-incompetent host depended on traits that allowed (i) highly efficient transmission from acutely-infected hosts, (ii) long-lived acute or ‘carrier’ host infections, and/or (iii) transmission amongst co-feeding ticks. Minimum values for transmission efficiency to ticks, and duration of host infectivity, necessary for microparasite persistence, were always higher when nymphal and larval ticks were seasonally asynchronous than when these instars were synchronous. Thus, traits influencing duration of host infectivity, transmission efficiency to ticks and co-feeding transmission are likely to be dominant determinants of fitness inI. scapularis-borne microparasites in northeastern North America due to abiotic forcings influencingI. scapularisseasonality.

Host specialization is a key issue in infectious disease research because patterns of cross-species transmission affect parasite dispersal., We examined the degree of host specialization of different strains of Borrelia burgdorferi, the tickborne pathogen that causes Lyme borreliosis in the northeastern United States. We first assessed the genetic population structures of B. burgdorferi in ticks obtained from different mammalian host species and in questing ticks sampled in a woodland ecosystem in Connecticut. By comparing the patterns found in our study with data from another cross-sectional study, we demonstrate that B. burgdorferi is a generalist microparasite and conclude that efficient cross-species transmission of B. burgdorferi is a key feature that has allowed the rapid spread of Lyme borreliosis across the northeastern United States.

The development of novel antiviral drugs against hepatitis C is a challenging and competitive area of research. Progress of this research has been hampered due to the quasispecies nature of the hepatitis C virus, the absence of cellular infection models and the lack of easily accessible and highly representative animal models. The current combination therapy consisting of interferon-α and ribavirin mainly acts by supporting host cell defence. These therapeutics are the prototypic representatives of indirect antiviral agents as they act on cellular targets. However, the therapy is not a cure, when considered from the long-term perspective, for almost half of the chronically infected patients. This draws attention to the urgent need for more efficient treatments. Novel anti-hepatitis C treatments under study are directed against a number of so-called direct antiviral targets such as polymerases and proteases, which are encoded by the virus. Although such direct antiviral approaches have proven to be successful in several viral indications, there is a risk of resistant viruses developing. In order to avoid resistance, the development of indirect antiviral compounds has to be intensified. These act on host cell targets either by boosting the immune response or by blocking the virus host cell interaction. A particularly interesting approach is the development of inhibitors that interfere with signal transduction, such as protein kinase inhibitors. The purpose of this review is to stress the importance of developing indirect antiviral agents that act on host cell targets. In doing so, a large source of potential targets and mechanisms can be exploited, thus increasing the likelihood of success. Ultimately, combination therapies consisting of drugs against direct and indirect viral targets will most probably provide the solution to fighting and eradicating hepatitis C virus in patients.

Glia represents the most numerous group of nervous system cells and CNS development and function depend on glial cells. We developed a purified Muller glia culture to investigate the expression of several neurotransmitter markers on these cells, such as dopaminergic, cholinergic, GABAergic and peptidergic receptors or enzymes, based on functional assays measuring second messenger levels or Western blot for specific proteins. Purified Muller cell culture was obtained from 8-day-old (E8) embryonic chick. Glial cells cultured for 15 days (E8C15) expressed D1A and D1B receptors mRNAs, but not D1D, as detected by RT-PCR. The binding of [3H]-SCH 23390 revealed an amount of expressed receptors around 40 fmol/mg protein. Dopamine (100 microM), PACAP (50 nM) and forskolin (10 microM) induced a 50-, 30- and 40-fold cAMP accumulation on glial cells, respectively, but not ip3 production. The dopamine-promoted cAMP accumulation was blocked by 2 microM SCH 23390. Carbachol stimulated a 3-fold ip3 accumulation. Western blot analysis also revealed the expression of tyrosine hydroxylase, L-dopa decarboxylase, PAC1 receptor, GAD67 and beta2-nicotinic receptor subunit by these cells. These results indicate that several components of neurotransmitter signaling and metabolism are found in cultured Muller cells.

More than a decade after a study on the transmission cycle of Borrelia burgdorferi sensu lato in the Siebengebirge, a nature reserve near Bonn, Germany, questing nymphal and adult Ixodes ricinus ticks were collected again in three selected areas of the same low mountain range and examined for infection with B. burgdorferi sensu lato. Between May and October 2001, a total of 1,754 ticks were collected by blanket dragging; 374 ticks were analyzed for B. burgdorferi sensu lato by both an immunofluorescence assay (IFA) and at least two different PCR tests, whereas 171 ticks were analyzed by PCR only. By combining all assays, an average of 14% of the ticks tested positive for B. burgdorferi sensu lato, 5.5, 15.8, and 21.8% in the three collection areas. Of the nymphs and adults examined, 12.9 and 21.1%, respectively, were found to be spirochete infected. A lower total infection prevalence was obtained by IFA (14.4%) than by a nested PCR approach (16.5%), but both were higher than that obtained by a simple PCR approach (11.9%). Compared with data collected over a decade ago, the mean infection prevalence of B. burgdorferi sensu lato in the ticks was significantly higher for all three biotopes, whereas a similar pattern of habitat-specific infection prevalence was observed. Genotyping of B. burgdorferi sensu lato revealed high relative prevalences of B. valaisiana (identified in 43.1% of infected ticks) and B. garinii (32.3%), whereas B. afzelii (12.3%) and B. burgdorferi sensu stricto (1.5%) were relatively rare. We conclude that B. burgdorferi sensu lato infection has increased in this region over the last 15 years due to presently unknown changes in ecological conditions, perhaps related to climate change or wildlife management.

In Europe, 6 of the 11 genospecies of Borrelia burgdorferi sensu lato are prevalent in questing Ixodes ricinus ticks. In most parts of Central Europe, B. afzelii , B. garinii , and B. valaisiana are the most frequent species, whereas B. burgdorferi sensu stricto, B. bissettii , and B. lusitaniae are rare. Previously, it has been shown that B. afzelii is associated with European rodents. Therefore, the aim of this study was to identify reservoir hosts of B. garinii and B. valaisiana in Slovakia. Songbirds were captured in a woodland near Bratislava and investigated for engorged ticks. Questing I. ricinus ticks were collected in the same region. Both tick pools were analyzed for spirochete infections by PCR, followed by DNA-DNA hybridization and, for a subsample, by nucleotide sequencing. Three of the 17 captured songbird species were infested with spirochete-infected ticks. Spirochetes in ticks that had fed on birds were genotyped as B. garinii and B. valaisiana , whereas questing ticks were infected with B. afzelii , B. garinii , and B. valaisiana . Furthermore, identical ospA alleles of B. garinii were found in ticks that had fed on the birds and in questing ticks. The data show that songbirds are reservoir hosts of B. garinii and B. valaisiana but not of B. afzelii . This and previous studies confirm that B. burgdorferi sensu lato is host associated and that this bacterial species complex contains different ecotypes.

Borrelia burgdorferi sensu lato (s.l.) is maintained in nature by complex zoonotic transmission cycles, involving a large variety of vertebrates as hosts and hard ticks of the genus Ixodes as vectors. Recent studies suggest that the genospecies of B. burgdorferi s.l. and sometimes their subtypes are propagated by different spectra of hosts, mainly birds and rodents. In order to test the concept of host-association, we analysed the relationships between Borrelia genospecies, rodent hosts and I. ricinus ticks in an endemic focus of Lyme borreliosis in western Slovakia. Rodents and questing ticks were collected at a forested lowland locality near Bratislava. Tick infestation levels on rodents were determined, and spirochaete infections in ticks and in ear punch biopsies were analysed by PCR followed by genotyping. Mice were more heavily infested with ticks than bank voles, and a higher proportion of mice was infected with spirochaetes than voles. However, the infectivity of voles was much higher than that of mice. The vast majority of infections detected in the skin and in ticks feeding on the rodents represented B. afzelii. In contrast, more than half of all infections in questing ticks collected in the same region of Slovakia were identified as B. valaisiana and B. garinii. In conclusion, whilst the study reveals that mice and voles play different quantitative roles in the ecology of Lyme borreliosis, it demonstrates that B. afzelii is specifically maintained by European rodents, validating the concept of host-association of B. burgdorferi s.l.

Phosphorylation of hepatitis B virus (HBV) core protein has recently been shown to be a prerequisite for pregenomic RNA encapsidation into viral capsids, but the host cell kinases mediating this essential step of the HBV replication cycle have not been identified. We detected two kinases of 95 and 115 kDa in HuH-7 total cell lysates which interacted specifically with the HBV core protein and phosphorylated its arginine-rich C-terminal domain. The 95-kDa kinase was purified and characterized as SR protein-specific kinase 1 (SRPK1) by mass spectrometry. Based on this finding, the 115-kDa kinase could be identified as the related kinase SRPK2 by immunoblot analysis. In vitro, both SRPKs phosphorylated HBV core protein on the same serine residues which are found to be phosphorylated in vivo. Moreover, the major cellular HBV core kinase activity detected in the total cell lysate showed biochemical properties identical to those of SRPK1 and SRPK2, as examined by measuring binding to a panel of chromatography media. We also clearly demonstrate that neither the cyclin-dependent kinases Cdc2 and Cdk2 nor protein kinase C, previously implicated in HBV core protein phosphorylation, can account for the HBV core protein kinase activity. We conclude that both SRPK1 and SRPK2 are most likely the cellular protein kinases mediating HBV core protein phosphorylation during viral infection and therefore represent important host cell targets for therapeutic intervention in HBV infection.

Borrelia burgdorferi sensu lato (s.l.), the tick-borne agent of Lyme borreliosis, is a bacterial species complex comprising 11 genospecies. Here, we discuss whether the delineation of genospecies is ecologically relevant. We provide evidence that B. burgdorferi s.l. is structured ecologically into distinct clusters that are host specific. An immunological model for niche adaptation is proposed that suggests the operation of complement-mediated selection in the midgut of the feeding tick. We conclude that vertebrate hosts rather than tick species are the key to Lyme borreliosis spirochaete diversity.

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in individual adult Ixodes ricinus ticks from Europe by direct PCR amplification of spirochetal DNA followed by genospecies-specific hybridization. Analysis of mixed infections in the ticks showed that B . garinii and B . valaisiana segregate from B . afzelii . This and previous findings suggest that host complement interacts with spirochetes in the tick, thereby playing an important role in the ecology of Lyme borreliosis.

LYME borreliosis, also called Lyme disease, is the most prevalent vectorborne disease of human beings in the temperate zones of the northern hemisphere ([Steere and others 2004][1]). It also occurs in domestic animals, including dogs, cats and horses ([Stanek and others 2002][2]). Lyme borreliosis

The genetic diversity of Borrelia burgdorferi sensu lato was assessed in a focus of Lyme borreliosis in southern Britain dominated by game birds. Ticks, rodents, and pheasants were analyzed for spirochete infections by PCR targeting the 23S-5S rRNA genes, followed by genotyping by the reverse line blot method. In questing Ixodes ricinus ticks, three genospecies of B. burgdorferi sensu lato were detected, with the highest prevalences found for Borrelia garinii and Borrelia valaisiana. B. burgdorferi sensu stricto was rare (Borrelia afzelii was not detected in any of the samples. More than 50% of engorged nymphs collected from pheasants were infected with borreliae, mainly B. garinii and/or B. valaisiana . Although 19% of the rodents harbored B. burgdorferi sensu stricto and/or B. garinii in internal organs, only B. burgdorferi sensu stricto was transmitted to xenodiagnostic tick larvae (it was transmitted to 1% of the larvae). The data indicate that different genospecies of B. burgdorferi sensu lato can be maintained in nature by distinct transmission cycles involving the same vector tick species but different vertebrate host species. Wildlife management may have an influence on the relative risk of different clinical forms of Lyme borreliosis.

The sensitivity of Borrelia burgdorferi sensu lato to animal sera was analyzed. Complement-mediated borreliacidal effects were observed with particular combinations of host serum and Borrelia genospecies. The species-specific pattern of viability and/or lysis is highly consistent with the pattern of reservoir competence of hosts for B. burgdorferi sensu lato, suggesting a key role of complement in the global ecology of Lyme borreliosis.

As observed in humans, immune responses in naturally infected reservoir hosts of Borrelia burgdorferi sensu lato rarely target the outer surface proteins (Osp) A and B of Lyme disease spirochaetes. The absence of protective immunity in such hosts following tick-borne infection allows them to play an effective role in the maintenance of Lyme borreliosis in nature. Therefore, the question was addressed whether one of the most prominent natural reservoir host species of B. burgdorferi s.l. in Europe, the yellow-necked mouse (Apodemus flavicollis), may lack the ability to elicit transmission-blocking antibodies to Lyme borreliosis spirochaetes. Yellow-necked mice were immunized with a recombinant lipidated OspA from B. burgdorferi sensu stricto or with high numbers of UV-irradiated whole spirochaetes. All immunized mice, but not untreated controls, developed polyclonal humoral immune responses to OspA (31 kDa). Serum antibodies of animals vaccinated with the recombinant OspA contained high levels of antibody to an epitope of OspA, defined by the monoclonal antibody LA-2, whereas only low levels of LA-2 equivalent antibodies could be detected in sera from animals immunized with killed spirochaetes. Ixodes ricinus ticks infected with B. burgdorferi s.s. lost their spirochaete load after feeding on animals with high levels of LA-2 equivalent antibody; ticks feeding on animals which had only low or undetectable serum levels of LA-2 equivalent antibodies retained their spirochaete infection. Furthermore, animals with high levels of LA-2 equivalent antibody were protected against spirochaete infection. Our study shows that natural mouse reservoir hosts are highly competent to generate transmission-blocking antibodies after vaccination with a lipidated recombinant OspA and indicates that antibodies to the LA-2 epitope play a key role in the destruction of B. burgdorferi s.s. within feeding Ixodes ricinus ticks.

Various genetic or toxin-induced mouse models are frequently used for investigation of early PD pathology. Although olfactory impairment is known to precede motor symptoms by years, it is not known whether it is caused by impairments in the brain, the olfactory epithelium, or both. In this study, we investigated the olfactory function in three genetic Parkinson's disease (PD) mouse models and mice treated with MPTP intraperitoneally and intranasally. To investigate olfactory function, we performed electro-olfactogram recordings (EOGs) and an olfactory behavior test (cookie-finding test). We show that neither a parkin knockout mouse strain, nor intraperitoneal MPTP treated animals display any olfactory impairment in EOG recordings and the applied behavior test. We also found no difference in the responses of the olfactory epithelium to odorants in a mouse strain over-expressing doubly mutated \(\alpha\)-synuclein, while this mouse strain was not suitable to test olfaction in a cookie-finding test as it displays a mobility impairment. A transgenic mouse expressing mutated \(\alpha\)-synuclein in dopaminergic neurons performed equal to control animals in the cookie-finding test. Further we show that intranasal MPTP application can cause functional damage of the olfactory epithelium.

Summary The European rodents Clethrionomys glareolus (bank vole) and Apodemus flavicollis (yellow-necked-mouse) are important hosts of the tick species Ixodes ricinus, the main European vector of Borrelia burgdorferi. We have addressed the question whether or not these tick hosts develop resistance to I. ricinus larvae. C. glareolus and A. flavicollis were exposed to 40 I. ricinus larvae for five consecutive times at two week intervals. Resistance was tested by the following parameters: percentage of ticks fully engorged, time of attachment, engorgement index, percentage of recovered ticks and proportion of larvae moulting to nymphs. Repeated infestation ofC. glareolus resulted in progressive and significant reductions in the percentage of fully engorged ticks, the time of attachment of partially engorged ticks, the scutal index of partially engorged ticks and the moulting success. In contrast, repeatedly infested A. flavicollis did not acquire resistance to larval I. ricinus. Effects of resistance in C glareolus could be partially disrupted by treatment with the immunosuppressive agent, cyclosporin A (CsA), indicating that T helper cells participate in the immune responses to tick bites. The data suggest that acquired immunity to I. ricinus larvae in C. glareolus is a density-dependent factor regulating natural tick burdens and that it may have an impact on the transmission cycle of B. burgdorferi in Central Europe.

Pannexin 1 (Panx1) represents a class of vertebrate membrane channels, bearing significant sequence homology with the invertebrate gap junction proteins, the innexins and more distant similarities in the membrane topologies and pharmacological sensitivities with gap junction proteins of the connexin family. In the nervous system, cooperation among pannexin channels, adenosine receptors, and \(K_{ATP}\) channels modulating neuronal excitability via ATP and adenosine has been recognized, but little is known about the significance in vivo. However, the localization of Panx1 at postsynaptic sites in hippocampal neurons and astrocytes in close proximity together with the fundamental role of ATP and adenosine for CNS metabolism and cell signaling underscore the potential relevance of this channel to synaptic plasticity and higher brain functions. Here, we report increased excitability and potently enhanced early and persistent LTP responses in the CA1 region of acute slice preparations from adult \(Panx1^{−/−}\) mice. Adenosine application and N-methyl-D-aspartate receptor (NMDAR)-blocking normalized this phenotype, suggesting that absence of Panx1 causes chronic extracellular ATP/adenosine depletion, thus facilitating postsynaptic NMDAR activation. Compensatory transcriptional up-regulation of metabotropic glutamate receptor 4 (grm4) accompanies these adaptive changes. The physiological modification, promoted by loss of Panx1, led to distinct behavioral alterations, enhancing anxiety and impairing object recognition and spatial learning in \(Panx1^{−/−}\) mice. We conclude that ATP release through Panx1 channels plays a critical role in maintaining synaptic strength and plasticity in CA1 neurons of the adult hippocampus. This result provides the rationale for in-depth analysis of Panx1 function and adenosine based therapies in CNS disorders.

Background Chordate evolution is a history of innovations that is marked by physical and behavioral specializations, which led to the development of a variety of forms from a single ancestral group. Among other important characteristics, vertebrates obtained a well developed brain, anterior sensory structures, a closed circulatory system and gills or lungs as blood oxygenation systems. The duplication of pre-existing genes had profound evolutionary implications for the developmental complexity in vertebrates, since mutations modifying the function of a duplicated protein can lead to novel functions, improving the evolutionary success. Results We analyzed here the evolution of the GPRC5 family of G protein-coupled receptors by comprehensive similarity searches and found that the receptors are only present in chordates and that the size of the receptor family expanded, likely due to genome duplication events in the early history of vertebrate evolution. We propose that a single GPRC5 receptor coding gene originated in a stem chordate ancestor and gave rise by duplication events to a gene family comprising three receptor types (GPRC5A-C) in vertebrates, and a fourth homologue present only in mammals (GPRC5D). Additional duplications of GPRC5B and GPRC5C sequences occurred in teleost fishes. The finding that the expression patterns of the receptors are evolutionarily conserved indicates an important biological function of these receptors. Moreover, we found that expression of GPRC5B is regulated by vitamin A in vivo, confirming previous findings that linked receptor expression to retinoic acid levels in tumor cell lines and strengthening the link between the receptor expression and the development of a complex nervous system in chordates, known to be dependent on retinoic acid signaling. Conclusions GPRC5 receptors, a class of G protein-coupled receptors with unique sequence characteristics, may represent a molecular novelty that helped non-chordates to become chordates.

Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a “shifting” site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.

The geographic patterns of transmission opportunities of vector-borne zoonoses are determined by a complex interplay between the migration patterns of the host and the vector. Here we examine the impact of host migration on the spread of a tick-borne zoonotic disease, using Lyme Borreliosis (LB) spirochaetal species in Europe. We demonstrate that the migration of the LB species is dependent on and limited by the migration of their respective hosts. We note that populations of Borrelia spp. associated with birds (Borrelia garinii and B. valaisiana) show limited geographic structuring between countries compared with those associated with small mammals (Borrelia afzelii), and we argue that this can be explained by higher rates of migration in avian hosts. We also show the presence of B. afzelii strains in England and, through the use of the multi-locus sequence analysis scheme, reveal that the strains are highly structured. This pattern in English sites is very different from that observed at the continental sites, and we propose that these may be recent introductions.

Plasmid profiles of 2 Portuguese Borrelia lusitaniae strains, one isolated from a human patient and the other one from an Ixodes ricinus tick, were obtained by pulsed-field gel electrophoresis to evaluate the plasmid diversity in each strain. Overall, a maximum of 6 plasmids were detected that ranged from 19 kb to 76 kb, revealing completely different plasmid profiles from those previously described for other genospecies of B. burgdorferi sensu lato, the causative agents of Lyme borreliosis. The plasmid location of the ospA gene was investigated by hybridization, allowing its allocation to the plasmid of 70 kb instead of the 54 kb linear plasmid described for B. burgdorferi sensu stricto strains.

Calcium-activated chloride channels (CaCCs) are involved in many physiological processes, including sensory signal transduction, but only little is known to date about their structure and function. We performed a proteome analysis of the olfactory epithelium (OE) membrane proteome and identified so far uncharacterized membrane proteins as candidate channels. One of the most abundant membrane proteins in olfactory sensory neurons (OSNs) was Tmem16b, a member of a recently identified family of CaCCs. In addition to former studies performed on Tmem16b, we show here that Tmem16b expression is highly specific for the OE, in contrast to the closely related Tmem16a, which shows a broad expression pattern in secretory epithelial cells. Native Tmem16b is localized in the cilia of the OSNs, which is in agreement with previous electrophysiological recordings.

Chronic Chagasic patient immunoglobulins (CChP-IgGs) recognize an acidic amino acid cluster at the second extracellular loop (el2) of cardiac M(2)-muscarinic acetylcholine receptors (M(2)AChRs). These residues correspond to a common binding site for various allosteric agents. We characterized the nature of the M(2)AChR/CChP-IgG interaction in functional and radioligand binding experiments applying the same mainstream strategies previously used for the characterization of other allosteric agents. Dose-response curves of acetylcholine effect on heart rate were constructed with data from isolated heart experiments in the presence of CChP or normal blood donor (NBD) sera. In these experiments, CChP sera but not NBD sera increased the efficacy of agonist action by augmenting the onset of bradyarrhythmias and inducing a Hill slope of 2.5. This effect was blocked by gallamine, an M(2)AChR allosteric antagonist. Correspondingly, CChP-IgGs increased acetylcholine affinity twofold and showed negative cooperativity for [(3)H]-N-methyl scopolamine ([(3)H]-NMS) in allosterism binding assays. A peptide corresponding to the M(2)AChR-el2 blocked this effect. Furthermore, dissociation assays showed that the effect of gallamine on the [(3)H]-NMS off-rate was reverted by CChP-IgGs. Finally, concentration-effect curves for the allosteric delay of W84 on [(3)H]-NMS dissociation right shifted from an IC(50) of 33 nmol/L to 78 nmol/L, 992 nmol/L, and 1670 nmol/L in the presence of 6.7 x 10(- 8), 1.33 x 10(- 7), and 2.0 x 10(- 7) mol/L of anti-el2 affinity-purified CChP-IgGs. Taken together, these findings confirmed a competitive interplay of these ligands at the common allosteric site and revealed the novel allosteric nature of the interaction of CChP-IgGs at the M(2)AChRs as a positive cooperativity effect on acetylcholine action.