Botulinum neurotoxin type-A enters a non-recycling pool of synaptic vesicles

Neuronal communication relies on synaptic vesicles undergoing regulated exocytosis and recycling for multiple rounds of fusion. Whether all synaptic vesicles have identical protein content has been challenged, suggesting that their recycling ability may differ greatly. Botulinum neurotoxin type-A (BoNT/A) is a highly potent neurotoxin that is internalized in synaptic vesicles at motor nerve terminals and induces flaccid paralysis. Recently, BoNT/A was also shown to undergo retrograde transport, suggesting it might enter a specific pool of synaptic vesicles with a retrograde trafficking fate. Using high-resolution microscopy techniques including electron microscopy and single molecule imaging, we found that the BoNT/A binding domain is internalized within a subset of vesicles that only partially co-localize with cholera toxin B-subunit and have markedly reduced VAMP2 immunoreactivity. Synaptic vesicles loaded with pHrodo-BoNT/A-Hc exhibited a significantly reduced ability to fuse with the plasma membrane in mouse hippocampal nerve terminals when compared with pHrodo-dextran-containing synaptic vesicles and pHrodo-labeled anti-GFP nanobodies bound to VAMP2-pHluorin or vGlut-pHluorin. Similar results were also obtained at the amphibian neuromuscular junction. These results reveal that BoNT/A is internalized in a subpopulation of synaptic vesicles that are not destined to recycle, highlighting the existence of significant molecular and functional heterogeneity between synaptic vesicles.

José Penna y Salvador Mazza en tiempos del cólera : salud, inmigración y legitimidad política en la Argentina de 1910

Resumen: Este artículo estudia las teorías sobre el origen, contagio y control del cólera en el siglo XIX, los intentos de las autoridades de la Argentina por contrarrestar estas epidemias y por último, la campaña anticolérica de 1910. Hasta ese momento, las medidas preventivas habían priorizado la vigilancia, desinfección y aislamiento de viviendas, objetos y personas infectadas. Pero el reciente descubrimiento de la transmisión el cólera por individuos asintomáticos hizo que en 1910 el Departamento Nacional de Higiene (DNH) impusiese un sistema de análisis bacteriológico obligatorio. En particular, el artículo examina las ideas y actividades de José Penna, quien en 1910 se desempeñaba como director del DGN y de Salvador Mazza. Un médico recién recibido, este último estuvo a cargo del laboratorio bacteriológico del lazareto de Martín García donde se sometía a estudio a todos los pasajeros de tercera clase provenientes de zonas infectadas de cólera. El DNH presentó la campaña anticolérica de 1910 como resultado de la experiencia acumulada durante el siglo XIX, del progreso científico y administrativo de la Argentina y de los esfuerzos de las autoridades por proteger a la nación. En un momento en que la elite argentina luchaba para mantener su dominio, tanto reprimiendo como buscando co-optar a la oposición, las cuestiones de salud pública constituyeron un elemento importante de la retórica política.

Death or taxes? The political economy of sanitation expenditure in nineteenth-century Britain

This thesis consists of three papers studying the relationship between democratic reform, expenditure on sanitation public goods and mortality in Britain in the second half of the nineteenth century. During this period decisions over spending on critical public goods such as water supply and sewer systems were made by locally elected town councils, leading to extensive variation in the level of spending across the country. This dissertation uses new historical data to examine the political factors determining that variation, and the consequences for mortality rates.

The first substantive chapter describes the spread of government sanitation expenditure, and analyzes the factors that determined towns' willingness to invest. The results show the importance of towns' financial constraints, both in terms of the available tax base and access to borrowing, in limiting the level of expenditure. This suggests that greater involvement by Westminster could have been very effective in expediting sanitary investment. There is little evidence, however, that democratic reform was an important driver of greater expenditure.

Chapter 3 analyzes the effect of extending voting rights to the poor on government public goods spending. A simple model predicts that the rich and the poor will desire lower levels of public goods expenditure than the middle class, and so extensions of the right to vote to the poor will be associated with lower spending. This prediction is tested using plausibly exogenous variation in the extent of the franchise. The results strongly support the theoretical prediction: expenditure increased following relatively small extensions of the franchise, but fell once more than approximately 50% of the adult male population held the right to vote.

Chapter 4 tests whether the sanitary expenditure was effective in combating the high mortality rates following the Industrial Revolution. The results show that increases in urban expenditure on sanitation-water supply, sewer systems and streets-was extremely effective in reducing mortality from cholera and diarrhea.

Surveying bacterial human pathogens in shrimp culture in Thailand

Any presence of bacterial human pathogen in shrimp products may be of public health concern. This note concludes that Salmonella do not appear to constitute a part of the microbial flora where shrimp culture is practiced in Thailand. Vibrio cholerae 01, the cause of cholera are rarely recovered from the environment with no isolates containing genes encoding cholera toxin. Further studies are needed to describe the prevalence of bacterial human pathogens in shrimp culture, especially determination of possible postharvest cross-contamnation with these pathogens

Avaliação microbiológica, detecção e susceptibilidade a antimicrobianos de potenciais enteropatógenos das famílias Enterobacteriaceae e Vibrionaceae em mexilhões Perna perna da praia de Itaipu, Niterói-RJ

Por serem organismos filtradores, os mexilhões devem ser extraídos para consumo somente de águas com padrões microbiológicos regulamentados pelo Conselho Nacional do Meio Ambiente, uma vez que intoxicações alimentares de origem bacteriana são as consequências mais comuns relacionadas ao consumo destes moluscos. Após a retirada dos costões, estes organismos passam por processos de fervura, lavagem, acondicionamento em sacos plásticos e transporte até a chegada ao mercado onde são comercializados; etapas estas, realizadas sem cuidados assépticos. Objetivando avaliar a qualidade microbiológica de mexilhões Perna perna coletados na praia de Itaipu, Niterói, RJ; após a sua fervura; e comercializados no Mercado São Pedro, foram realizadas contagens de coliformes totais e termotolerantes e pesquisa de Escherichia coli, Salmonella spp. e Vibrio spp. por metodologia convencional e molecular em amostras obtidas destes locais. Pelo teste de disco-difusão foi observado o perfil de resistência das cepas isoladas. Do total de amostras analisadas (27) apenas 3 dos mexilhões que sofreram processo de aferventação, 1 do comercializado e 3 do in natura, se encontravam dentro dos padrões aceitáveis pela legislação. Não houve diferença significativa entre as contagens de coliformes termotolerantes dos diferentes mexilhões analisados, mas sim entre os períodos seco e chuvoso. Somente uma das nove coletas de água mostrou-se própria para o cultivo de mexilhões (até 14 CF/mL). Foram isoladas e caracterizadas fisiológicamente, 77 estirpes da espécie E. coli, sendo confirmadas molecularmente por PCR os sorotipos EPEC, STEC e EAEC; 4 cepas Salmonella spp. sendo apenas uma confirmada por PCR; e das 57 cepas caracterizadas como Vibrio spp., 51 foram confirmadas por PCR, sendo 46 Vibrio spp., 2 V. cholerae, 1 V. vulnificus, 1 V. parahaemolyticus e 1 V. mimicus. Entre as estirpes de E. coli, 13% apresentaram multirresistência e 15,6% apresentaram resistência múltipla. A estirpe de Salmonella spp. se mostrou sensível a todos os antimicrobiados testados. Das estirpes de Vibrio spp. testadas, 68,6% apresentaram multirresistência e 72,5% apresentaram resistência múltipla. A partir da pesquisa de genes direto do caldo de enriquecimento foi possível detectar todos os genes pesquisados, com exceção para os sorotipos de Salmonella e V. cholerae. Baseado nos resultados do presente trabalho pode-se inferir que os mexilhões, amplamente comercializados no município de Itaipú, podem se constituir em risco para a saúde pública dos consumidores no Rio de Janeiro, necessitando dos órgãos competentes uma eficiente fiscalização nos pontos de venda e cultivo destes moluscos.

Antibacterial activity of a marine bacterium against pathogenic and environmental isolates of vibrio species

The marine environment covers three quarters of the surface of the planet is estimated to be home to more than 80% of life and yet it remains largely unexplored. The rich diversity of marine flora and fauna and its adaptation to the harsh marine environment coupled with new developments in biotechnology, has opened up a new exciting vista for extraction of bioactive products of use in medicine. In this study inhibitory activity of a marine bacterium isolated from gut of ribbonfish was studied against pathogenic and environmental isolates of Vibrio species. This strain was identified as Pseudomonas stutzeri and it was found active against V. harveyi (luminescent bacteria), V. cholerae, V. alginolyticus, V. damseal, V. fluvialis. The antibacterial substance produced by Pseudomonas stutzeri was soluble in organic solvent and closely bound to external surface of bacterial cells. Reduction of the absorbance of the V. cholera cell suspension was observed when log phase cells of V. cholerae were treated with MIC and 4xMIC concentration of crude extract of Pseudomonas stutzeri.

Preparation and characterization of a novel pyrrole-benzophenone copolymerized silica nanocomposite as a reagent in a visual immunologic-agglutination test.

Biological sensing is explored through novel stable colloidal dispersions of pyrrole-benzophenone and pyrrole copolymerized silica (PPy-SiO(2)-PPyBPh) nanocomposites, which allow covalent linking of biological molecules through light mediation. The mechanism of nanocomposite attachment to a model protein is studied by gold labeled cholera toxin B (CTB) to enhance the contrast in electron microscopy imaging. The biological test itself is carried out without gold labeling, i.e., using CTB only. The protein is shown to be covalently bound through the benzophenone groups. When the reactive PPy-SiO(2)-PPyBPh-CTB nanocomposite is exposed to specific recognition anti-CTB immunoglobulins, a qualitative visual agglutination assay occurs spontaneously, producing as a positive test, PPy-SiO(2)-PPyBPh-CTB-anti-CTB, in less than 1 h, while the control solution of the PPy-SiO(2)-PPyBPh-CTB alone remained well-dispersed during the same period. These dispersions were characterized by cryogenic transmission microscopy (cryo-TEM), scanning electron microscopy (SEM), FTIR and X-ray photoelectron spectroscopy (XPS).

Self-assembled monolayer growth of phospholipids on hydrophobic surface toward mimetic biomembranes: Scanning probe microscopy study

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used simultaneously to analyze a model membrane bilayer structure consisting of a phospholipid outer monolayer deposited onto organosilane-derivatized mica surfaces, which were constructed by using painting and self-assembly methods. The phospholipid used as outer monolayer was dimyristoylphosphatidylcholine (DMPC). The hydrocarbon-covered substrate that formed the inner half bilayer was composed of a self-assembly monolayer (SAM) of octadecyltrichloroorganosilane (OTS) on mica. SAMs of DMPC were formed by exposing hydrophobic mica to a solution of DMPC in decane/isobutanol and subsequently immersing into pure water. AFM images of samples immersed in solution for varying exposure times showed that before forming a complete monolayer the molecules aggregated into dense islands (2.2-2.6 nm high) on the surface. The islands had a compact and rounded morphology. LFM, coupled with topographic data obtained with the atomic force mode, had made possible the distinction between DMPC and OTS. The rate constant of DMPC growth was calculated. This is the first systematic study of the SAM formation of DMPC by AFM and LFM imaging. It reveals more direct information about the film morphology than previous studies with conventional surface analytical techniques such as infrared spectroscopy, X-ray, or fluorescence microscopy.

Adenovirus F protein as a delivery vehicle for botulinum B.

BACKGROUND: Immunization with recombinant carboxyl-terminal domain of the heavy chain (Hc domain) of botulinum neurotoxin (BoNT) stimulates protective immunity against native BoNT challenge. Most studies developing a botulism vaccine have focused on the whole Hc; however, since the principal protective epitopes are located within beta-trefoil domain (Hcbetatre), we hypothesize that immunization with the Hcbetatre domain is sufficient to confer protective immunity. In addition, enhancing its uptake subsequent to nasal delivery prompted development of an alternative vaccine strategy, and we hypothesize that the addition of targeting moiety adenovirus 2 fiber protein (Ad2F) may enhance such uptake during vaccination. RESULTS: The Hcbetatre serotype B immunogen was genetically fused to Ad2F (Hcbetatre/B-Ad2F), and its immunogenicity was tested in mice. In combination with the mucosal adjuvant, cholera toxin (CT), enhanced mucosal IgA and serum IgG Ab titers were induced by nasal Hcbetatre-Ad2F relative to Hcbetatre alone; however, similar Ab titers were obtained upon intramuscular immunization. These BoNT/B-specific Abs induced by nasal immunization were generally supported in large part by Th2 cells, as opposed to Hcbetatre-immunized mice that showed more mixed Th1 and Th2 cells. Using a mouse neutralization assay, sera from animals immunized with Hcbetatre and Hcbetatre-Ad2F protected mice against 2.0 LD50. CONCLUSION: These results demonstrate that Hcbetatre-based immunogens are highly immunogenic, especially when genetically fused to Ad2F, and Ad2F can be exploited as a vaccine delivery platform to the mucosa.

Lack of evidence for ectopic sprouting of genetically labeled Aβ touch afferents in inflammatory and neuropathic trigeminal pain.

BACKGROUND: Mechanical and in particular tactile allodynia is a hallmark of chronic pain in which innocuous touch becomes painful. Previous cholera toxin B (CTB)-based neural tracing experiments and electrophysiology studies had suggested that aberrant axon sprouting from touch sensory afferents into pain-processing laminae after injury is a possible anatomical substrate underlying mechanical allodynia. This hypothesis was later challenged by experiments using intra-axonal labeling of A-fiber neurons, as well as single-neuron labeling of electrophysiologically identified sensory neurons. However, no studies have used genetically labeled neurons to examine this issue, and most studies were performed on spinal but not trigeminal sensory neurons which are the relevant neurons for orofacial pain, where allodynia oftentimes plays a dominant clinical role. FINDINGS: We recently discovered that parvalbumin::Cre (Pv::Cre) labels two types of Aβ touch neurons in trigeminal ganglion. Using a Pv::CreER driver and a Cre-dependent reporter mouse, we specifically labeled these Aβ trigeminal touch afferents by timed taxomifen injection prior to inflammation or infraorbital nerve injury (ION transection). We then examined the peripheral and central projections of labeled axons into the brainstem caudalis nucleus after injuries vs controls. We found no evidence for ectopic sprouting of Pv::CreER labeled trigeminal Aβ axons into the superficial trigeminal noci-receptive laminae. Furthermore, there was also no evidence for peripheral sprouting. CONCLUSIONS: CreER-based labeling prior to injury precluded the issue of phenotypic changes of neurons after injury. Our results suggest that touch allodynia in chronic orofacial pain is unlikely caused by ectopic sprouting of Aβ trigeminal afferents.

Heat-labile enterotoxin: beyond G(m1) binding.

Enterotoxigenic Escherichia coli (ETEC) is a significant source of morbidity and mortality worldwide. One major virulence factor released by ETEC is the heat-labile enterotoxin LT, which is structurally and functionally similar to cholera toxin. LT consists of five B subunits carrying a single catalytically active A subunit. LTB binds the monosialoganglioside G(M1), the toxin's host receptor, but interactions with A-type blood sugars and E. coli lipopolysaccharide have also been identified within the past decade. Here, we review the regulation, assembly, and binding properties of the LT B-subunit pentamer and discuss the possible roles of its numerous molecular interactions.

Pseudomonas aeruginosa vesicles associate with and are internalized by human lung epithelial cells.

BACKGROUND: Pseudomonas aeruginosa is the major pathogen associated with chronic and ultimately fatal lung infections in patients with cystic fibrosis (CF). To investigate how P. aeruginosa-derived vesicles may contribute to lung disease, we explored their ability to associate with human lung cells. RESULTS: Purified vesicles associated with lung cells and were internalized in a time- and dose-dependent manner. Vesicles from a CF isolate exhibited a 3- to 4-fold greater association with lung cells than vesicles from the lab strain PAO1. Vesicle internalization was temperature-dependent and was inhibited by hypertonic sucrose and cyclodextrins. Surface-bound vesicles rarely colocalized with clathrin. Internalized vesicles colocalized with the endoplasmic reticulum (ER) marker, TRAPalpha, as well as with ER-localized pools of cholera toxin and transferrin. CF isolates of P. aeruginosa abundantly secrete PaAP (PA2939), an aminopeptidase that associates with the surface of vesicles. Vesicles from a PaAP knockout strain exhibited a 40% decrease in cell association. Likewise, vesicles from PAO1 overexpressing PaAP displayed a significant increase in cell association. CONCLUSION: These data reveal that PaAP promotes the association of vesicles with lung cells. Taken together, these results suggest that P. aeruginosa vesicles can interact with and be internalized by lung epithelial cells and contribute to the inflammatory response during infection.

Langerhans Cells Prime IL-17-Producing T Cells and Dampen Genital Cytotoxic Responses following Mucosal Immunization

Langerhans cells (LCs) are dendritic cells (DCs) localized in stratified epithelia, such as those overlaying skin, buccal mucosa, and vagina. The contribution of LCs to the promotion or control of immunity initiated at epithelial sites remains debated. We report in this paper that an immunogen comprising OVA linked to the B subunit of cholera toxin, used as delivery vector, was efficient to generate CTLs after vaginal immunization. Using Lang-EGFP mice, we evaluated the contribution of distinct DC subsets to the generation of CD4 and CD8 T cell responses. We demonstrate that the vaginal epithelium, unlike the skin epidermis, includes a minor population of LCs and a major subset of langerin(-) DCs. Intravaginally administered Ag is taken up by LCs and langerin(-) DCs and carried up to draining lymph nodes, where both subsets prime CD8 T cells, unlike blood-derived DCs, although with distinct capabilities. LCs prime CD8 T cells with a cytokine profile dominated by IL-17, whereas Lang(-) DCs induce IFN-gamma-producing T cells. Using Lang-DTR-EGFP mice to ensure a transient ablation of LCs, we found that these cells not only are dispensable for the generation of genital CTL responses but also downregulate these responses, by a mechanism that may involve IL-10 and IL-17 cytokines. This finding has implications for the development of mucosal vaccines and immunotherapeutic strategies designed for the targeting of DCs.

Old Town Glasgow: Squalor, Agitation and City Improvements

In 1862, Glasgow Corporation initiated the first of a series of three legislative acts which would become known collectively as the City Improvements Acts. Despite having some influence on the nature of the built fabric on the expanding city as a whole, the most extensive consequences of these acts was reserved for one specific area of the city, the remnants of the medieval Old Town. As the city had expanded towards all points of the compass in a regular, grid-iron structure throughout the nineteenth century, the Old Town remained singularly as a densely wrought fabric of medieval wynds, vennels, oblique passageways and accelerated tenementalisation. Here, as the rest of the city began to assume the form of an ordered entity, visible and classifiable, one could still find and addresses such as ‘Bridgegate, No. 29, backland, stair first left, three up, right lobby, door facing’ (quoted in Pacione, 1995).

Unsurprisingly, this place, where proximity to the midden (dung-heap) was considered an enviable position, was seen by the authorities as a major health hazard and a source not only of cholera, but also of the more alarming typhoid epidemic of 1842. Accordingly, the demolitions which occurred in the backlands of the Old Town under the first of the acts, the Glasgow Police Act of 1862, were justified on health and medical grounds. But disease was not the only social problem thought to issue from this district. Reports from social reformers including Fredrick Engels suggested that the decay of the area’s physical fabric could be extended to the moral profile of its inhabitants. This was in such a state of degeneracy that there were calls for a nearby military barracks to be relocated to more salubrious climes because troops were routinely coming into contact ‘with the most dissolute and profligate portion of the population’ (Peter Clonston, Lord Provost, June 1861). Perhaps more worrying for the city fathers, however, was that the barracks’ arsenal was seen as a potential source of arms for the militant and often illegal cotton workers’ unions and organisations who inhabited the Old Town as well as the districts to the east. In fact, the Old Town and East End had been the site of numerous working class actions and riots since 1787, including a strike of 60,000 workers in 1820, 100,000 in 1838, and the so-called Bread Riots of 1848 where shouts of ‘Vive La Revolution’ were reported in the Gallowgate.

The events in Paris in 1848 precipitated Baron Hausmann’s interventions into that city. The boulevards were in turn visited by members of Glasgow Corporation and ultimately, it can be argued, provided an example for Old Town Glasgow. This paper suggests that the city improvement acts carried a similarly complex and pervasive agenda, one which embodied not only health, class conflict and sexual morality but also the more local condition of sectarianism. And, like in Paris, these were played out spatially in a extensive reconfiguration of the urban fabric of the Old Town which, through the creation of new streets and a railway yard, not only made it more amenable to large scale military manoeuvres but also, opened up the area to capitalist accumulation. By the end of the works, the medieval heritage of the Old Town had been almost completely razed, the working class and Catholic East End had, through the insertion of the railway yard, been isolated from the city centre and approximately 70,000 people had been made homeless.

Nasal immunization of mice with human papillomavirus type 16 virus-like particles elicits neutralizing antibodies in mucosal secretions.

To specifically induce a mucosal antibody response to purified human papillomavirus type 16 (HPV16) virus-like particles (VLP), we immunized female BALB/c mice orally, intranasally, and/or parenterally and evaluated cholera toxin (CT) as a mucosal adjuvant. Anti-HPV16 VLP immunoglobulin G (IgG) and IgA titers in serum, saliva, and genital secretions were measured by enzyme-linked immunosorbent assay (ELISA). Systemic immunizations alone induced HPV16 VLP-specific IgG in serum and, to a lesser extent, in genital secretions but no secretory IgA. Oral immunization, even in the presence of CT, was inefficient. However, three nasal immunizations with 5 microgram of VLP given at weekly intervals to anesthetized mice induced high (>10(4)) and long-lasting (>15 weeks) titers of anti-HPV16 VLP antibodies in all samples, including IgA and IgG in saliva and genital secretions. CT enhanced the VLP-specific antibody response 10-fold in serum and to a lesser extent in saliva and genital secretions. Nasal immunization of conscious mice compared to anesthetized mice was inefficient and correlated with the absence of uptake of a marker into the lung. However, a 1-microgram VLP systemic priming followed by two 5-microgram VLP intranasal boosts in conscious mice induced both HPV16 VLP-specific IgG and IgA in secretions, although the titers were lower than in anesthetized mice given three intranasal immunizations. Antibodies in serum, saliva, and genital secretions of immunized mice were strongly neutralizing in vitro (50% neutralization with ELISA titers of 65 to 125). The mucosal and systemic/mucosal HPV16 VLP immunization protocols that induced significant titers of neutralizing IgG and secretory IgA in mucosal secretions in mice may be relevant to genital HPV VLP-based human vaccine trials.