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Find video protocols related to scientific articles indexed in Pubmed.
Production of Water-Soluble Few-Layer Graphene Mesosheets by Dry Milling with Hydrophobic Drug.
Langmuir
PUBLISHED: 11-20-2014
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A novel, fast and easy mechano-chemistry-based (dry milling) method has been developed to exfoliate graphene with hydrophobic drugs generating few layer graphene mesosheets (< 10 nm in thickness and ~ 1 µm in width). The electronic properties of the graphitic structure were partially preserved after the milling treatment compared to Graphene Oxide (GO) prepared by Hummers' method. Several characterization techniques such as thermogravimetric analysis (TGA), Raman spectroscopy, atomic force microscopy (AFM), Electron Microscopy (EM) and molecular dynamics simulation were used to characterize this material. The drug-exfoliated mesosheets were pharmacologically inactive offering a new approach for making water-soluble few-layer graphene mesosheets upon dry milling with hydrophobic drugs, mainly used as exfoliating agents.
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Pathogenicity mechanisms and host response during oral Candida albicans infections.
Expert Rev Anti Infect Ther
PUBLISHED: 05-07-2014
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Oral candidiasis remains one of the most common forms of Candida infections and occurs if the balance between host, Candida and microbiota is disturbed, e.g., by broad spectrum antibiotics or immunosuppression. In recent years, identification of fungal factors contributing to host cell damage and new insights into host defense mechanisms have significantly extended our understanding of the pathogenesis of oral candidiasis. In this review, we will provide an overview of the pathogenicity mechanisms during oral Candida infections and discuss some approaches by which this knowledge could be transferred into therapeutic approaches.
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Oral and vaginal epithelial cell lines bind and transfer cell-free infectious HIV-1 to permissive cells but are not productively infected.
PLoS ONE
PUBLISHED: 01-01-2014
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The majority of HIV-1 infections worldwide are acquired via mucosal surfaces. However, unlike the vaginal mucosa, the issue of whether the oral mucosa can act as a portal of entry for HIV-1 infection remains controversial. To address potential differences with regard to the fate of HIV-1 after exposure to oral and vaginal epithelium, we utilized two epithelial cell lines representative of buccal (TR146) and pharyngeal (FaDu) sites of the oral cavity and compared them with a cell line derived from vaginal epithelium (A431) in order to determine (i) HIV-1 receptor gene and protein expression, (ii) whether HIV-1 genome integration into epithelial cells occurs, (iii) whether productive viral infection ensues, and (iv) whether infectious virus can be transferred to permissive cells. Using flow cytometry to measure captured virus by HIV-1 gp120 protein detection and western blot to detect HIV-1 p24 gag protein, we demonstrate that buccal, pharyngeal and vaginal epithelial cells capture CXCR4- and CCR5-utilising virus, probably via non-canonical receptors. Both oral and vaginal epithelial cells are able to transfer infectious virus to permissive cells either directly through cell-cell attachment or via transcytosis of HIV-1 across epithelial cells. However, HIV-1 integration, as measured by real-time PCR and presence of early gene mRNA transcripts and de novo protein production were not detected in either epithelial cell type. Importantly, both oral and vaginal epithelial cells were able to support integration and productive infection if HIV-1 entered via the endocytic pathway driven by VSV-G. Our data demonstrate that under normal conditions productive HIV-1 infection of epithelial cells leading to progeny virion production is unlikely, but that epithelial cells can act as mediators of systemic viral dissemination through attachment and transfer of HIV-1 to permissive cells.
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Protection against epithelial damage during Candida albicans infection is mediated by PI3K/Akt and mTOR signaling.
J. Infect. Dis.
PUBLISHED: 12-19-2013
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Background.?The ability of epithelial cells (ECs) to discriminate between commensal and pathogenic microbes is essential for healthy living. Key to these interactions are mucosal epithelial responses to pathogen-induced damage.Methods.?Using reconstituted oral epithelium we assessed epithelial gene transcriptional responses to C. albicans infection by microarray. Signal pathway activation was monitored by Western blot and Transcription factor ELISA, and their role in C. albicans-induced damage protection determined using chemical inhibitors.Results.?Transcript profiling demonstrated early up-regulation of epithelial genes involved in immune responses. Many of these genes constituted components of signaling pathways, but only NF-?B, MAPK and PI3K/Akt pathways were functionally activated. We demonstrate that PI3K/Akt signaling is independent of NF-?B and MAPK signaling and plays a key role in epithelial immune activation and damage protection via mTOR activation.Conclusion.?PI3K/Akt/mTOR signaling may play a critical role in protecting epithelial cells from damage during mucosal fungal infections independently of NF-?B or MAPK signaling.
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Clotrimazole dampens vaginal inflammation and neutrophil infiltration in response to Candida albicans infection.
Antimicrob. Agents Chemother.
PUBLISHED: 07-29-2013
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The pathology of vulvovaginal candidiasis (VVC) caused by Candida albicans is associated with a nonprotective inflammatory response and is frequently treated with clotrimazole. We investigated the mechanisms by which clotrimazole resolves VVC. Low levels of clotrimazole, which do not block fungal growth, inhibit expression of a "danger response" transcription factor, c-Fos, block production of proinflammatory cytokines, and inhibit neutrophil infiltration to the site of infection.
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Candida albicans cell wall glycosylation may be indirectly required for activation of epithelial cell proinflammatory responses.
Infect. Immun.
PUBLISHED: 09-19-2011
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Oral epithelial cells discriminate between the yeast and hyphal forms of Candida albicans via the mitogen-activated protein kinase (MAPK) signaling pathway. This occurs through phosphorylation of the MAPK phosphatase MKP1 and activation of the c-Fos transcription factor by the hyphal form. Given that fungal cell wall polysaccharides are critical in host recognition and immune activation in myeloid cells, we sought to determine whether ?-glucan and N- or O-glycosylation was important in activating the MAPK/MKP1/c-Fos hypha-mediated response mechanism and proinflammatory cytokines in oral epithelial cells. Using a series of ?-glucan and N- and O-mannan mutants, we found that N-mannosylation (via ?och1 and ?pmr1 mutants) and O-mannosylation (via ?pmt1 and ?mnt1 ?mnt2 mutants), but not phosphomannan (via a ?mnn4 mutant) or ?-1,2 mannosylation (via ?bmt1 to ?bmt6 mutants), were required for MKP1/c-Fos activation, proinflammatory cytokine production, and cell damage induction. However, the N- and O-mannan mutants showed reduced adhesion or lack of initial hypha formation at 2 h, resulting in little MKP1/c-Fos activation, or restricted hypha formation/pseudohyphal formation at 24 h, resulting in minimal proinflammatory cytokine production and cell damage. Further, the ?-1,6-mannose backbone of the N-linked outer chain (corresponding to a ?mnn9 mutant) may be required for epithelial adhesion, while the ?-1,2-mannose component of phospholipomannan (corresponding to a ?mit1 mutant) may contribute to epithelial cell damage. ?-Glucan appeared to play no role in adhesion, epithelial activation, or cell damage. In summary, N- and O-mannosylation defects affect the ability of C. albicans to induce proinflammatory cytokines and damage in oral epithelial cells, but this may be due to indirect effects on fungal pathogenicity rather than mannose residues being direct activators of the MAPK/MKP1/c-Fos hypha-mediated immune response.
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Candida albicans yeast and hyphae are discriminated by MAPK signaling in vaginal epithelial cells.
PLoS ONE
PUBLISHED: 07-01-2011
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We previously reported that a bi-phasic innate immune MAPK response, constituting activation of the mitogen-activated protein kinase (MAPK) phosphatase MKP1 and c-Fos transcription factor, discriminates between the yeast and hyphal forms of Candida albicans in oral epithelial cells (ECs). Since the vast majority of mucosal Candida infections are vaginal, we sought to determine whether a similar bi-phasic MAPK-based immune response was activated by C. albicans in vaginal ECs. Here, we demonstrate that vaginal ECs orchestrate an innate response to C. albicans via NF-?B and MAPK signaling pathways. However, unlike in oral ECs, the first MAPK response, defined by c-Jun transcription factor activation, is delayed until 2 h in vaginal ECs but is still independent of hypha formation. The second or late MAPK response, constituting MKP1 and c-Fos transcription factor activation, is identical to oral ECs and is dependent upon both hypha formation and fungal burdens. NF-?B activation is immediate but independent of morphology. Furthermore, the proinflammatory response in vaginal ECs is different to oral ECs, with an absence of G-CSF and CCL20 and low level IL-6 production. Therefore, differences exist in how C. albicans activates signaling mechanisms in oral and vaginal ECs; however, the activation of MAPK-based pathways that discriminate between yeast and hyphal forms is retained between these mucosal sites. We conclude that this MAPK-based signaling pathway is a common mechanism enabling different human epithelial tissues to orchestrate innate immune responses specifically against C. albicans hyphae.
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Activation of MAPK/c-Fos induced responses in oral epithelial cells is specific to Candida albicans and Candida dubliniensis hyphae.
Med. Microbiol. Immunol.
PUBLISHED: 05-27-2011
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Oral epithelial cells detect the human pathogenic fungus Candida albicans via NF-?B and a bi-phasic mitogen-activated protein kinase (MAPK) signaling response. However, discrimination between C. albicans yeast and hyphal forms is mediated only by the MAPK pathway, which constitutes activation of the MAPK phosphatase MKP1 and the c-Fos transcription factor and is targeted against the hyphal form. Given that C. albicans is not the only Candida species capable of filamentation or causing mucosal infections, we sought to determine whether this MAPK/MKP1/c-Fos mediated response mechanism was activated by other pathogenic Candida species, including C. dubliniensis, C. tropicalis, C. parapsilosis, C. glabrata and C. krusei. Although all Candida species activated the NF-?B signaling pathway, only C. albicans and C. dubliniensis were capable of inducing MKP1 and c-Fos activation, which directly correlated with hypha formation. However, only C. albicans strongly induced cytokine production (G-CSF, GM-CSF, IL-6 and IL-1?) and cell damage. Candida dubliniensis, C. tropicalis and C. parapsilosis were also capable of inducing IL-1? and this correlated with mild cell damage and was dependent upon fungal burdens. Our data demonstrate that activation of the MAPK/MKP1/c-Fos pathway in oral epithelial cells is specific to C. dubliniensis and C. albicans hyphae.
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Candida albicans interactions with epithelial cells and mucosal immunity.
Microbes Infect.
PUBLISHED: 05-05-2011
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Candida albicans interactions with epithelial cells are critical for commensal growth, fungal pathogenicity and host defence. This review will outline our current understanding of C. albicans-epithelial interactions and will discuss how this may lead to the induction of a protective mucosal immune response.
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Mucosal immunity and Candida albicans infection.
Clin. Dev. Immunol.
PUBLISHED: 02-01-2011
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Interactions between mucosal surfaces and microbial microbiota are key to host defense, health, and disease. These surfaces are exposed to high numbers of microbes and must be capable of distinguishing between those that are beneficial or avirulent and those that will invade and cause disease. Our understanding of the mechanisms involved in these discriminatory processes has recently begun to expand as new studies bring to light the importance of epithelial cells and novel immune cell subsets such as T(h)17 T cells in these processes. Elucidating how these mechanisms function will improve our understanding of many diverse diseases and improve our ability to treat patients suffering from these conditions. In our voyage to discover these mechanisms, mucosal interactions with opportunistic commensal organisms such as the fungus Candida albicans provide insights that are invaluable. Here, we review current knowledge of the interactions between C. albicans and epithelial surfaces and how this may shape our understanding of microbial-mucosal interactions.
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A biphasic innate immune MAPK response discriminates between the yeast and hyphal forms of Candida albicans in epithelial cells.
Cell Host Microbe
PUBLISHED: 03-18-2010
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Discriminating between commensal and pathogenic states of opportunistic pathogens is critical for host mucosal defense and homeostasis. The opportunistic human fungal pathogen Candida albicans is also a constituent of the normal oral flora and grows either as yeasts or hyphae. We demonstrate that oral epithelial cells orchestrate an innate response to C. albicans via NF-?B and a biphasic MAPK response. Activation of NF-?B and the first MAPK phase, constituting c-Jun activation, is independent of morphology and due to fungal cell wall recognition. Activation of the second MAPK phase, constituting MKP1 and c-Fos activation, is dependent upon hypha formation and fungal burdens and correlates with proinflammatory responses. Such biphasic response may allow epithelial tissues to remain quiescent under low fungal burdens while responding specifically and strongly to damage-inducing hyphae when burdens increase. MAPK/MKP1/c-Fos activation may represent a "danger response" pathway that is critical for identifying and responding to the pathogenic switch of commensal microbes.
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Glycosylation of Candida albicans cell wall proteins is critical for induction of innate immune responses and apoptosis of epithelial cells.
PLoS ONE
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C. albicans is one of the most common fungal pathogen of humans, causing local and superficial mucosal infections in immunocompromised individuals. Given that the key structure mediating host-C. albicans interactions is the fungal cell wall, we aimed to identify features of the cell wall inducing epithelial responses and be associated with fungal pathogenesis. We demonstrate here the importance of cell wall protein glycosylation in epithelial immune activation with a predominant role for the highly branched N-glycosylation residues. Moreover, these glycan moieties induce growth arrest and apoptosis of epithelial cells. Using an in vitro model of oral candidosis we demonstrate, that apoptosis induction by C. albicans wild-type occurs in early stage of infection and strongly depends on intact cell wall protein glycosylation. These novel findings demonstrate that glycosylation of the C. albicans cell wall proteins appears essential for modulation of epithelial immunity and apoptosis induction, both of which may promote fungal pathogenesis in vivo.
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The mycobiome: influencing IBD severity.
Cell Host Microbe
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The etiology and maintenance of inflammatory bowel disease (IBD) is the subject of much speculation. Iliev et al. (2012) impose a change in our views of the gut microbiome and catapult the fungal "mycobiome" center-stage in the exploration of IBD.
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Evaluation of the role of Candida albicans agglutinin-like sequence (Als) proteins in human oral epithelial cell interactions.
PLoS ONE
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The fungus C. albicans uses adhesins to interact with human epithelial surfaces in the processes of colonization and pathogenesis. The C. albicans ALS (agglutinin-like sequence) gene family encodes eight large cell-surface glycoproteins (Als1-Als7 and Als9) that have adhesive function. This study utilized C. albicans ?als mutant strains to investigate the role of the Als family in oral epithelial cell adhesion and damage, cytokine induction and activation of a MAPK-based (MKP1/c-Fos) signaling pathway that discriminates between yeast and hyphae. Of the eight ?als mutants tested, only the ?als3 strain showed significant reductions in oral epithelial cell adhesion and damage, and cytokine production. High fungal:epithelial cell multiplicities of infection were able to rescue the cell damage and cytokine production phenotypes, demonstrating the importance of fungal burden in mucosal infections. Despite its adhesion, damage and cytokine induction phenotypes, the ?als3 strain induced MKP1 phosphorylation and c-Fos production to a similar extent as control cells. Our data demonstrate that Als3 is involved directly in epithelial adhesion but indirectly in cell damage and cytokine induction, and is not the factor targeted by oral epithelial cells to discriminate between the yeast and hyphal form of C. albicans.
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Murine model of concurrent oral and vaginal Candida albicans colonisation.
Methods Mol. Biol.
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Investigations into the complex interaction between the fungal pathogen Candida albicans and its human host require the use of animals as in vivo models. A major advance is the creation of a low-oestrogen murine model of concurrent oral and vaginal C. albicans colonisation that resembles human candidal carriage at both mucosal sites. Weekly intramuscular (5 ?g) and subcutaneous (5 ?g) oestrogen administration was determined as optimal, enhancing oral colonisation but essential for vaginal colonisation. Using a clinical C. albicans oral isolate, persistent colonisation for up to 6 weeks can be achieved at both sites in two strains of mice (BALB/c and C57BL/6). This concurrent model of mucosal colonisation reduces the numbers of experimental mice by half, and opens up new avenues of research in assessing potential mucosal vaccine candidates and in studying delicate host-pathogen interactions during the most natural state of C. albicans epithelial colonisation.
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Analysis of host-cell responses by immunoblotting, ELISA, and real-time PCR.
Methods Mol. Biol.
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Intracellular responses to external pathogens/stimuli are crucial to the hosts response to infection. The methods used to analyse these responses fall into many categories. Activation of proteins as part of a signal cascade can be screened for using conventional immunoblotting techniques or immunoprecipitation to identify the presence of modified proteins or protein complexes. Transcription factor activity can be screened for by EMSA or ELISAs to identify DNA binding of these factors. Finally, expression of activated genes can be quantified using real-time PCR methods. Here, we will show how to perform these assays and discuss the relative merits of each.
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What is Visualize?

JoVE Visualize is a tool created to match the last 5 years of PubMed publications to methods in JoVE's video library.

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We use abstracts found on PubMed and match them to JoVE videos to create a list of 10 to 30 related methods videos.

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In developing our video relationships, we compare around 5 million PubMed articles to our library of over 4,500 methods videos. In some cases the language used in the PubMed abstracts makes matching that content to a JoVE video difficult. In other cases, there happens not to be any content in our video library that is relevant to the topic of a given abstract. In these cases, our algorithms are trying their best to display videos with relevant content, which can sometimes result in matched videos with only a slight relation.