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Find video protocols related to scientific articles indexed in Pubmed.
Purification of intracellular bacteria: isolation of viable Brucella abortus from host cells.
Methods Mol. Biol.
PUBLISHED: 08-31-2014
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The pathogenesis of brucellosis depends on the ability of bacteria from the genus Brucella to invade and replicate within animal cells. To understand the molecular pathways used by Brucella spp. to reach its intracellular niche, robust and reproducible bacteria purification protocols that provide enough material for biochemical and molecular biology studies are essential. Here, we describe a detailed methodology designed to extract and purify viable brucellae from mammalian host cells at different time periods of their intracellular cycle. The yield of proteins and nucleic acids is sufficient to perform immunochemical analysis, genetic studies, transcriptomics, and proteomics among others.
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Brucella ceti infection in dolphins from the Western Mediterranean sea.
BMC Vet. Res.
PUBLISHED: 03-02-2014
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Background Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis¿, subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea.ResultsWe describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses.ConclusionsAll the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.
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Retrospective and prospective perspectives on zoonotic brucellosis.
Front Microbiol
PUBLISHED: 01-01-2014
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Members of the genus Brucella are pathogenic bacteria exceedingly well adapted to their hosts. The bacterium is transmitted by direct contact within the same host species or accidentally to secondary hosts, such as humans. Human brucellosis is strongly linked to the management of domesticated animals and ingestion of their products. Since the domestication of ungulates and dogs in the Fertile Crescent and Asia in 12000 and 33000 ya, respectively, a steady supply of well adapted emergent Brucella pathogens causing zoonotic disease has been provided. Likewise, anthropogenic modification of wild life may have also impacted host susceptibility and Brucella selection. Domestication and human influence on wild life animals are not neutral phenomena. Consequently, Brucella organisms have followed their hosts' fate and have been selected under conditions that favor high transmission rate. The "arm race" between Brucella and their preferred hosts has been driven by genetic adaptation of the bacterium confronted with the evolving immune defenses of the host. Management conditions, such as clustering, selection, culling, and vaccination of Brucella preferred hosts have profound influences in the outcome of brucellosis and in the selection of Brucella organisms. Countries that have controlled brucellosis systematically used reliable smooth live vaccines, consistent immunization protocols, adequate diagnostic tests, broad vaccination coverage and sustained removal of the infected animals. To ignore and misuse tools and strategies already available for the control of brucellosis may promote the emergence of new Brucella variants. The unrestricted use of low-efficacy vaccines may promote a "false sense of security" and works towards selection of Brucella with higher virulence and transmission potential.
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Neutrophils exert a suppressive effect on Th1 responses to intracellular pathogen Brucella abortus.
PLoS Pathog.
PUBLISHED: 02-14-2013
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Polymorphonuclear neutrophils (PMNs) are the first line of defense against microbial pathogens. In addition to their role in innate immunity, PMNs may also regulate events related to adaptive immunity. To investigate the influence of PMNs in the immune response during chronic bacterial infections, we explored the course of brucellosis in antibody PMN-depleted C57BL/6 mice and in neutropenic mutant Genista mouse model. We demonstrate that at later times of infection, Brucella abortus is killed more efficiently in the absence of PMNs than in their presence. The higher bacterial removal was concomitant to the: i) comparatively reduced spleen swelling; ii) augmented infiltration of epithelioid histiocytes corresponding to macrophages/dendritic cells (DCs); iii) higher recruitment of monocytes and monocyte/DCs phenotype; iv) significant activation of B and T lymphocytes, and v) increased levels of INF-? and negligible levels of IL4 indicating a balance of Th1 over Th2 response. These results reveal that PMNs have an unexpected influence in dampening the immune response against intracellular Brucella infection and strengthen the notion that PMNs actively participate in regulatory circuits shaping both innate and adaptive immunity.
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An evolutionary strategy for a stealthy intracellular Brucella pathogen.
Immunol. Rev.
PUBLISHED: 02-26-2011
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Brucella is an intracellular bacterial pathogen that causes abortion and infertility in mammals and leads to a debilitating febrile illness that can progress into a long lasting disease with severe complications in humans. Its virulence depends on survival and replication properties in host cells. In this review, we describe the stealthy strategy used by Brucella to escape recognition of the innate immunity and the means by which this bacterium evades intracellular destruction. We also discuss the development of adaptive immunity and its modulation during brucellosis that in course leads to chronic infections. Brucella has developed specific strategies to influence antigen presentation mediated by cells. There is increasing evidence that Brucella also modulates signaling events during host adaptive immune responses.
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Brucella abortus ornithine lipids are dispensable outer membrane components devoid of a marked pathogen-associated molecular pattern.
PLoS ONE
PUBLISHED: 01-07-2011
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The brucellae are ?-Proteobacteria facultative intracellular parasites that cause an important zoonosis. These bacteria escape early detection by innate immunity, an ability associated to the absence of marked pathogen-associated molecular patterns in the cell envelope lipopolysaccharide, lipoproteins and flagellin. We show here that, in contrast to the outer membrane ornithine lipids (OL) of other Gram negative bacteria, Brucella abortus OL lack a marked pathogen-associated molecular pattern activity. We identified two OL genes (olsB and olsA) and by generating the corresponding mutants found that olsB deficient B. abortus did not synthesize OL or their lyso-OL precursors. Liposomes constructed with B. abortus OL did not trigger IL-6 or TNF-? release by macrophages whereas those constructed with Bordetella pertussis OL and the olsB mutant lipids as carriers were highly active. The OL deficiency in the olsB mutant did not promote proinflammatory responses or generated attenuation in mice. In addition, OL deficiency did not increase sensitivity to polymyxins, normal serum or complement consumption, or alter the permeability to antibiotics and dyes. Taken together, these observations indicate that OL have become dispensable in the extant brucellae and are consistent within the trend observed in ?-Proteobacteria animal pathogens to reduce and eventually eliminate the envelope components susceptible of recognition by innate immunity.
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The two-component system BvrR/BvrS regulates the expression of the type IV secretion system VirB in Brucella abortus.
J. Bacteriol.
PUBLISHED: 09-10-2010
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The pathogenesis of Brucella is related to the ability to multiply intracellularly, an event controlled by the two-component system BvrR/BvrS (TCS BvrRS) and the type IV secretion machinery VirB (T4SS VirB). We have hypothesized that the TCS BvrRS transcriptionally regulates the T4SS VirB. To test this hypothesis, we have compared the levels of VirB proteins in the wild-type strain Brucella abortus 2308 and mutant strains devoid of the sensor and regulator genes (bvrS and bvrR mutants, respectively). While the bvrR and bvrS mutants showed low levels of the VirB1, VirB5, VirB8, and VirB9 proteins, the same proteins were overexpressed in the bvrR mutant complemented with a plasmid carrying a functional bvrR gene. Quantitation of virB5 mRNA confirmed these data and indicated that the influence of the TCS BvrRS on the T4SS VirB occurs at the transcriptional level. The expression of the transcriptional activator VjbR also depended on the TCS BvrRS. In addition, we demonstrate a direct interaction between the promoter region of the VirB operon and the response regulator BvrR. Altogether these data demonstrate that the TCS BvrRS controls the expression of the T4SS VirB through direct and indirect mechanisms.
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Synthetic peptides derived from the C-terminal region of Lys49 phospholipase A2 homologues from viperidae snake venoms: biomimetic activities and potential applications.
Curr. Pharm. Des.
PUBLISHED: 07-09-2010
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Lys49-phospholipase A(2) homologues constitute a large family of toxins present in the venoms of viperid snake species, which despite lacking catalytic activity, cause significant skeletal muscle necrosis. The main structural determinants of this toxic effect have been experimentally mapped to a region near their C-terminus (115-129), which combines cationic and hydrophobic/aromatic amino acid residues. Short (13-mer) synthetic peptides representing this C-terminal region can mimick several of the effects of Lys49 PLA(2) homologues. In addition to their ability to damage muscle cells, these peptides display antibacterial, antiendotoxic, antifungal, antiparasite, and antitumor activities, as well as VEGF-receptor 2 (KDR)-binding and heparin-binding properties. Modifications of their sequences have shown possibilities to enhance their effects upon prokaryotic cells, while decreasing toxicity for eukaryotic cells. This review presents an updated summary on the biomimetic actions exerted by such peptides, and highlights their potential value as molecular tools or as drug leads in diverse biomedical areas.
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Proteomics-based confirmation of protein expression and correction of annotation errors in the Brucella abortus genome.
BMC Genomics
PUBLISHED: 05-12-2010
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Brucellosis is a major bacterial zoonosis affecting domestic livestock and wild mammals, as well as humans around the globe. While conducting proteomics studies to better understand Brucella abortus virulence, we consolidated the proteomic data collected and compared it to publically available genomic data.
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The use of green fluorescent protein as a marker for Brucella vaccines.
Vaccine
PUBLISHED: 04-24-2010
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Brucellosis is an important malady of productive and wildlife animals and a worldwide zoonosis. The use of effective vaccines and the corresponding diagnostic tests that allow differentiating infected from vaccinated animals are essential tools to control the disease. For this, a prototype of Brucella abortus S19 vaccine expressing green fluorescent protein (S19-GFP) was constructed. The S19-GFP was readily identified under ultraviolet light by macroscopic and microscopic examination and maintained all the biochemical characteristics of the parental S19 vaccine. S19-GFP replicated ex vivo and in vivo, and protected mice against challenge with virulent B. abortus to the same extent as the isogenic S19. An immunoenzymatic assay designed to measure anti-GFP antibodies allowed the discrimination between mice vaccinated with S19-GFP and those immunized with S19. Both vaccines raised antibodies against lipopolysaccharide molecule to similar levels. This experimental model constitutes a "proof of concept" for the use of Brucella-GFP vaccines and associated diagnostic tests to distinguish vaccinated from naturally Brucella infected animals.
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Helicobacter pylori: bacterial factors and the role of cytokines in the immune response.
Curr. Microbiol.
PUBLISHED: 08-08-2009
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Helicobacter pylori is a gram-negative micro-aerophilic bacterium that is widely distributed geographically and causes chronic gastritis, peptic ulcers, gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. Bacterial virulence factors play an important role, since the virulent strains are more aggressive and increase the risk of developing severe clinical manifestations; in addition, other determinant factors are the nutritional state and the immune response of the host. Studies on humans, non-human primates, and rodents have reported that regulating proteins of the Th1 phenotype predominate in the immune response to the bacterial infection. The cytokines produced by this phenotype, are not very effective in eradicating the microorganism and furthermore, contribute to gastro-duodenal pathogenesis. Gastric inflammation in patients infected with H. pylori has been characterized by increased production of IL-1, IL-6, IL-12, IL-18, TNF-alpha, and IFN-gamma. Many prophylactic and therapeutic strategies have been researched using experimental animals. The utilization and effectiveness of vaccination on humans requires more study.
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The differential interaction of Brucella and ochrobactrum with innate immunity reveals traits related to the evolution of stealthy pathogens.
PLoS ONE
PUBLISHED: 04-24-2009
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During evolution, innate immunity has been tuned to recognize pathogen-associated molecular patterns. However, some alpha-Proteobacteria are stealthy intracellular pathogens not readily detected by this system. Brucella members follow this strategy and are highly virulent, but other Brucellaceae like Ochrobactrum are rhizosphere inhabitants and only opportunistic pathogens. To gain insight into the emergence of the stealthy strategy, we compared these two phylogenetically close but biologically divergent bacteria.
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Serological diagnosis of Brucella infections in odontocetes.
Clin. Vaccine Immunol.
PUBLISHED: 04-22-2009
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Brucella ceti causes disease in Odontoceti. The absence of control serum collections and the diversity of cetaceans have hampered the standardization of serological tests for the diagnosis of cetacean brucellosis. Without a "gold" standard for sensitivity and specificity determination, an alternative approach was followed. We designed an indirect enzyme-linked immunosorbent assay (iELISA) that recognizes immunoglobulins G (IgGs) from 17 odontocete species as a single group. For the standardization, we used Brucella melitensis and Brucella abortus lipopolysaccharides, serum samples from seven resident odontocetes with no history of infectious disease displaying negative rose bengal test (RBT) reactions, and serum samples from seven dolphins infected with B. ceti. We compared the performance of the iELISA with those of the protein G ELISA (gELISA), the competitive ELISA (cELISA), and the immunofluorescence (IF) and dot blot (DB) tests, using 179 odontocete serum samples and RBT as the reference. The diagnostic potential based on sensitivity and specificity of the iELISA was superior to that of gELISA and cELISA. The correlation and agreement between the iELISA and the gELISA were relatively good (R(i/g)2 = 0.65 and kappa(i/g) = 0.66, respectively), while the correlation and agreement of these two ELISAs with cELISA were low (R(i/c)2 = 0.46, R(g/c)2 = 0.37 and kappa(i/c) = 0.62, kappa(g/c) = 0.42). In spite of using the same anti-odontocete IgG antibody, the iELISA was more specific than were the IF and DB tests. An association between high antibody titers and the presence of neurological symptoms in dolphins was observed. The prediction is that iELISA based on broadly cross-reacting anti-dolphin IgG antibody would be a reliable test for the diagnosis of brucellosis in odontocetes, including families not covered in this study.
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Intracellular adaptation of Brucella abortus.
J. Proteome Res.
PUBLISHED: 02-17-2009
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Macrophages were infected with virulent Brucella abortus strain 2308 or attenuated strain 19. Intracellular bacteria were recovered at different times after infection and their proteomes compared. The virulent strain initially reduced most biosynthesis and altered its respiration; adaptations reversed later in infection. The attenuated strain was unable to match the magnitude of the virulent strains adjustments. The results provide insight into mechanisms utilized by Brucella to establish intracellular infections.
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Brucella ? 1,2 cyclic glucan is an activator of human and mouse dendritic cells.
PLoS Pathog.
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Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella ? 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella ? 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8(+) T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4(+) and CD8(+) T cell responses including cross-presentation by different human DC subsets. Brucella ? 1,2 cyclic glucans increased the memory CD4(+) T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies.
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Brucella ceti and brucellosis in cetaceans.
Front Cell Infect Microbiol
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Since the first case of brucellosis detected in a dolphin aborted fetus, an increasing number of Brucella ceti isolates has been reported in members of the two suborders of cetaceans: Mysticeti and Odontoceti. Serological surveys have shown that cetacean brucellosis may be distributed worldwide in the oceans. Although all B. ceti isolates have been included within the same species, three different groups have been recognized according to their preferred host, bacteriological properties, and distinct genetic traits: B. ceti dolphin type, B. ceti porpoise type, and B. ceti human type. It seems that B. ceti porpoise type is more closely related to B. ceti human isolates and B. pinnipedialis group, while B. ceti dolphin type seems ancestral to them. Based on comparative phylogenetic analysis, it is feasible that the B. ceti ancestor radiated in a terrestrial artiodactyl host close to the Raoellidae family about 58 million years ago. The more likely mode of transmission of B. ceti seems to be through sexual intercourse, maternal feeding, aborted fetuses, placental tissues, vertical transmission from mother to the fetus or through fish or helminth reservoirs. The B. ceti dolphin and porpoise types seem to display variable virulence in land animal models and low infectivity for humans. However, brucellosis in some dolphins and porpoises has been demonstrated to be a severe chronic disease, displaying significant clinical and pathological signs related to abortions, male infertility, neurobrucellosis, cardiopathies, bone and skin lesions, strandings, and death.
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The lipopolysaccharide core of Brucella abortus acts as a shield against innate immunity recognition.
PLoS Pathog.
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Innate immunity recognizes bacterial molecules bearing pathogen-associated molecular patterns to launch inflammatory responses leading to the activation of adaptive immunity. However, the lipopolysaccharide (LPS) of the gram-negative bacterium Brucella lacks a marked pathogen-associated molecular pattern, and it has been postulated that this delays the development of immunity, creating a gap that is critical for the bacterium to reach the intracellular replicative niche. We found that a B. abortus mutant in the wadC gene displayed a disrupted LPS core while keeping both the LPS O-polysaccharide and lipid A. In mice, the wadC mutant induced proinflammatory responses and was attenuated. In addition, it was sensitive to killing by non-immune serum and bactericidal peptides and did not multiply in dendritic cells being targeted to lysosomal compartments. In contrast to wild type B. abortus, the wadC mutant induced dendritic cell maturation and secretion of pro-inflammatory cytokines. All these properties were reproduced by the wadC mutant purified LPS in a TLR4-dependent manner. Moreover, the core-mutated LPS displayed an increased binding to MD-2, the TLR4 co-receptor leading to subsequent increase in intracellular signaling. Here we show that Brucella escapes recognition in early stages of infection by expressing a shield against recognition by innate immunity in its LPS core and identify a novel virulence mechanism in intracellular pathogenic gram-negative bacteria. These results also encourage for an improvement in the generation of novel bacterial vaccines.
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What have we learned from brucellosis in the mouse model?
Vet. Res.
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Brucellosis is a zoonosis caused by Brucella species. Brucellosis research in natural hosts is often precluded by practical, economical and ethical reasons and mice are widely used. However, mice are not natural Brucella hosts and the course of murine brucellosis depends on bacterial strain virulence, dose and inoculation route as well as breed, genetic background, age, sex and physiological statu of mice. Therefore, meaningful experiments require a definition of these variables. Brucella spleen replication profiles are highly reproducible and course in four phases: i), onset or spleen colonization (first 48?h); ii), acute phase, from the third day to the time when bacteria reach maximal numbers; iii), chronic steady phase, where bacterial numbers plateaus; and iv), chronic declining phase, during which brucellae are eliminated. This pattern displays clear physiopathological signs and is sensitive to small virulence variations, making possible to assess attenuation when fully virulent bacteria are used as controls. Similarly, immunity studies using mice with known defects are possible. Mutations affecting INF-?, TLR9, Myd88, T?? and TNF-? favor Brucella replication; whereas IL-1?, IL-18, TLR4, TLR5, TLR2, NOD1, NOD2, GM-CSF, IL/17r, Rip2, TRIF, NK or Nramp1 deficiencies have no noticeable effects. Splenomegaly development is also useful: it correlates with IFN-? and IL-12 levels and with Brucella strain virulence. The genetic background is also important: Brucella-resistant mice (C57BL) yield lower splenic bacterial replication and less splenomegaly than susceptible breeds. When inoculum is increased, a saturating dose above which bacterial numbers per organ do not augment, is reached. Unlike many gram-negative bacteria, lethal doses are large (? 108 bacteria/mouse) and normally higher than the saturating dose. Persistence is a useful virulence/attenuation index and is used in vaccine (Residual Virulence) quality control. Vaccine candidates are also often tested in mice by determining splenic Brucella numbers after challenging with appropriate virulent brucellae doses at precise post-vaccination times. Since most live or killed Brucella vaccines provide some protection in mice, controls immunized with reference vaccines (S19 or Rev1) are critical. Finally, mice have been successfully used to evaluate brucellosis therapies. It is concluded that, when used properly, the mouse is a valuable brucellosis model.
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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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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.