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Find video protocols related to scientific articles indexed in Pubmed.
Crystal structure of kiwellin, a major cell-wall protein from kiwifruit.
J. Struct. Biol.
PUBLISHED: 06-23-2014
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Kiwellin is a cysteine-rich, cell wall-associated protein with no known structural homologues. It is one of the most abundant proteins in kiwifruit (Actinidia spp.), and has been shown to be recognised by IgE of some patients allergic to kiwifruit. Cleavage of kiwellin into an N-terminal 4 kDa peptide called kissper and a core domain called KiTH is mediated by actinidin in vitro, and isolation of the kissper peptide from green-fleshed kiwifruit extracts suggested it may result from in vivo processing of kiwellin. In solution, kissper is highly flexible and displays pore-forming activity in synthetic lipid-bilayers. We present here the 2.05 Å resolution crystal structure of full-length kiwellin, purified from its native source, Actinidia chinensis (gold-fleshed kiwifruit). The structure confirms the modularity of the protein and the intrinsic flexibility of kissper and reveals that KiTH harbours a double-psi ?-barrel fold hooked to an N-terminal ? hairpin. Comparisons with structurally-related proteins suggest that a deep gorge located at the protein surface forms a binding site for endogenous ligands.
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Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.
Eur. J. Immunol.
PUBLISHED: 03-31-2014
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Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.
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A slow-forming isopeptide bond in the structure of the major pilin SpaD from Corynebacterium diphtheriae has implications for pilus assembly.
Acta Crystallogr. D Biol. Crystallogr.
PUBLISHED: 01-20-2014
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The Gram-positive organism Corynebacterium diphtheriae, the cause of diphtheria in humans, expresses pili on its surface which it uses for adhesion and colonization of its host. These pili are covalent protein polymers composed of three types of pilin subunit that are assembled by specific sortase enzymes. A structural analysis of the major pilin SpaD, which forms the polymeric backbone of one of the three types of pilus expressed by C. diphtheriae, is reported. Mass-spectral and crystallographic analysis shows that SpaD contains three internal Lys-Asn isopeptide bonds. One of these, shown by mass spectrometry to be located in the N-terminal D1 domain of the protein, only forms slowly, implying an energy barrier to bond formation. Two crystal structures, of the full-length three-domain protein at 2.5 Å resolution and of a two-domain (D2-D3) construct at 1.87 Å resolution, show that each of the three Ig-like domains contains a single Lys-Asn isopeptide-bond cross-link, assumed to give mechanical stability as in other such pili. Additional stabilizing features include a disulfide bond in the D3 domain and a calcium-binding loop in D2. The N-terminal D1 domain is more flexible than the others and, by analogy with other major pilins of this type, the slow formation of its isopeptide bond can be attributed to its location adjacent to the lysine used in sortase-mediated polymerization during pilus assembly.
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Plant antimicrobial peptides snakin-1 and snakin-2: chemical synthesis and insights into the disulfide connectivity.
Chemistry
PUBLISHED: 01-19-2014
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Antimicrobial peptides and proteins represent an important class of plant defensive compounds against pathogens and provide a rich source of lead compounds in the field of drug discovery. We describe the effective preparation of the cysteine-rich snakin-1 and -2 antimicrobial peptides by using a combination of solid-phase synthesis and native chemical ligation. A subsequent cysteine/cystine mediated oxidative folding to form the six internal disulfide bonds concurrently gave the folded proteins in 40-50?% yield. By comparative evaluation of mass spectrometry, HPLC, biological data and trypsin digest mapping of folded synthetic snakin-2 compared to natural snakin-2, we demonstrated that synthetic snakin-2 possesses full antifungal activity and displayed similar chromatographic behaviour to natural snakin-2. Trypsin digest analysis allowed tentative assignment of three of the purported six disulfide bonds.
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Lower fetuin-A, retinol binding protein 4 and several metabolites after gastric bypass compared to sleeve gastrectomy in patients with type 2 diabetes.
PLoS ONE
PUBLISHED: 01-01-2014
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Bypass of foregut secreted factors promoting insulin resistance is hypothesized to be one of the mechanisms by which resolution of type 2 diabetes (T2D) follows roux-en-y gastric bypass (GBP) surgery.
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Structural model for the covalent adhesion of the Streptococcus pyogenes pilus through a thioester bond.
J. Biol. Chem.
PUBLISHED: 11-12-2013
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The human pathogen Streptococcus pyogenes produces pili that are essential for adhesion to host surface receptors. Cpa, the adhesin at the pilus tip, was recently shown to have a thioester-containing domain. The thioester bond is believed to be important in adhesion, implying a mechanism of covalent attachment analogous to that used by human complement factors. Here, we characterize a second active thioester-containing domain on Cpa, termed CpaN. Expression of CpaN in E. coli gave covalently-linked dimers. These were shown by X-ray crystallography and mass spectrometry to comprise two CpaN molecules crosslinked by the polyamine spermidine, following reaction with the thioester bonds. This cross-linked CpaN dimer provides a model for the covalent attachment of Cpa to target receptors and thus the streptococcal pilus to host cells. Similar thioester domains were identified in cell-wall proteins of other Gram-positive pathogens, suggesting that thioester domains are more widely used and provide a mechanism of adhesion, by covalent bonding to target molecules on host cells, that mimics that used by the human complement system to eliminate pathogens.
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Two optimization strategies of multi-stage design in clinical proteomic studies.
Stat Appl Genet Mol Biol
PUBLISHED: 03-19-2013
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We evaluated statistical approaches to facilitate and improve multi-stage designs for clinical proteomic studies which plan to transit from laboratory discovery to clinical utility. To find the design with the greatest expected number of true discoveries under constraints on cost and false discovery, the operating characteristics of the multi-stage study were optimized as a function of sample sizes and nominal type-I error rates at each stage. A nested simulated annealing algorithm was used to find the best solution in the bounded spaces constructed by multiple design parameters. This approach is demonstrated to be feasible and lead to efficient designs. The use of biological grouping information in the study design was also investigated using synthetic datasets based on a cardiac proteomic study, and an actual dataset from a clinical immunology proteomic study. When different protein patterns presented, performance improved when the grouping was informative, with little loss in performance when the grouping was uninformative.
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A simple and rapid method for identifying and semi-quantifying peptide hormones in isolated pancreatic islets by direct-tissue matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Rapid Commun. Mass Spectrom.
PUBLISHED: 10-18-2011
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We describe a new, simple, robust and efficient method based on direct-tissue matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry that enables consistent semi-quantitation of peptide hormones in isolated pancreatic islets from normal and diabetic rodents. Prominent signals were measured that corresponded to all the main peptide hormones present in islet-endocrine cells: (?-cells) glucagon, glicentin-related polypeptide/GRPP; (?-cells) insulin I, insulin II, C-peptide I, C-peptide II, amylin; (?-cells) somatostatin-14; and (PP-cells), and pancreatic polypeptide. The signal ratios coincided with known relative hormone abundances. The method demonstrated that severe insulin deficiency is accompanied by elevated levels of all non-?-cell-hormones in diabetic rat islets, consistent with alleviation of paracrine suppression of hormone production by non-?-cells. It was also effective in characterizing hormonal phenotype in hemizygous human-amylin transgenic mice that express human and mouse amylin in approx. equimolar quantities. Finally, the method demonstrated utility in basic peptide-hormone discovery by identifying a prominent new Gcg-gene-derived peptide (theoretical monoisotopic molecular weight 3263.5?Da), closely related to but distinct from GRPP, in diabetic islets. This peptide, whose sequence is HAPQDTEENARSFPASQTEPLEDPNQINE in Rattus norvegicus, could be a peptide hormone whose roles in physiology and metabolic disease warrant further investigation. This method provides a powerful new approach that could provide important new insights into the physiology and regulation of peptide hormones in islets and other endocrine tissues. It has potentially wide-ranging applications that encompass endocrinology, pharmacology, phenotypic analysis in genetic models of metabolic disease, and hormone discovery, and could also effectively limit the numbers of animals required for such studies.
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Structural analyses of a purine biosynthetic enzyme from Mycobacterium tuberculosis reveal a novel bound nucleotide.
J. Biol. Chem.
PUBLISHED: 09-28-2011
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Enzymes of the de novo purine biosynthetic pathway have been identified as essential for the growth and survival of Mycobacterium tuberculosis and thus have potential for the development of anti-tuberculosis drugs. The final two steps of this pathway are carried out by the bifunctional enzyme 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/inosine monophosphate cyclohydrolase (ATIC), also known as PurH. This enzyme has already been the target of anti-cancer drug development. We have determined the crystal structures of the M. tuberculosis ATIC (Rv0957) both with and without the substrate 5-aminoimidazole-4-carboxamide ribonucleotide, at resolutions of 2.5 and 2.2 ?, respectively. As for other ATIC enzymes, the protein is folded into two domains, the N-terminal domain (residues 1-212) containing the cyclohydrolase active site and the C-terminal domain (residues 222-523) containing the formyltransferase active site. An adventitiously bound nucleotide was found in the cyclohydrolase active site in both structures and was identified by NMR and mass spectral analysis as a novel 5-formyl derivative of an earlier intermediate in the biosynthetic pathway 4-carboxy-5-aminoimidazole ribonucleotide. This result and other studies suggest that this novel nucleotide is a cyclohydrolase inhibitor. The dimer formed by M. tuberculosis ATIC is different from those seen for human and avian ATICs, but it has a similar ?50-? separation of the two active sites of the bifunctional enzyme. Evidence in M. tuberculosis ATIC for reactivity of half-the-sites in the cyclohydrolase domains can be attributed to ligand-induced movements that propagate across the dimer interface and may be a common feature of ATIC enzymes.
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A unique case of neural amyloidoma diagnosed by mass spectrometry of formalin-fixed tissue using a novel preparative technique.
Amyloid
PUBLISHED: 08-24-2011
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We report here a unique amyloidoma of the radial nerve which could not be subtyped by available techniques, including immunohistochemistry and standard clinical and laboratory evaluation. In order to identify the amyloid monomer, we developed a novel preparative procedure designed to optimize conditions for liquid chromatography tandem mass spectrometry analysis of formalin-fixed/paraffin-embedded (FFPE) tissue. Subsequent mass spectrometric analysis clearly identified kappa light chain as the monomer, with no evidence of lambda light chain. Manual interpretation of the matched spectra revealed no evidence of polyclonality. This study also enabled detailed characterisation of twelve likely amyloid matrix components. Finally, our analysis revealed extensive hydroxylation of collagen type I but, unexpectedly, an almost complete lack of hydroxylated residues in the normally heavily-hydroxylated collagen type VI chains, pointing to structural/functional alterations of collagen VI in this matrix that could have contributed to the pathogenesis of this very unusual tumour. Given the high quality of the data here acquired using a standard quadrupole-time of flight tandem mass spectrometer of modest performance, the robust and straightforward preparative method described constitutes a competitive alternative to more involved approaches using state-of-the-art equipment.
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Illuminating the molecular basis of diabetic arteriopathy: a proteomic comparison of aortic tissue from diabetic and healthy rats.
Proteomics
PUBLISHED: 08-14-2010
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Arterial disease is a major diabetic complication, yet the component molecular mechanisms of diabetic arteriopathy remain poorly understood. In order to identify major proteins/pathways implicated in diabetic arteriopathy, we studied the effect of 16-wk untreated streptozotocin-induced diabetes on the rat aortic proteome. Specific protein levels in isolated aortas were compared in six discrete, pair-wise (streptozotocin-diabetic and non-diabetic age-matched controls) experiments in which individual proteins were identified and quantified by iTRAQ combined with LC-MS/MS. A total of 398 unique non-redundant proteins were identified in at least one experiment and 208 were detected in three or more. Between-group comparisons revealed significant changes or trends towards changes in relative abundance of 51 proteins (25 increased, 26 decreased). Differences in levels of selected proteins were supported by Western blotting and/or enzyme assays. The most prominent diabetes-associated changes were in groups of proteins linked to oxidative stress responses and the structure/function of myofibrils and microfilaments. Indexes of mitochondrial content were measurably lower in aortic tissue from diabetic animals. Functional cluster analysis also showed decreased levels of glycolytic enzymes and mitochondrial electron transport system-complex components. These findings newly implicate several proteins/functional pathways in the pathogenesis of arteriosclerosis/diabetic arteriopathy.
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Crystal structure of the minor pilin FctB reveals determinants of Group A streptococcal pilus anchoring.
J. Biol. Chem.
PUBLISHED: 04-27-2010
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Cell surface pili are polymeric protein assemblies that enable bacteria to adhere to surfaces and to specific host tissues. The pili expressed by Gram-positive bacteria constitute a unique paradigm in which sortase-mediated covalent linkages join successive pilin subunits like beads on a string. These pili are formed from two or three distinct types of pilin subunit, typically encoded in small gene clusters, often with their cognate sortases. In Group A streptococci (GAS), a major pilin forms the polymeric backbone, whereas two minor pilins are located at the tip and the base. Here, we report the 1.9-A resolution crystal structure of the GAS basal pilin FctB, revealing an immunoglobulin (Ig)-like N-terminal domain with an extended proline-rich tail. Unexpected structural homology between the FctB Ig-like domain and the N-terminal domain of the GAS shaft pilin helps explain the use of the same sortase for polymerization of the shaft and its attachment to FctB. It also enabled the identification, from mass spectral data, of the lysine residue involved in the covalent linkage of FctB to the shaft. The proline-rich tail forms a polyproline-II helix that appears to be a common feature of the basal (cell wall-anchoring) pilins. Together, our results indicate distinct structural elements in the pilin proteins that play a role in selecting for the appropriate sortases and thereby help orchestrate the ordered assembly of the pilus.
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Changes in the mesenteric lymph proteome induced by hemorrhagic shock.
Shock
PUBLISHED: 02-18-2010
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Biologically active factors produced by the intestine and transported by the aqueous and protein fraction of mesenteric lymph are now thought to contribute significantly to the development of distant organ failure in hemorrhagic shock. Despite the likely relevance of the protein composition of mesenteric lymph conditioned by hemorrhagic shock, there is no detailed description of its proteome. The aim of this study was to provide the first comprehensive description of the proteome of hemorrhagic shock-conditioned mesenteric lymph. Mesenteric lymph was collected from 16 male Wistar rats randomized to group 1 (n = 8) sham control and group 2 (n = 8) with hemorrhagic shock. The lymph was subjected to proteomic analysis using iTRAQ and liquid chromatography-tandem mass spectrometry. Sixty of the 245 proteins had a significant increase in their relative abundance in the hemorrhagic shock group. A bioinformatics approach highlighted the importance of the key gene ontology pathways relating to response to injury and metabolic responses as changing most significantly in shock. Using an interactome, we identified several highly connected proteins: 14-3-3 Zeta, 14-3-3 epsilon, actin, aldolase A, calmodulin, cofilin 1, cystatin C, fatty acid-binding protein 4, profilin 1, prolyl 4-hydrolase, peptidylprolyl isomerase, and transgelin. This study provides the first detailed description of protein changes in hemorrhagic shock-conditioned mesenteric lymph, and using a bioinformatics approach, we identified several targets for possible further research.
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Quantitative proteomic profiling identifies new renal targets of copper(II)-selective chelation in the reversal of diabetic nephropathy in rats.
Proteomics
PUBLISHED: 07-28-2009
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This study aimed to identify new diabetic nephropathy (DN)-related proteins and renal targets of the copper(II)-selective chelator, triethylenetetramine (TETA) in streptozotocin-diabetic rats. We used the recently developed iTRAQ technology to compare renal protein profiles among non-diabetic, diabetic, and TETA-treated diabetic rats. In diabetic kidneys, tubulointerstitial nephritis antigen (TINag), voltage-dependent anion-selective channel (VDAC) 1, and VDAC2 were up-regulated in parallel with alterations in expression of proteins with functions in oxidative stress and oxidative phosphorylation (OxPhos) pathways. By contrast, mitochondrial HSP 60, Cu/Zn-superoxide dismutase, glutathione S-transferase alpha3 and aquaporin-1 were down-regulated in diabetic kidneys. Following TETA treatment, levels of D-amino acid oxidase-1, epoxide hydrolase-1, aquaporin-1, and a number of mitochondrial proteins were normalized, with concomitant amelioration of albuminuria. Changes in levels of TINag, collagen VIalpha1, actinin 4alpha, apoptosis-inducing factor 1, cytochrome C, histone H3, VDAC1, and aquaporin-1 were confirmed by Western blotting or immunohistochemistry. Changes in expression of proteins related to tubulointerstitial function, podocyte structure, and mitochondrial apoptosis are implicated in the mechanism of DN and their reversal by TETA. These findings are consistent with the hypothesis that this new experimental therapy may be useful for treatment of DN.
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The proteome of mesenteric lymph during acute pancreatitis and implications for treatment.
JOP
PUBLISHED: 03-17-2009
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The protein fraction of mesenteric lymph during acute pancreatitis and other critical illness is thought to contain toxic factors. However, we do not have a complete description of the mesenteric lymph proteome during acute pancreatitis.
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Proteins associated with immunopurified granules from a model pancreatic islet beta-cell system: proteomic snapshot of an endocrine secretory granule.
J. Proteome Res.
PUBLISHED: 02-28-2009
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beta-Cell granules contain proteins involved in fuel regulation, which when altered, contribute to metabolic disorders including diabetes mellitus. We analyzed proteins present in purified granules from the INS-1E beta-cell model. Fifty-one component proteins were identified by LC-MS/MS including hormones, granins, protein processing components, cellular trafficking components, enzymes implicated in cellular metabolism and chaperone proteins. These findings may increase understanding of granule secretion and the processes leading to protein aggregation and beta-cell death in type-2 diabetes.
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Isopeptide bonds in bacterial pili and their characterization by X-ray crystallography and mass spectrometry.
Biopolymers
PUBLISHED: 02-20-2009
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Pili are long, filamentous protein assemblies which extend from the surfaces of many bacteria, and mediate their adhesion to host cells and other matrices. For pathogenic bacteria they are critical to colonization and infection. Whereas the pili of gram-negative bacteria are formed by noncovalent association of their pilin subunits, those of gram-positive bacteria are assembled with the aid of sortase enzymes that mediate the formation of covalent isopeptide bonds between successive pilin subunits. Sequence comparisons, mutagenesis and crystallography have implicated specific lysine residues in the formation of these intermolecular bonds and mass spectral analyses of native and modified pili have now provided definitive proof of these linkages. Crystallographic studies of pilin subunits have also led to the unexpected discovery of internal isopeptide crosslinks formed between lysine and asparagine residues. These, too, have been confirmed by mass spectrometry.
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Purification, crystallization and preliminary crystallographic analysis of the adhesion domain of Epf from Streptococcus pyogenes.
Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun.
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The extracellular protein Epf from Streptococcus pyogenes is important for streptococcal adhesion to human epithelial cells. However, Epf has no sequence identity to any protein of known structure or function. Thus, several predicted domains of the 205?kDa protein Epf were cloned separately and expressed in Escherichia coli. The N-terminal domain of Epf was crystallized in space groups P2(1) and P2(1)2(1)2(1) in the presence of the protease chymotrypsin. Mass spectrometry showed that the species crystallized corresponded to a fragment comprising residues 52-357 of Epf. Complete data sets were collected to 2.0 and 1.6?Å resolution, respectively, at the Australian Synchrotron.
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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.

Video X seems to be unrelated to Abstract Y...

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.