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Find video protocols related to scientific articles indexed in Pubmed.
Catastrophic antiphospholipid syndrome and heparin-induced thrombocytopenia-related diseases or chance association?
Blood Coagul. Fibrinolysis
PUBLISHED: 10-12-2014
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Antiphospholipid syndrome (APS) and heparin-induced thrombocytopenia (HIT) are thrombotic disorders due to specific autoimmune-mediated antibodies. Catastrophic APS (CAPS), also known as Asherman's syndrome, is a life-threatening severe form of APS. Diagnostic criteria for CAPS include the development of a thrombotic event of three or more organs in less than a week with the presence of antiphospholipid antibodies and microvascular thrombosis on histology. Thrombocytopenia is seen in more than 60% of cases of CAPS. HIT is a life-threatening disorder with the clinical presentation of thrombocytopenia and arterial or venous thrombosis in patients who develop antibodies to heparin and platelet factor 4 typically within 10 days after starting heparin treatment. Due to the multiple similarities in clinical features and pathophysiology of CAPS and HIT, it has been postulated that these two antibody-mediated disorders may be related. We report two cases in which patients diagnosed with CAPS developed HIT very soon during the same admission as well as a case of a patient initially diagnosed with HIT who presented with CAPS years later.
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Manufacturing and characterization of a recombinant adeno-associated virus type 8 reference standard material.
Hum. Gene Ther.
PUBLISHED: 10-03-2014
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Abstract Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. The rAAV8RSM was produced using transient transfection, and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to 16 laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50×10(11) pt/ml; CI, 4.26×10(11) to 6.75×10(11) pt/ml), vector genomes (mean, 5.75×10(11) vg/ml; CI, 3.05×10(11) to 1.09×10(12) vg/ml), and infectious units (mean, 1.26×10(9) IU/ml; CI, 6.46×10(8) to 2.51×10(9) IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time when the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
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Effective concentration and detection of cryptosporidium, giardia, and the microsporidia from environmental matrices.
J Pathog
PUBLISHED: 05-31-2014
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Cryptosporidium spp., Giardia spp., and members of Microsporidia are enteropathogenic parasites of humans and animals, producing asymptomatic to severe intestinal infections. To circumvent various impediments associated with current detection methods, we tested a method providing multistage purification and separation in a single, confined step. Standard real-time PCR was used as a detection method. Samples spiked with C. parvum and G. intestinalis were split for comparison to standard Method 1623. Results were equivalent to immunomagnetic procedures for Cryptosporidium, and Giardia. Overall percent recovery for Cryptosporidium with Method 1623 averaged 26.89% (std 21.44%; min = 0%; max = 73%) and was similar but less variable for qPCR method at an estimated average of 27.67 (std 17.65%; min = 5%; max = 63%). For Giardia, Method 1623 had an overall average recovery of 27.11% (std 17.98%; min = 1%; max = 58%), while multistage purification and qPCR had an estimated lower overall recovery at 18.58% (std 13.95%; min = 0%; max = 35%). Microsporidia were also readily detected with an estimated recovery of 46.81% overall (std 17.66%; min = 18%; max = 70%) for E. intestinalis and 38.90% (std 14.36%; min = 13%; max = 62%) for E. bieneusi.
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Medical homes and cost and utilization among high-risk patients.
Am J Manag Care
PUBLISHED: 04-30-2014
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Evaluate the effects of the patient-centered medical home (PCMH) model on medical costs and utilization in the nonpediatric population, particularly among high-risk patients. StuDY DESIGN: Longitudinal case-control design, comparing per member per month (PMPM) cost and utilization per 1000 patients for members enrolled in PCMH and non-PCMH practices from 2009 to 2011.
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PAH concentrations in Coquina (Donax spp.) on a sandy beach shoreline impacted by a marine oil spill.
Mar. Pollut. Bull.
PUBLISHED: 04-04-2014
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The BP MC252 well failure in the Gulf of Mexico, April 2010 caused concern for crude oil and polycyclic aromatic hydrocarbon (PAHs) exposure along the sandy beaches of the Florida Panhandle. We began collections of Coquina clams (Donax spp.) from the surf zone of Florida Panhandle beaches to monitor PAH contamination to compliment analysis of surf zone sand samples. These clams had higher levels of PAHs relative to ambient sand, and this allowed us to continue to monitor PAH levels after sand concentrations fell below limits of detection. PAH levels in the Coquina tissues were highly variable, perhaps indicative of the heterogeneous distribution of oil and tar on the beaches and exposure to tar particles. Overall, PAH levels decreased continuously in both sand and Coquina tissues, reaching limits of detection within one and two years respectively after oil landed on Florida Panhandle beaches. Our work suggests these surf zone molluscs may be used to monitor pollutant exposure along high energy sandy beach shorelines.
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Patient-centered medical home impact on health plan members with diabetes.
J Public Health Manag Pract
PUBLISHED: 04-03-2014
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To compare costs and utilization for patients with diabetes enrolled in patient-centered medical home (PCMH) practices and non-PCMH practices.
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Keratinocytes produce IL-6 in response to desmoglein 1 cleavage by Staphylococcus aureus exfoliative toxin A.
Immunol. Res.
PUBLISHED: 11-30-2013
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Many skin infections are caused by Staphylococcus aureus, a bacterial pathogen that produces virulence factors associated with these conditions such as exfoliative toxins A and B (ETA, ETB) and the leukotoxin Panton-Valentine leukocidin (PVL). Herein, we examine the potential of skin-infecting S. aureus to produce virulence factors and their impact on the local immune response. Toxin gene profiles were generated from 188 S. aureus isolated as single infecting organisms from skin lesions and demonstrated a higher potential to express ETA, ETB, and PVL than community isolates (p < 0.001). Within the study isolate group, the prevalence of genes encoding PVL was higher among methicillin-resistant S. aureus (MRSA; n = 49), while genes encoding ETs were more prevalent in methicillin-susceptible S. aureus (MSSA; n = 139). When lesion-associated white blood cell (WBC) counts were dichotomized into high- or low-WBC-count-associated bacteria, the gene for ETA was found to be associated with a low WBC count among MSSA (p = 0.001). The ETA-induced mouse model of staphylococcal scalded skin syndrome was used to investigate the link between ETA and cytokine production. Elevated IL-6 levels in the serum and increased expression of IL-6 mRNA in the skin were detected in response to ETA exposure. These findings were recapitulated in vitro using primary human keratinocytes. Thus, S. aureus may influence the local immune response via ETA cleavage of desmoglein 1 and the induction of cutaneous IL-6 expression.
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Case Manager as Therapy Extender for Cognitive Behavior Therapy of Serious Mental Illness: A Case Report.
Community Ment Health J
PUBLISHED: 06-03-2013
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Cognitive behavior therapy (CBT) is an evidence-based intervention for individuals with serious mental illness and potentiates standard medication management. Americans receiving publicly funded treatment for serious mental illnesses have limited access to CBT and hence we need to devise innovative ways of providing access to this important intervention. We present a case of a man who had severe disability, was medication resistant, and diagnosed with Obsessive Compulsive Disorder and Major Depressive Disorder. After being home bound for many years he was provided CBT utilizing his existing case manager as a therapy extender. The specific roles of the primary therapist and case manager as well as the improvement in quality of life of the individual are delineated. This case report opens up the possibility of further studying case managers as therapy extenders for treating serious mental illnesses.
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PCR-based detection of gene transfer vectors: application to gene doping surveillance.
Anal Bioanal Chem
PUBLISHED: 05-20-2013
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Athletes who illicitly use drugs to enhance their athletic performance are at risk of being banned from sports competitions. Consequently, some athletes may seek new doping methods that they expect to be capable of circumventing detection. With advances in gene transfer vector design and therapeutic gene transfer, and demonstrations of safety and therapeutic benefit in humans, there is an increased probability of the pursuit of gene doping by athletes. In anticipation of the potential for gene doping, assays have been established to directly detect complementary DNA of genes that are top candidates for use in doping, as well as vector control elements. The development of molecular assays that are capable of exposing gene doping in sports can serve as a deterrent and may also identify athletes who have illicitly used gene transfer for performance enhancement. PCR-based methods to detect foreign DNA with high reliability, sensitivity, and specificity include TaqMan real-time PCR, nested PCR, and internal threshold control PCR.
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Surface renewal: an advanced micrometeorological method for measuring and processing field-scale energy flux density data.
J Vis Exp
PUBLISHED: 04-21-2013
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Advanced micrometeorological methods have become increasingly important in soil, crop, and environmental sciences. For many scientists without formal training in atmospheric science, these techniques are relatively inaccessible. Surface renewal and other flux measurement methods require an understanding of boundary layer meteorology and extensive training in instrumentation and multiple data management programs. To improve accessibility of these techniques, we describe the underlying theory of surface renewal measurements, demonstrate how to set up a field station for surface renewal with eddy covariance calibration, and utilize our open-source turnkey data logger program to perform flux data acquisition and processing. The new turnkey program returns to the user a simple data table with the corrected fluxes and quality control parameters, and eliminates the need for researchers to shuttle between multiple processing programs to obtain the final flux data. An example of data generated from these measurements demonstrates how crop water use is measured with this technique. The output information is useful to growers for making irrigation decisions in a variety of agricultural ecosystems. These stations are currently deployed in numerous field experiments by researchers in our group and the California Department of Water Resources in the following crops: rice, wine and raisin grape vineyards, alfalfa, almond, walnut, peach, lemon, avocado, and corn.
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rAAV vector product characterization and stability studies.
Methods Mol. Biol.
PUBLISHED: 10-29-2011
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Recombinant adeno-associated viral (rAAV) vectors mediate the safe and long-term correction of genetic diseases following a single administration. Preclinical studies in animal models and human trials have shown rAAV vector persistence and safety. In some trials, sustained or transient transgene expression has been demonstrated in humans treated for alpha-1 antitrypsin deficiency, LPL deficiency, hemophilia B and cystic fibrosis, and sustained correction of inherited blindness has been reported by three groups. For human use, rAAV vectors are manufactured and tested in compliance with current Good Manufacturing Practices as outlined in the Code of Federal Regulations (21CFR) or European Good Manufacturing Practices (Eudralex, Volume 4, GMP Guidelines, 2003/94/CE and 91/356/EEC). Manufacturing control, as well as product quality is evaluated by quality control testing and all manufacturing, facilities, and testing activities are reviewed by the quality assurance department. In-process specifications are set and in-process testing is conducted to confirm that the manufacturing process is controlled, aseptic, and performs consistently. Final product is tested to ensure release specifications are met for identity, safety, purity, potency, and stability.
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Adeno-associated viral vector-mediated transgene expression is independent of DNA methylation in primate liver and skeletal muscle.
PLoS ONE
PUBLISHED: 04-21-2011
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Recombinant adeno-associated viral (rAAV) vectors can support long-term transgene expression in quiescent tissues. Intramuscular (i.m.) administration of a single-stranded AAV vector (ssAAV) in the nonhuman primate (NHP) results in a peak protein level at 2-3 months, followed by a decrease over several months before reaching a steady-state. To investigate transgene expression and vector genome persistence, we previously demonstrated that rAAV vector genomes associate with histones and form a chromatin structure in NHP skeletal muscle more than one year after injection. In the mammalian nucleus, chromatin remodeling via epigenetic modifications plays key role in transcriptional regulation. Among those, CpG hyper-methylation of promoters is a known hallmark of gene silencing. To assess the involvement of DNA methylation on the transgene expression, we injected NHP via the i.m. or the intravenous (i.v.) route with a recombinant ssAAV2/1 vector. The expression cassette contains the transgene under the transcriptional control of the constitutive Rous Sarcoma Virus promoter (RSVp). Total DNA isolated from NHP muscle and liver biopsies from 1 to 37 months post-injection was treated with sodium bisulfite and subsequently analyzed by pyrosequencing. No significant CpG methylation of the RSVp was found in rAAV virions or in vector DNA isolated from NHP transduced tissues. Direct de novo DNA methylation appears not to be involved in repressing transgene expression in NHP after gene transfer mediated by ssAAV vectors. The study presented here examines host/vector interactions and the impact on transgene expression in a clinically relevant model.
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Largemouth bass (Micropterus salmoides) and striped mullet (Mugil cephalus) as vectors of contaminants to human consumers in northwest Florida.
Mar. Environ. Res.
PUBLISHED: 03-28-2011
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The health benefits of regular consumption of fish and seafood have been espoused for many years. However, fish are also a potential source of environmental contaminants that have well known adverse effects on human health. We investigated the consumption risks for largemouth bass (Micropterus salmoides; n = 104) and striped mullet (Mugil cephalus; n = 170), two commonly harvested and consumed fish species inhabiting fresh and estuarine waters in northwest Florida. Skinless fillets were analyzed for total mercury, inorganic arsenic, polychlorinated dibenzo-p-dioxins and polychlorinated dibenzofurans (PCDD/F), polychlorinated biphenyls (PCBs), and organochlorine pesticides. Contaminant levels were compared to screening values (SV) calculated using U.S. Environmental Protection Agency (EPA) recommendations for establishing consumption advisories. Largemouth bass were found to contain high levels of total mercury at all sampling locations (0.37-0.89 ug/g) and one location exhibited elevated total PCBs (39.4 ng/g). All of the samples exceeded Florida fish consumption advisory trigger levels for total mercury and one location exceeded the U.S. EPA SV for total PCBs. As a result of the high mercury levels, the non-cancer health risks (hazard index-HI) for bass were above 1 for all locations. Striped mullet from several locations with known point sources contained elevated levels of PCBs (overall range 3.4-59.3 ng/g). However, total mercury levels in mullet were low. Eight of the 16 mullet sampling locations exceeded the U.S. EPA SV for total PCBs and two locations exceeded an HI of 1 due to elevated PCBs. Despite the elevated levels of total PCBs in some samples, only two locations exceeded the acceptable cancer risk range and therefore cancer health risks from consumption of bass and mullet were determined to be low at most sampling locations.
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Characterization of a recombinant adeno-associated virus type 2 Reference Standard Material.
Hum. Gene Ther.
PUBLISHED: 09-16-2010
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A recombinant adeno-associated virus serotype 2 Reference Standard Material (rAAV2 RSM) has been produced and characterized with the purpose of providing a reference standard for particle titer, vector genome titer, and infectious titer for AAV2 gene transfer vectors. Production and purification of the reference material were carried out by helper virus-free transient transfection and chromatographic purification. The purified bulk material was vialed, confirmed negative for microbial contamination, and then distributed for characterization along with standard assay protocols and assay reagents to 16 laboratories worldwide. Using statistical transformation and modeling of the raw data, mean titers and confidence intervals were determined for capsid particles ({X}, 9.18?x?10¹¹ particles/ml; 95% confidence interval [CI], 7.89?x?10¹¹ to 1.05?x?10¹² particles/ml), vector genomes ({X}, 3.28?x?10¹? vector genomes/ml; 95% CI, 2.70?x?10¹? to 4.75?x?10¹? vector genomes/ml), transducing units ({X}, 5.09?x?10? transducing units/ml; 95% CI, 2.00?x?10? to 9.60?x?10? transducing units/ml), and infectious units ({X}, 4.37?x?10? TCID?? IU/ml; 95% CI, 2.06?x?10? to 9.26?x?10? TCID?? IU/ml). Further analysis confirmed the identity of the reference material as AAV2 and the purity relative to nonvector proteins as greater than 94%. One obvious trend in the quantitative data was the degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This relatively poor degree of interlaboratory precision and accuracy was apparent even though attempts were made to standardize the assays by providing detailed protocols and common reagents. This is the first time that such variation between laboratories has been thoroughly documented and the findings emphasize the need in the field for universal reference standards. The rAAV2 RSM has been deposited with the American Type Culture Collection and is available to the scientific community to calibrate laboratory-specific internal titer standards. Anticipated uses of the rAAV2 RSM are discussed.
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Role of cyclic AMP-dependent kinase response element-binding protein in recombinant adeno-associated virus-mediated transduction of heart muscle cells.
Hum. Gene Ther.
PUBLISHED: 06-09-2009
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Recombinant adeno-associated virus (rAAV) vectors represent a promising approach to gene delivery for clinical use. Published data indicate that rAAV vector genomes persist in vivo as episomal chromatin in the skeletal muscle of nonhuman primates. In this study, we assessed the interconnection between the transcription factor cyclic AMP response element-binding protein (CREB) and recombinant AAV serotype 2 vector genomes after transduction in vitro and in vivo. rAAV-mediated myocyte transduction was potently blocked in the hearts of mice expressing CREB-S133A, which is a CREB-S133A dominant-negative mutant. Isoproterenol, a strong CREB activator, prominently increased rAAV transduction and the increase was abrogated by silencing the CREB gene with small interfering RNA. In addition, rAAV infection of muscle cells mildly but significantly induced CREB protein phosphorylation at serine-133, and was capable of stimulating CREB-dependent transcription from a reporter plasmid. Using chromatin immunoprecipitation and immunoblotting assays, both CREB and p300 were found to physically associate with two different rAAV genomes. Accordingly, CREB/p300 appears to have a role in rAAV transduction to establish active vector transcription in heart muscle cells.
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Adeno-associated virus capsid serotype identification: Analytical methods development and application.
J. Virol. Methods
PUBLISHED: 03-11-2009
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Mass spectrometry (MS) has been utilized to address the need for a rapid and reliable assay to confirm the capsid serotype identity of recombinant AAV gene transfer vectors. The differences in the primary amino acid sequence of AAV serotypes generate a unique set of fragments with different masses upon proteolytic digestion, and by comparing the fragment masses against common and custom databases, reliable capsid serotype identification is achieved. Highly homologous serotypes, such as AAV1, AAV2, and AAV8, can be distinguished from each other, as well as from less homologous serotypes such as AAV4, and AAV5. Furthermore, analysis of the MS data for wild-type AAV4 compared to an AAV4 capsid with a single amino acid mutation demonstrates the sensitivity of the method and validates the relevance of the method in the context of retinal gene transfer. With an expanding repertoire of AAV serotypes, physicochemical methods for capsid analysis, such as MS, are highly desirable and do not require product-specific analytical reagents such as monoclonal antibodies. A MS-based capsid identity test is suitable for cGMP lot release testing of rAAV gene transfer products and will help ensure patient protection.
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Gene therapy for aromatic L-amino acid decarboxylase deficiency.
Sci Transl Med
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Aromatic L-amino acid decarboxylase (AADC) is required for the synthesis of the neurotransmitters dopamine and serotonin. Children with defects in the AADC gene show compromised development, particularly in motor function. Drug therapy has only marginal effects on some of the symptoms and does not change early childhood mortality. Here, we performed adeno-associated viral vector-mediated gene transfer of the human AADC gene bilaterally into the putamen of four patients 4 to 6 years of age. All of the patients showed improvements in motor performance: One patient was able to stand 16 months after gene transfer, and the other three patients achieved supported sitting 6 to 15 months after gene transfer. Choreic dyskinesia was observed in all patients, but this resolved after several months. Positron emission tomography revealed increased uptake by the putamen of 6-[(18)F]fluorodopa, a tracer for AADC. Cerebrospinal fluid analysis showed increased dopamine and serotonin levels after gene transfer. Thus, gene therapy targeting primary AADC deficiency is well tolerated and leads to improved motor function.
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Development and utility of an internal threshold control (ITC) real-time PCR assay for exogenous DNA detection.
PLoS ONE
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Sensitive and specific tests for detecting exogenous DNA molecules are useful for infectious disease diagnosis, gene therapy clinical trial safety, and gene doping surveillance. Taqman real-time PCR using specific sequence probes provides an effective approach to accurately and quantitatively detect exogenous DNA. However, one of the major challenges in these analyses is to eliminate false positive signals caused by either non-targeted exogenous or endogenous DNA sequences, or false negative signals caused by impurities that inhibit PCR. Although multiplex Taqman PCR assays have been applied to address these problems by adding extra primer-probe sets targeted to endogenous DNA sequences, the differences between targets can lead to different detection efficiencies. To avoid these complications, a Taqman PCR-based approach that incorporates an internal threshold control (ITC) has been developed. In this single reaction format, the target sequence and ITC template are co-amplified by the same primers, but are detected by different probes each with a unique fluorescent dye. Sample DNA, a prescribed number of ITC template molecules set near the limit of sensitivity, a single pair of primers, target probe and ITC probe are added to one reaction. Fluorescence emission signals are obtained simultaneously to determine the cycle thresholds (Ct) for amplification of the target and ITC sequences. The comparison of the target Ct with the ITC Ct indicates if a sample is a true positive for the target (i.e. Ct less than or equal to the ITC Ct) or negative (i.e. Ct greater than the ITC Ct). The utility of this approach was demonstrated in a nonhuman primate model of rAAV vector mediated gene doping in vivo and in human genomic DNA spiked with plasmid DNA.
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Integration frequency and intermolecular recombination of rAAV vectors in non-human primate skeletal muscle and liver.
Mol. Ther.
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The comprehensive characterization of recombinant adeno-associated viral (rAAV) integration frequency and persistence for assessing rAAV vector biosafety in gene therapy is severely limited due to the predominance of episomal rAAV vector genomes maintained in vivo. Introducing rAAV insertional standards (rAIS), we show that linear amplification-mediated (LAM)-PCR and deep sequencing can be used for validated measurement of rAAV integration frequencies. Integration of rAAV2/1 or rAAV2/8, following intramuscular (IM) or regional intravenous (RI) administration of therapeutically relevant vector doses in nine adult non-human primates (NHP), occurs at low frequency between 10(-4) and 10(-5) both in NHP liver and muscle, but with no preference for specific genomic loci. High resolution mapping of inverted terminal repeat (ITR) breakpoints in concatemeric and integrated vector genomes reveals distinct vector recombination hotspots, including large deletions of up to 3 kb. Moreover, retrieval of integrated rAAV genomes indicated approximately threefold increase in liver compared to muscle. This molecular analysis of rAAV persistence in NHP provides a promising basis for a reliable genotoxic risk assessment of rAAV in clinical trials.
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Recombinant adeno-associated viral vector reference standards.
Meth. Enzymol.
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Reference standard materials (RSMs) exist for a variety of biologics including vaccines but are not readily available for gene therapy vectors. To date, a recombinant adeno-associated virus serotype 2 RSM (rAAV2 RSM) has been produced and characterized and was made available to the scientific community in 2010. In addition, a rAAV8 RSM has been produced and will be characterized in the coming months. The use of these reference materials by members of the gene therapy field facilitates the calibration of individual laboratory vector-specific internal standards and the eventual comparison of preclinical and clinical data based on common dosage units. Normalization of data to determine therapeutic dose ranges of rAAV vectors for each particular tissue target and disease indication is important information that can enhance the safety and protection of patients.
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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.