JoVE Visualize What is visualize?
Stop Reading. Start Watching.
Advanced Search
Stop Reading. Start Watching.
Regular Search
Find video protocols related to scientific articles indexed in Pubmed.
Phase II Trial of Stereotactic Body Radiation Therapy Combined With Erlotinib for Patients With Limited but Progressive Metastatic Non-Small-Cell Lung Cancer.
J. Clin. Oncol.
PUBLISHED: 10-29-2014
Show Abstract
Hide Abstract
Patients with stage IV non-small-cell lung cancer (NSCLC) who progress through first-line therapy have poor progression-free survival (PFS) and overall survival (OS), most commonly failing in original sites of gross disease. Cytoreduction with stereotactic body radiation therapy (SBRT) may help systemic agents delay relapse.
Related JoVE Video
Predatory fish sounds can alter crab foraging behaviour and influence bivalve abundance.
Proc. Biol. Sci.
PUBLISHED: 06-20-2014
Show Abstract
Hide Abstract
The risk of predation can have large effects on ecological communities via changes in prey behaviour, morphology and reproduction. Although prey can use a variety of sensory signals to detect predation risk, relatively little is known regarding the effects of predator acoustic cues on prey foraging behaviour. Here we show that an ecologically important marine crab species can detect sound across a range of frequencies, probably in response to particle acceleration. Further, crabs suppress their resource consumption in the presence of experimental acoustic stimuli from multiple predatory fish species, and the sign and strength of this response is similar to that elicited by water-borne chemical cues. When acoustic and chemical cues were combined, consumption differed from expectations based on independent cue effects, suggesting redundancies among cue types. These results highlight that predator acoustic cues may influence prey behaviour across a range of vertebrate and invertebrate taxa, with the potential for cascading effects on resource abundance.
Related JoVE Video
Malignant Infiltration of the Liver Presenting as Acute Liver Failure.
Clin. Gastroenterol. Hepatol.
PUBLISHED: 04-03-2014
Show Abstract
Hide Abstract
There have been few reports of acute liver failure (ALF, with encephalopathy and coagulopathy) due to infiltration of the liver by malignant cells. We describe a case series of 27 patients with ALF caused by malignancy. We examined a large, multi-center ALF registry (1910 patients; mean age, 47.1±13.9 years) and found only 27 cases (1.4%) of ALF attributed to malignancy. Twenty cases (74%) presented with abdominal pain and 11 with ascites. The malignancies included lymphoma or leukemia (33%), breast cancer, (30%), and colon cancer (7%); 90% of the patients with lymphoma or leukemia had no history of cancer, compared to 25% of patients with breast cancer. Overall, 44% of the patients had evidence of liver masses by imaging. Diagnosis was confirmed by biopsy in 15 (55%) and autopsy for 6 cases. Twenty-four patients (89%) died within 3 weeks of ALF.
Related JoVE Video
The biogeography of trophic cascades on US oyster reefs.
Ecol. Lett.
PUBLISHED: 02-18-2014
Show Abstract
Hide Abstract
Predators can indirectly benefit prey populations by suppressing mid-trophic level consumers, but often the strength and outcome of trophic cascades are uncertain. We manipulated oyster reef communities to test the generality of potential causal factors across a 1000-km region. Densities of oyster consumers were weakly influenced by predators at all sites. In contrast, consumer foraging behaviour in the presence of predators varied considerably, and these behavioural effects altered the trophic cascade across space. Variability in the behavioural cascade was linked to regional gradients in oyster recruitment to and sediment accumulation on reefs. Specifically, asynchronous gradients in these factors influenced whether the benefits of suppressed consumer foraging on oyster recruits exceeded costs of sediment accumulation resulting from decreased consumer activity. Thus, although predation on consumers remains consistent, predator influences on behaviour do not; rather, they interact with environmental gradients to cause biogeographic variability in the net strength of trophic cascades.
Related JoVE Video
An amino acid depleted cell-free protein synthesis system for the incorporation of non-canonical amino acid analogs into proteins.
J. Biotechnol.
PUBLISHED: 02-08-2014
Show Abstract
Hide Abstract
Residue-specific incorporation of non-canonical amino acids into proteins is usually performed in vivo using amino acid auxotrophic strains and replacing the natural amino acid with an unnatural amino acid analog. Herein, we present an efficient amino acid depleted cell-free protein synthesis system that can be used to study residue-specific replacement of a natural amino acid by an unnatural amino acid analog. This system combines a simple methodology and high protein expression titers with a high-efficiency analog substitution into a target protein. To demonstrate the productivity and efficacy of a cell-free synthesis system for residue-specific incorporation of unnatural amino acids in vitro, we use this system to show that 5-fluorotryptophan and 6-fluorotryptophan substituted streptavidin retain the ability to bind biotin despite protein-wide replacement of a natural amino acid for the amino acid analog. We envisage this amino acid depleted cell-free synthesis system being an economical and convenient format for the high-throughput screening of a myriad of amino acid analogs with a variety of protein targets for the study and functional characterization of proteins substituted with unnatural amino acids when compared to the currently employed in vivo methodologies.
Related JoVE Video
Directed evolution of genetic parts and circuits by compartmentalized partnered replication.
Nat. Biotechnol.
PUBLISHED: 07-25-2013
Show Abstract
Hide Abstract
Most existing directed evolution methods, both in vivo and in vitro, suffer from inadvertent selective pressures (i.e., altering organism fitness), resulting in the evolution of products with unintended or suboptimal function. To overcome these barriers, here we present compartmentalized partnered replication (CPR). In this approach, synthetic circuits are linked to the production of Taq DNA polymerase so that evolved circuits that most efficiently drive Taq DNA polymerase production are enriched by exponential amplification during a subsequent emulsion PCR step. We apply CPR to evolve a T7 RNA polymerase variant that recognizes an orthogonal promoter and to reengineer the tryptophanyl tRNA-synthetase:suppressor tRNA pair from Saccharomyces cerevisiae to efficiently and site-specifically incorporate an unnatural amino acid into proteins. In both cases, the CPR-evolved parts were more orthogonal and/or more active than variants evolved using other methods. CPR should be useful for evolving any genetic part or circuit that can be linked to Taq DNA polymerase expression.
Related JoVE Video
Host and parasite recruitment correlated at a regional scale.
Oecologia
PUBLISHED: 06-11-2013
Show Abstract
Hide Abstract
Drivers of large-scale variability in parasite prevalence are not well understood. For logistical reasons, explorations of spatial patterns in parasites are often performed as observational studies. However, to understand the mechanisms that underlie these spatial patterns, standardized and controlled comparisons are needed. Here, we examined spatial variability in infection of an important fishery species and ecosystem engineer, the oyster (Crassostrea virginica) by its pea crab parasite (Zaops ostreus) across 700 km of the southeastern USA coastline. To minimize the influence of host genetics on infection patterns, we obtained juvenile oysters from a homogeneous source stock and raised them in situ for 3 months at multiple sites with similar environmental characteristics. We found that prevalence of pea crab infection varied between 24 and 73 % across sites, but not systematically across latitude. Of all measured environmental variables, oyster recruitment correlated most strongly (and positively) with pea crab infection, explaining 92 % of the variability in infection across sites. Our data ostensibly suggest that regional processes driving variation in oyster recruitment similarly affect the recruitment of one of its common parasites.
Related JoVE Video
Genetic relatedness influences plant biomass accumulation in eelgrass (Zostera marina).
Am. Nat.
PUBLISHED: 03-27-2013
Show Abstract
Hide Abstract
In multispecies assemblages, phylogenetic relatedness often predicts total community biomass. In assemblages dominated by a single species, increasing the number of genotypes increases total production, but the role of genetic relatedness is unknown. We used data from three published experiments and a field survey of eelgrass (Zostera marina), a habitat-forming marine angiosperm, to examine the strength and direction of the relationship between genetic relatedness and plant biomass. The genetic relatedness of an assemblage strongly predicted its biomass, more so than the number of genotypes. However, contrary to the pattern observed in multispecies assemblages, maximum biomass occurred in assemblages of more closely related individuals. The mechanisms underlying this pattern remain unclear; however, our data support a role for both trait differentiation and cooperation among kin. Many habitat-forming species interact intensely with conspecifics of varying relatedness; thus, genetic relatedness could influence the functioning of ecosystems dominated by such species.
Related JoVE Video
Residue-specific incorporation of unnatural amino acids into proteins in vitro and in vivo.
Methods Mol. Biol.
PUBLISHED: 02-21-2013
Show Abstract
Hide Abstract
The incorporation of noncanonical (unnatural) amino acids into proteins offers researchers the ability to augment the biochemical functionality of proteins for a myriad of applications including bioorthogonal conjugation, biophysical and structural studies, and the enhancement or de novo creation of novel enzymatic activities. The augmentation of a protein throughout its coding sequence by global residue-specific incorporation of unnatural amino acid analogs is an attractive technique for studying both the utility of individual chemistries available through unnatural amino acids and the general effects of unnatural amino acid substitution on protein structure and function. Herein we describe protocols to introduce unnatural amino acids into proteins using the Escherichia coli translation system either in vivo or in vitro. Special attention is paid to obtaining high levels of incorporation while maintaining high yields of protein expression.
Related JoVE Video
Molecular deconvolution of the monoclonal antibodies that comprise the polyclonal serum response.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 02-04-2013
Show Abstract
Hide Abstract
We have developed and validated a methodology for determining the antibody composition of the polyclonal serum response after immunization. Pepsin-digested serum IgGs were subjected to standard antigen-affinity chromatography, and resulting elution, wash, and flow-through fractions were analyzed by bottom-up, liquid chromatography-high-resolution tandem mass spectrometry. Identification of individual monoclonal antibodies required the generation of a database of IgG variable gene (V-gene) sequences constructed by NextGen sequencing of mature B cells. Antibody V-gene sequences are characterized by short complementarity determining regions (CDRs) of high diversity adjacent to framework regions shared across thousands of IgGs, greatly complicating the identification of antigen-specific IgGs from proteomically observed peptides. By mapping peptides marking unique V(H) CDRH3 sequences, we identified a set of V-genes heavily enriched in the affinity chromatography elution, constituting the serum polyclonal response. After booster immunization in a rabbit, we find that the antigen-specific serum immune response is oligoclonal, comprising antibodies encoding 34 different CDRH3s that group into 30 distinct antibody V(H) clonotypes. Of these 34 CDRH3s, 12 account for ?60% of the antigen-specific CDRH3 peptide mass spectral counts. For comparison, antibodies with 18 different CDRH3s (12 clonotypes) were represented in the antigen-specific IgG fraction from an unimmunized rabbit that fortuitously displayed a moderate titer for BSA. Proteomically identified antibodies were synthesized and shown to display subnanomolar affinities. The ability to deconvolute the polyclonal serum response is likely to be of key importance for analyzing antibody responses after vaccination and for more completely understanding adaptive immune responses in health and disease.
Related JoVE Video
Structure-based non-canonical amino acid design to covalently crosslink an antibody-antigen complex.
J. Struct. Biol.
PUBLISHED: 01-28-2013
Show Abstract
Hide Abstract
Engineering antibodies to utilize non-canonical amino acids (NCAA) should greatly expand the utility of an already important biological reagent. In particular, introducing crosslinking reagents into antibody complementarity determining regions (CDRs) should provide a means to covalently crosslink residues at the antibody-antigen interface. Unfortunately, finding the optimum position for crosslinking two proteins is often a matter of iterative guessing, even when the interface is known in atomic detail. Computer-aided antibody design can potentially greatly restrict the number of variants that must be explored in order to identify successful crosslinking sites. We have therefore used Rosetta to guide the introduction of an oxidizable crosslinking NCAA, l-3,4-dihydroxyphenylalanine (l-DOPA), into the CDRs of the anti-protective antigen scFv antibody M18, and have measured crosslinking to its cognate antigen, domain 4 of the anthrax protective antigen. Computed crosslinking distance, solvent accessibility, and interface energetics were three factors considered that could impact the efficiency of l-DOPA-mediated crosslinking. In the end, 10 variants were synthesized, and crosslinking efficiencies were generally 10% or higher, with the best variant crosslinking to 52% of the available antigen. The results suggest that computational analysis can be used in a pipeline for engineering crosslinking antibodies. The rules learned from l-DOPA crosslinking of antibodies may also be generalizable to the formation of other crosslinked interfaces and complexes.
Related JoVE Video
Loss of blue carbon from coastal salt marshes following habitat disturbance.
PLoS ONE
PUBLISHED: 01-01-2013
Show Abstract
Hide Abstract
Increased recognition of the global importance of salt marshes as blue carbon (C) sinks has led to concern that salt marshes could release large amounts of stored C into the atmosphere (as CO2) if they continue undergoing disturbance, thereby accelerating climate change. Empirical evidence of C release following salt marsh habitat loss due to disturbance is rare, yet such information is essential for inclusion of salt marshes in greenhouse gas emission reduction and offset schemes. Here we investigated the stability of salt marsh (Spartinaalterniflora) sediment C levels following seagrass (Thallasiatestudinum) wrack accumulation; a form of disturbance common throughout the world that removes large areas of plant biomass in salt marshes. At our study site (St Joseph Bay, Florida, USA), we recorded 296 patches (7.5 ± 2.3 m(2) mean area ± SE) of vegetation loss (aged 3-12 months) in a salt marsh meadow the size of a soccer field (7 275 m(2)). Within these disturbed patches, levels of organic C in the subsurface zone (1-5 cm depth) were ~30% lower than the surrounding undisturbed meadow. Subsequent analyses showed that the decline in subsurface C levels in disturbed patches was due to loss of below-ground plant (salt marsh) biomass, which otherwise forms the main component of the long-term refractory C stock. We conclude that disturbance to salt marsh habitat due to wrack accumulation can cause significant release of below-ground C; which could shift salt marshes from C sinks to C sources, depending on the intensity and scale of disturbance. This mechanism of C release is likely to increase in the future due to sea level rise; which could increase wrack production due to increasing storminess, and will facilitate delivery of wrack into salt marsh zones due to higher and more frequent inundation.
Related JoVE Video
Impact of treatment sequence of multimodal therapy for advanced oral cavity cancer with mandible invasion.
Otolaryngol Head Neck Surg
PUBLISHED: 08-02-2011
Show Abstract
Hide Abstract
To determine the effect of treatment sequence of multimodal therapy for clinically advanced squamous cell carcinoma (SCC) of the oral cavity (OC) with mandible invasion.
Related JoVE Video
Gene synthesis: methods and applications.
Meth. Enzymol.
PUBLISHED: 05-24-2011
Show Abstract
Hide Abstract
DNA synthesis techniques and technologies are quickly becoming a cornerstone of modern molecular biology and play a pivotal role in the field of synthetic biology. The ability to synthesize whole genes, novel genetic pathways, and even entire genomes is no longer the dream it was 30 years ago. Using little more than a thermocycler, commercially synthesized oligonucleotides, and DNA polymerases, a standard molecular biology laboratory can synthesize several kilobase pairs of synthetic DNA in a week using existing techniques. Herein, we review the techniques used in the generation of synthetic DNA, from the chemical synthesis of oligonucleotides to their assembly into long, custom sequences. Software and websites to facilitate the execution of these approaches are explored, and applications of DNA synthesis techniques to gene expression and synthetic biology are discussed. Finally, an example of automated gene synthesis from our own laboratory is provided.
Related JoVE Video
Rational design of an orthogonal tryptophanyl nonsense suppressor tRNA.
Nucleic Acids Res.
PUBLISHED: 06-22-2010
Show Abstract
Hide Abstract
While a number of aminoacyl tRNA synthetase (aaRS):tRNA pairs have been engineered to alter or expand the genetic code, only the Methanococcus jannaschii tyrosyl tRNA synthetase and tRNA have been used extensively in bacteria, limiting the types and numbers of unnatural amino acids that can be utilized at any one time to expand the genetic code. In order to expand the number and type of aaRS/tRNA pairs available for engineering bacterial genetic codes, we have developed an orthogonal tryptophanyl tRNA synthetase and tRNA pair, derived from Saccharomyces cerevisiae. In the process of developing an amber suppressor tRNA, we discovered that the Escherichia coli lysyl tRNA synthetase was responsible for misacylating the initial amber suppressor version of the yeast tryptophanyl tRNA. It was discovered that modification of the G:C content of the anticodon stem and therefore reducing the structural flexibility of this stem eliminated misacylation by the E. coli lysyl tRNA synthetase, and led to the development of a functional, orthogonal suppressor pair that should prove useful for the incorporation of bulky, unnatural amino acids into the genetic code. Our results provide insight into the role of tRNA flexibility in molecular recognition and the engineering and evolution of tRNA specificity.
Related JoVE Video
Monoclonal antibodies isolated without screening by analyzing the variable-gene repertoire of plasma cells.
Nat. Biotechnol.
PUBLISHED: 06-11-2010
Show Abstract
Hide Abstract
Isolation of antigen-specific monoclonal antibodies (mAbs) and antibody fragments relies on high-throughput screening of immortalized B cells or recombinant antibody libraries. We bypassed the screening step by using high-throughput DNA sequencing and bioinformatic analysis to mine antibody variable region (V)-gene repertoires from bone marrow plasma cells (BMPC) of immunized mice. BMPCs, which cannot be immortalized, produce the vast majority of circulating antibodies. We found that the V-gene repertoire of BMPCs becomes highly polarized after immunization, with the most abundant sequences represented at frequencies between approximately 1% and >10% of the total repertoire. We paired the most abundant variable heavy (V(H)) and variable light (V(L)) genes based on their relative frequencies, reconstructed them using automated gene synthesis, and expressed recombinant antibodies in bacteria or mammalian cells. Antibodies generated in this manner from six mice, each immunized with one of three antigens were overwhelmingly antigen specific (21/27 or 78%). Those generated from a mouse with high serum titers had nanomolar binding affinities.
Related JoVE Video
Accelerating loss of seagrasses across the globe threatens coastal ecosystems.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-08-2009
Show Abstract
Hide Abstract
Coastal ecosystems and the services they provide are adversely affected by a wide variety of human activities. In particular, seagrass meadows are negatively affected by impacts accruing from the billion or more people who live within 50 km of them. Seagrass meadows provide important ecosystem services, including an estimated $1.9 trillion per year in the form of nutrient cycling; an order of magnitude enhancement of coral reef fish productivity; a habitat for thousands of fish, bird, and invertebrate species; and a major food source for endangered dugong, manatee, and green turtle. Although individual impacts from coastal development, degraded water quality, and climate change have been documented, there has been no quantitative global assessment of seagrass loss until now. Our comprehensive global assessment of 215 studies found that seagrasses have been disappearing at a rate of 110 km(2) yr(-1) since 1980 and that 29% of the known areal extent has disappeared since seagrass areas were initially recorded in 1879. Furthermore, rates of decline have accelerated from a median of 0.9% yr(-1) before 1940 to 7% yr(-1) since 1990. Seagrass loss rates are comparable to those reported for mangroves, coral reefs, and tropical rainforests and place seagrass meadows among the most threatened ecosystems on earth.
Related JoVE Video
Ecological impacts of genotypic diversity in the clonal seagrass Zostera marina.
Ecology
PUBLISHED: 06-23-2009
Show Abstract
Hide Abstract
Genetic diversity, like species diversity, can have important consequences for communities and ecosystems. However, little is known about whether the effects of genetic diversity demonstrated in experimental assemblages are of sufficient strength to generate patterns in natural systems. We conducted a survey of eelgrass (Zostera marina) to examine the correlation between eelgrass clonal diversity and two metrics of community structure across two seasons: shoot density (reflective of habitat quality) and biomass of epiphytic algae (as a measure of food resource availability). Eelgrass clonal diversity was not related to epiphyte biomass in either winter or summer. Interestingly, there was a positive relationship between eelgrass clonal diversity and shoot density only in the winter, when eelgrass experiences stress from abiotic and biotic factors. The magnitude of this correlation was similar to that of other factors known to affect shoot density such as tidal elevation or position in the bed. In contrast, summer shoot density and diversity were uncorrelated. This natural pattern is consistent with previous experimental results in which diversity positively affected shoot density only during periods of abiotic or biotic stress, suggesting that the effects of clonal diversity are sufficiently strong to influence shoot density in the field, despite the presence of potentially confounding environmental gradients.
Related JoVE Video
A phase II study of concurrent chemoradiation with weekly docetaxel, carboplatin, and radiation therapy followed by consolidation chemotherapy with docetaxel and carboplatin for locally advanced inoperable non-small cell lung cancer (NSCLC).
J Thorac Oncol
PUBLISHED: 05-01-2009
Show Abstract
Hide Abstract
The current standard of care for good performance status patients with locally advanced non-small cell lung carcinoma is concurrent chemoradiation, although a clearly superior regimen has not been identified. Docetaxel has been shown to possess good single-agent activity against non-small cell lung cancer (NSCLC) and radiosensitizing properties, both alone and synergistically with carboplatin. We undertook this phase II study to determine the safety and efficacy of weekly docetaxel-carboplatin and concurrent radiation therapy followed by docetaxel-carboplatin consolidation for the treatment of locally advanced NSCLC.
Related JoVE Video
Morphological and physiological variation among seagrass (Zostera marina) genotypes.
Oecologia
PUBLISHED: 01-09-2009
Show Abstract
Hide Abstract
Intraspecific variation in habitat-forming species can have important ecological consequences at the population, community, and ecosystem level. However, the contribution of genetic variation among individuals to these effects is seldom documented. We quantified morphological and physiological variation among genotypes of a marine foundation species, the seagrass Zostera marina. We grew replicate shoots of eight genetically distinct Zostera individuals collected from Bodega Bay, California, in a common garden environment and then quantified shoot production and morphology, nutrient uptake, and key photosynthetic parameters. We found that genotypes differed in shoot production, biomass, and both root and shoot nutrient uptake rates, even when corrected for genotype-specific biomass differences. In addition, the rank order of uptake ability differed for ammonium and nitrate, indicating that genotypes may exhibit resource partitioning of different forms of nutrients. Our results suggest that both niche complementarity among genotypes and the sampling/selection effect could contribute to previously observed positive effects of seagrass clonal diversity on resource utilization and biomass production. Further, they highlight that genotypic variation in key traits of habitat-forming species could have measurable effects on community structure and function.
Related JoVE Video
Predator cue and prey density interactively influence indirect effects on basal resources in intertidal oyster reefs.
PLoS ONE
Show Abstract
Hide Abstract
Predators can influence prey abundance and traits by direct consumption, as well as by non-consumptive effects of visual, olfactory, or tactile cues. The strength of these non-consumptive effects (NCEs) can be influenced by a variety of factors, including predator foraging mode, temporal variation in predator cues, and the density of competing prey. Testing the relative importance of these factors for determining NCEs is critical to our understanding of predator-prey interactions in a variety of settings. We addressed this knowledge gap by conducting two mesocosm experiments in a tri-trophic intertidal oyster reef food web. More specifically, we tested how a predatory fish (hardhead catfish, Ariopsis felis) directly influenced their prey (mud crabs, Panopeus spp.) and indirectly affected basal resources (juvenile oysters, Crassostrea virginica), as well as whether these direct and indirect effects changed across a density gradient of competing prey. Per capita crab foraging rates were inversely influenced by crab density, but they were not affected by water-borne predator cues. As a result, direct consumptive effects on prey foraging rates were stronger than non-consumptive effects. In contrast, predator cue and crab density interactively influenced indirect predator effects on oyster mortality in two experiments, with trait-mediated and density-mediated effects of similar magnitude operating to enhance oyster abundance. Consistent differences between a variable predator cue environment and other predator cue treatments (no cue and constant cue) suggests that an understanding of the natural risk environment experienced by prey is critical to testing and interpreting trait-mediated indirect interactions. Further, the prey response to the risk environment may be highly dependent on prey density, particularly in prey populations with strong intra-specific interactions.
Related JoVE Video
Enrichment of error-free synthetic DNA sequences by CEL I nuclease.
Curr Protoc Mol Biol
Show Abstract
Hide Abstract
As the availability of DNA sequence information has grown, so has the need to replicate DNA sequences synthetically. Synthetically produced DNA sequences allow the researcher to exert greater control over model systems and allow for the combinatorial design and construction of novel metabolic and regulatory pathways, as well as optimized protein-coding sequences for biotechnological applications. This utility has made synthetically produced DNA a hallmark of the molecular biosciences and a mainstay of synthetic biology. However, synthetically produced DNA has a significant shortcoming in that it typically has an error rate that is orders of magnitude higher when compared to DNA sequences derived directly from a biological source. This relatively high error rate adds to the cost and labor necessary to obtain sequence-verified clones from synthetically produced DNA sequences. This unit describes a protocol to enrich error-free sequences from a population of error-rich DNA via treatment with CEL I (Surveyor) endonuclease. This method is a straightforward and quick way of reducing the error content of synthetic DNA pools and reliably reduces the error rates by >6-fold per round of treatment.
Related JoVE Video
Design and assembly of large synthetic DNA constructs.
Curr Protoc Mol Biol
Show Abstract
Hide Abstract
The availability of custom synthetic gene-length DNA products removes numerous bottlenecks in research efforts, making gene synthesis an increasingly common commercial service. However, the assembly of synthetic oligonucleotides into large, custom DNA constructs is not especially difficult, and performing "in-house" gene synthesis has time and cost advantages. This unit will treat both the concerns of design and physical assembly in gene synthesis, including how to design DNA sequences for synthesis and the design of overlapping oligonucleotide schemes to ensure facile assembly into the final product. Assembly is accomplished using a reliable series of PCR reactions, with a troubleshooting assembly protocol included, which not only assembles difficult sequences but allows identification of the source of a failure down to a pair of oligonucleotides.
Related JoVE Video
A neighboring plant species creates associational refuge for consumer and host.
Ecology
Show Abstract
Hide Abstract
Examples of plant-animal and plant-plant associational defenses are common across a variety of systems, yet the potential for them to occur in concert has not been explored. In salt marshes in the Gulf of Mexico, the marsh periwinkle (Littoraria irrorata) is an abundant and conspicuous member of the community, climbing up the stems of marsh plants to remain out of the water at high tide. Though Littoraria are thought to primarily utilize stems of marsh cordgrass Spartina alterniflora as a source of food and refuge, Littoraria were more abundant in mixed assemblages of Spartina and Juncus roemerianus than in Spartina-only areas at the same tidal height. Mesocosm experiments confirmed that Juncus provided a refuge for Littoraria, with predation by Callinectes sapidus (but not Melongena corona) reduced when Juncus was present. However, Littorarias utilization of Juncus as well as the effectiveness of Juncus as a refuge depended strongly on plant height: when Juncus was experimentally clipped to a shorter height than Spartina, snail abundance on Spartina and snail predation by crabs increased. Interestingly, this plant animal refuge led to a corresponding refuge for Spartina from Littoraria: Spartina plants lost less biomass to snail grazing when growing with Juncus in mesocosm and field experiments, and Spartina plants in natural assemblages were taller when growing with Juncus than when growing alone, even in the presence of abundant snails. This example highlights the potential importance of plant plant and plant-animal associational refuges in competitive plant assemblages.
Related JoVE Video
SCHEMA-designed variants of human Arginase I and II reveal sequence elements important to stability and catalysis.
ACS Synth Biol
Show Abstract
Hide Abstract
Arginases catalyze the divalent cation-dependent hydrolysis of L-arginine to urea and L-ornithine. There is significant interest in using arginase as a therapeutic antineogenic agent against L-arginine auxotrophic tumors and in enzyme replacement therapy for treating hyperargininemia. Both therapeutic applications require enzymes with sufficient stability under physiological conditions. To explore sequence elements that contribute to arginase stability we used SCHEMA-guided recombination to design a library of chimeric enzymes composed of sequence fragments from the two human isozymes Arginase I and II. We then developed a novel active learning algorithm that selects sequences from this library that are both highly informative and functional. Using high-throughput gene synthesis and our two-step active learning algorithm, we were able to rapidly create a small but highly informative set of seven enzymatically active chimeras that had an average variant distance of 40 mutations from the closest parent arginase. Within this set of sequences, linear regression was used to identify the sequence elements that contribute to the long-term stability of human arginase under physiological conditions. This approach revealed a striking correlation between the isoelectric point and the long-term stability of the enzyme to deactivation under physiological conditions.
Related JoVE Video
Structure-based design of supercharged, highly thermoresistant antibodies.
Chem. Biol.
Show Abstract
Hide Abstract
Mutation of surface residues to charged amino acids increases resistance to aggregation and can enable reversible unfolding. We have developed a protocol using the Rosetta computational design package that "supercharges" proteins while considering the energetic implications of each mutation. Using a homology model, a single-chain variable fragment antibody was designed that has a markedly enhanced resistance to thermal inactivation and displays an unanticipated ?30-fold improvement in affinity. Such supercharged antibodies should prove useful for assays in resource-limited settings and for developing reagents with improved shelf lives.
Related JoVE Video

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.

How does it work?

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.