High-Throughput Surface Plasmon Resonance Biosensors for Identifying Diverse Therapeutic Monoclonal Antibodies.

Identification of antibodies targeting diverse functional epitopes on an antigen is highly crucial for discovering effective therapeutic candidates. Employing a traditional stepwise antibody "screening funnel" as well as prioritizing affinity-based selections over epitope-based selections, result in lead antibody panels lacking epitope diversity. In the present study, we employed an array-based surface plasmon resonance (SPR) platform to perform high-throughput epitope binning analysis on a large number of monoclonal antibodies (mAbs) generated in the early drug discovery process. The mAb panel contained clones from different antibody generation techniques and diverse transgenic mouse strains. The epitope binning results were analyzed in unique ways using various visualizations in the form of dendrograms and network plots, which assisted in determining diversity and redundancy in the mAb sample set. The binning data were further integrated with affinity information to evaluate the performance of seven different transgenic mouse strains. The combination of epitope binning results with binding kinetics and sequence analysis provided an effective and efficient way of selecting high affinity antibodies representing a diverse set of sequence families and epitopes.

Temporal stability and molecular persistence of the bone marrow plasma cell antibody repertoire.

Plasma cells in human bone marrow (BM) are thought to be responsible for sustaining lifelong immunity, but its underlying basis is controversial. Here we use high-throughput sequence analysis of the same individual across 6.5 years to show that the BM plasma cell immunoglobulin heavy chain repertoire is remarkably stable over time. We find a nearly static bias in individual and combinatorial gene usage across time. Analysis of a second donor corroborates these observations. We also report the persistence of numerous BM plasma cell clonotypes (∼2%) identifiable at all points assayed across 6.5 years, supporting a model of serological memory based upon intrinsic longevity of human plasma cells. Donors were adolescents who completely recovered from neuroblastoma prior to the start of this study. Our work will facilitate differentiation between healthy and diseased antibody repertoires, by serving as a point of comparison with future deep-sequencing studies involving immune intervention.

Tempo and mode of genome evolution in a 50,000-generation experiment.

Adaptation by natural selection depends on the rates, effects and interactions of many mutations, making it difficult to determine what proportion of mutations in an evolving lineage are beneficial. Here we analysed 264 complete genomes from 12 Escherichia coli populations to characterize their dynamics over 50,000 generations. The populations that retained the ancestral mutation rate support a model in which most fixed mutations are beneficial, the fraction of beneficial mutations declines as fitness rises, and neutral mutations accumulate at a constant rate. We also compared these populations to mutation-accumulation lines evolved under a bottlenecking regime that minimizes selection. Nonsynonymous mutations, intergenic mutations, insertions and deletions are overrepresented in the long-term populations, further supporting the inference that most mutations that reached high frequency were favoured by selection. These results illuminate the shifting balance of forces that govern genome evolution in populations adapting to a new environment.

A census of human soluble protein complexes.

Cellular processes often depend on stable physical associations between proteins. Despite recent progress, knowledge of the composition of human protein complexes remains limited. To close this gap, we applied an integrative global proteomic profiling approach, based on chromatographic separation of cultured human cell extracts into more than one thousand biochemical fractions that were subsequently analyzed by quantitative tandem mass spectrometry, to systematically identify a network of 13,993 high-confidence physical interactions among 3,006 stably associated soluble human proteins. Most of the 622 putative protein complexes we report are linked to core biological processes and encompass both candidate disease genes and unannotated proteins to inform on mechanism. Strikingly, whereas larger multiprotein assemblies tend to be more extensively annotated and evolutionarily conserved, human protein complexes with five or fewer subunits are far more likely to be functionally unannotated or restricted to vertebrates, suggesting more recent functional innovations.

Successes and failures in modular genetic engineering.

Synthetic biology relies on engineering concepts such as abstraction, standardization, and decoupling to develop systems that address environmental, clinical, and industrial needs. Recent advances in applying modular design to system development have enabled creation of increasingly complex systems. However, several challenges to module and system development remain, including syntactic errors, semantic errors, parameter mismatches, contextual sensitivity, noise and evolution, and load and stress. To combat these challenges, researchers should develop a framework for describing and reasoning about biological information, design systems with modularity in mind, and investigate how to predictively describe the diverse sources and consequences of metabolic load and stress.

DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency.

Synthetic scaffolds that permit spatial and temporal organization of enzymes in living cells are a promising post-translational strategy for controlling the flow of information in both metabolic and signaling pathways. Here, we describe the use of plasmid DNA as a stable, robust and configurable scaffold for arranging biosynthetic enzymes in the cytoplasm of Escherichia coli. This involved conversion of individual enzymes into custom DNA-binding proteins by genetic fusion to zinc-finger domains that specifically bind unique DNA sequences. When expressed in cells that carried a rationally designed DNA scaffold comprising corresponding zinc finger binding sites, the titers of diverse metabolic products, including resveratrol, 1,2-propanediol and mevalonate were increased as a function of the scaffold architecture. These results highlight the utility of DNA scaffolds for assembling biosynthetic enzymes into functional metabolic structures. Beyond metabolism, we anticipate that DNA scaffolds may be useful in sequestering different types of enzymes for specifying the output of biological signaling pathways or for coordinating other assembly-line processes such as protein folding, degradation and post-translational modifications.

BglBricks: A flexible standard for biological part assembly.

BACKGROUND: Standard biological parts, such as BioBricks parts, provide the foundation for a new engineering discipline that enables the design and construction of synthetic biological systems with a variety of applications in bioenergy, new materials, therapeutics, and environmental remediation. Although the original BioBricks assembly standard has found widespread use, it has several shortcomings that limit its range of potential applications. In particular, the system is not suitable for the construction of protein fusions due to an unfavorable scar sequence that encodes an in-frame stop codon. RESULTS: Here, we present a similar but new composition standard, called BglBricks, that addresses the scar translation issue associated with the original standard. The new system employs BglII and BamHI restriction enzymes, robust cutters with an extensive history of use, and results in a 6-nucleotide scar sequence encoding glycine-serine, an innocuous peptide linker in most protein fusion applications. We demonstrate the utility of the new standard in three distinct applications, including the construction of constitutively active gene expression devices with a wide range of expression profiles, the construction of chimeric, multi-domain protein fusions, and the targeted integration of functional DNA sequences into specific loci of the E. coli genome. CONCLUSIONS: The BglBrick standard provides a new, more flexible platform from which to generate standard biological parts and automate DNA assembly. Work on BglBrick assembly reactions, as well as on the development of automation and bioinformatics tools, is currently underway. These tools will provide a foundation from which to transform genetic engineering from a technically intensive art into a purely design-based discipline.

Synthetic protein scaffolds provide modular control over metabolic flux.

Engineered metabolic pathways constructed from enzymes heterologous to the production host often suffer from flux imbalances, as they typically lack the regulatory mechanisms characteristic of natural metabolism. In an attempt to increase the effective concentration of each component of a pathway of interest, we built synthetic protein scaffolds that spatially recruit metabolic enzymes in a designable manner. Scaffolds bearing interaction domains from metazoan signaling proteins specifically accrue pathway enzymes tagged with their cognate peptide ligands. The natural modularity of these domains enabled us to optimize the stoichiometry of three mevalonate biosynthetic enzymes recruited to a synthetic complex and thereby achieve 77-fold improvement in product titer with low enzyme expression and reduced metabolic load. One of the same scaffolds was used to triple the yield of glucaric acid, despite high titers (0.5 g/l) without the synthetic complex. These strategies should prove generalizeable to other metabolic pathways and programmable for fine-tuning pathway flux.

Clinical microneedle injection of methyl nicotinate: stratum corneum penetration.

BACKGROUND/PURPOSE: In recent years, microneedles were proposed as a method to painlessly deliver drugs past the stratum corneum. Microneedles have been fabricated in several designs, but limited studies have tested microneedle injections in humans. In this work, we compare microneedle injections with topical application (TA) to investigate if microneedles enhance in vivo drug delivery past the stratum corneum. METHOD: In vitro tests were used to measure microneedle pressures and injection volumes. In vivo microneedle injections were performed on the volar forearm of 11 healthy volunteers. Two sets of microneedles, pointed and symmetric, were used to develop microneedle/syringe apparatuses that were used to inject approximately 1 microL of 0.1 M methyl nicotinate, and were compared against TA. A Laser Doppler Perfusion Monitor was used to record maximum blood flow and the time to maximum blood flow at the treatment sites. RESULTS: Pointed and symmetric microneedle-injected sites showed a significantly faster time to maximum blood flow than TA. The pointed microneedle injections also resulted in a higher maximum blood flux. Volunteers reported feeling pressure but no pain from the microneedles during the injections. CONCLUSION: The microneedles aid in bypassing the stratum corneum and enhance drug delivery through it. The design of the microneedle influences its delivery capabilities, because the pointed microneedles seem to be less susceptible to clogging during the injection.

Tribological testing of skin products: gender, age, and ethnicity on the volar forearm.

BACKGROUND/PURPOSE: Few studies have focused on the simultaneous measurement of the friction and electrical properties of skin. This work investigates the feasibility of using these measurements to differentiate between the effects of chemicals commonly applied to the skin. In addition, this study also compares the condition of the skin and its response to application of chemicals across gender, ethnicity, and age at the volar forearm. METHOD: Friction and electrical tests were performed on 59 healthy volunteers with the UMT Series Micro-Tribometer (UMT). A 13-mm-diameter copper cylindrical friction/electrical probe was pressed onto the skin with a weight of 20 g and moved across the skin at a constant velocity of 0.4 mm/s. Each volunteer served as his or her own control. The friction and electrical impedance measurements were performed for polyvinylidene chloride occlusion and for the application of glycerin and petrolatum. RESULTS: No differences were found across age, gender, or ethnicity at the volar forearm. Polyvinylidene chloride (PVDC) occlusion showed a small increase in the friction and a small decrease in the electrical impedance; petrolatum increased the friction by a greater amount but its effect on the impedance was comparable to PVDC occlusion; glycerin increased the friction by an amount comparable to petrolatum, but it decreased the impedance to a much greater degree than petrolatum or the PVDC occlusion. An amplitude/mean measurement of the friction curves of glycerin and petrolatum showed that glycerin has a significantly higher amplitude/mean than petrolatum. CONCLUSION: The properties of the volar forearm appear to be independent of age, gender, and ethnicity. Also, the simultaneous measurement of friction and electrical impedance was useful in differentiating between compounds administered to the skin.