Drug-regulated CD33-targeted CAR T cells control AML using clinically optimized rapamycin dosing.

Chimeric antigen receptor (CAR) designs that incorporate pharmacologic control are desirable; however, designs suitable for clinical translation are needed. We designed a fully human, rapamycin-regulated drug product for targeting CD33+ tumors called dimerizaing agent-regulated immunoreceptor complex (DARIC33). T cell products demonstrated target-specific and rapamycin-dependent cytokine release, transcriptional responses, cytotoxicity, and in vivo antileukemic activity in the presence of as little as 1 nM rapamycin.

People with obesity exhibit losses in muscle proteostasis that are partly improved by exercise training.

This pilot experiment examines if a loss in muscle proteostasis occurs in people with obesity and whether endurance exercise positively influences either the abundance profile or turnover rate of proteins in this population. Men with (n = 3) or without (n = 4) obesity were recruited and underwent a 14-d measurement protocol of daily deuterium oxide (DO) consumption and serial biopsies of vastus lateralis muscle. Men with obesity then completed 10-weeks of high-intensity interval training (HIIT), encompassing 3 sessions per week of cycle ergometer exercise with 1 min intervals at 100% maximum aerobic power interspersed by 1 min recovery periods.

A neural network method to predict task- and step-specific ground reaction force magnitudes from trunk accelerations during running activities.

Prediction of ground reaction force (GRF) magnitudes during running-based sports has several important applications, including optimal load prescription and injury prevention in athletes. Existing methods typically require information from multiple body-worn sensors, limiting their ecological validity, or aim to estimate discrete force parameters, limiting their ability to assess overall biomechanical load. This paper presents a neural network method to predict GRF time series from a single, commonly used, trunk-mounted accelerometer.

Biomechanical loading during running: can a two mass-spring-damper model be used to evaluate ground reaction forces for high-intensity tasks?

Running impact forces expose the body to biomechanical loads leading to beneficial adaptations, but also risk of injury. High-intensity running tasks, especially, are deemed highly demanding for the musculoskeletal system, but loads experienced during these actions are not well understood. To eventually predict GRF and understand the biomechanical loads experienced during such activities in greater detail, this study aimed to (1) examine the feasibility of using a simple two mass-spring-damper model, based on eight model parameters, to reproduce ground reaction forces (GRFs) for high-intensity running tasks and (2) verify whether the required model parameters were physically meaningful.

Dynamic proteome profiling of individual proteins in human skeletal muscle after a high-fat diet and resistance exercise.

It is generally accepted that muscle adaptation to resistance exercise (REX) training is underpinned by contraction-induced, increased rates of protein synthesis and dietary protein availability. By using dynamic proteome profiling (DPP), we investigated the contribution of both synthesis and breakdown to changes in abundance on a protein-by-protein basis in human skeletal muscle. Age-matched, overweight males consumed 9 d of a high-fat, low-carbohydrate diet during which time they either undertook 3 sessions of REX or performed no exercise.

Crystallographic analyses illustrate significant plasticity and efficient recoding of meganuclease target specificity.

The retargeting of protein-DNA specificity, outside of extremely modular DNA binding proteins such as TAL effectors, has generally proved to be quite challenging. Here, we describe structural analyses of five different extensively retargeted variants of a single homing endonuclease, that have been shown to function efficiently in ex vivo and in vivo applications. The redesigned proteins harbor mutations at up to 53 residues (18%) of their amino acid sequence, primarily distributed across the DNA binding surface, making them among the most significantly reengineered ligand-binding proteins to date.

Reprogramming MHC specificity by CRISPR-Cas9-assisted cassette exchange.

The development of programmable nucleases has enabled the application of new genome engineering strategies for cellular immunotherapy. While targeted nucleases have mostly been used to knock-out or knock-in genes in immune cells, the scarless exchange of entire immunogenomic alleles would be of great interest. In particular, reprogramming the polymorphic MHC locus could enable the creation of matched donors for allogeneic cellular transplantation.

Simulation of Invertebrate Aggregation Shows the Importance of Stable Personality over Diversity in Consensus Decision-Making.

Aggregation of many species of invertebrate is an example of a consensus decision, the success of which is central to survival. Personality is a stable form of behavioural diversity which has been observed in the aggregation process, but neither the reasons for its stability nor its effects on consensus decisions are well understood. By using an agent-based model of invertebrate aggregation, it is found that diverse personalities have only limited benefits to the experimental consensus decision-making process, but may have a more valuable role in natural settings.

Immunogenomic engineering of a plug-and-(dis)play hybridoma platform.

Hybridomas, fusions of primary mouse B cells and myelomas, are stable, rapidly-proliferating cell lines widely utilized for antibody screening and production. Antibody specificity of a hybridoma clone is determined by the immunoglobulin sequence of the primary B cell. Here we report a platform for rapid reprogramming of hybridoma antibody specificity by immunogenomic engineering.

Quantitative assessment of the robustness of next-generation sequencing of antibody variable gene repertoires from immunized mice.

Background: Next-generation sequencing (NGS) of antibody variable regions has emerged as a powerful tool in systems immunology by providing quantitative molecular information on polyclonal humoral immune responses. Reproducible and robust information on antibody repertoires is valuable for basic and applied immunology studies: thus, it is essential to establish the reliability of antibody NGS data.

Results: We isolated RNA from antibody-secreting cells (ASCs) from either 1 mouse or a pool of 9 immunized mice in order to simulate both normal and high diversity populations.

Comprehensive evaluation and optimization of amplicon library preparation methods for high-throughput antibody sequencing.

High-throughput sequencing (HTS) of antibody repertoire libraries has become a powerful tool in the field of systems immunology. However, numerous sources of bias in HTS workflows may affect the obtained antibody repertoire data. A crucial step in antibody library preparation is the addition of short platform-specific nucleotide adapter sequences.

Multi-copy genes that enhance the yield of mammalian G protein-coupled receptors in Escherichia coli.

Low yields of recombinant expression represent a major barrier to the physical characterization of membrane proteins. Here, we have identified genes that globally enhance the production of properly folded G protein-coupled receptors (GPCRs) in Escherichia coli. Libraries of bacterial chromosomal fragments were screened using two separate systems that monitor: (i) elevated fluorescence conferred by enhanced expression of GPCR-GFP fusions and (ii) increased binding of fluorescent ligand in cells producing more active receptor.

Systems approaches in global change and biogeochemistry research.

Systems approaches have great potential for application in predictive ecology. In this paper, we present a range of examples, where systems approaches are being developed and applied at a range of scales in the field of global change and biogeochemical cycling. Systems approaches range from Bayesian calibration techniques at plot scale, through data assimilation methods at regional to continental scales, to multi-disciplinary numerical model applications at country to global scales.

Modelling complex biological systems using an agent-based approach.

Many of the complex systems found in biology are comprised of numerous components, where interactions between individual agents result in the emergence of structures and function, typically in a highly dynamic manner. Often these entities have limited lifetimes but their interactions both with each other and their environment can have profound biological consequences. We will demonstrate how modelling these entities, and their interactions, can lead to a new approach to experimental biology bringing new insights and a deeper understanding of biological systems.

Volumetric diffusers: pseudorandom cylinder arrays on a periodic lattice.

Most conventional diffusers take the form of a surface based treatment, and as a result can only operate in hemispherical space. Placing a diffuser in the volume of a room might provide greater efficiency by allowing scattering into the whole space. A periodic cylinder array (or sonic crystal) produces periodicity lobes and uneven scattering.

Proteases that can distinguish among different post-translational forms of tyrosine engineered using multicolor flow cytometry.

We report the isolation of a highly active (k(cat)/K(M) approximately 10(5) M(-1)s(-1)) variant of the E. coli endopeptidase OmpT that selectively hydrolyzes peptides after 3-nitrotyrosine while effectively discriminating against similar peptides containing unmodified tyrosine (160-fold), sulfotyrosine (3600-fold), phosphotyrosine (>8000-fold), and phosphoserine (>8000-fold). The isolation of endopeptidase variants that can discriminate between substrates based on the post-translational modification of Tyr was made possible by implementing a multicolor flow cytometric assay for the screening of large mutant libraries.

Engineering next generation proteases.

The engineering of novel and precise sequence specificity into proteases will provide an important route to the development of exciting new tools for analytical, biotechnological, and therapeutic applications. Significant progress has been made in reprogramming protease specificity, largely because of the development of high-throughput assay technologies allowing the isolation of protease variants from large libraries. For example, using directed evolution as well as other approaches, proteases have been reprogrammed to cleave substrates containing a variety of amino acids in the P1 and P1' positions including a post-translationally modified tyrosine, a specificity not yet identified in any naturally occurring protease.

Introducing spatial information into predictive NF-kappaB modelling--an agent-based approach.

Nature is governed by local interactions among lower-level sub-units, whether at the cell, organ, organism, or colony level. Adaptive system behaviour emerges via these interactions, which integrate the activity of the sub-units. To understand the system level it is necessary to understand the underlying local interactions.

Synthetic antibody libraries focused towards peptide ligands.

Synthetic antibody libraries have proven immensely useful for the de novo isolation of antibodies without the need for animal immunization. Recently, focused libraries designed to recognize particular classes of ligands, such as haptens or proteins, have been employed to facilitate the selection of high-affinity antibodies. Focused libraries are built using V regions encoding combinations of canonical structures that resemble the structural features of antibodies that bind the desired class of ligands and sequence diversity is introduced at residues typically involved in recognition.

Formal agent-based modelling of intracellular chemical interactions.

Individual-based or agent-based models have proved useful in a variety of different biological contexts. This paper presents an agent-based model using a formal computational modelling approach to model a crucial biological system--the intracellular NF-kappaB signalling pathway. The pathway is vital to immune response regulation, and is fundamental to basic survival in a range of species.