TECHNOLOGY

Natural products span broad activities and applications; however, their access and production are often limited by native cellular sources. As a result, the heterologous production of a siderophore termed yersiniabactin (Ybt) was completed using the surrogate host Escherichia coli. Post-production and purification steps are complicated by the complex nature of most media used for cell growth, prompting the development in this work of an aqueous two-phase pre-purification system capable of rapidly and simply enhancing the concentration of the target Ybt compound.

Polyurethanes (PUs) are composed of soft and hard segments, and segmental interactions induce biphasic morphologies which can influence endothelial cell (EC) organization by regulating cell–matrix and cell–cell interactions. In this study, we explored this effect using poly(caprolactone) (PCL)-based PU, where the soft segment was composed of PCL and the hard segment was composed of hexamethylene diisocyanate (HDI) and L-tyrosine-based dipeptide (DTH). The composition of the PUs was varied by altering the PCL molecular weight and correspondingly, different phase morphologies were observed. Organization and functional state of ECs on these PUs showed that composition and phase morphology of PU have a significant effect on cellular response. The ECs formed an organized network with cord-like structures which resulted in interconnected loops when soft and hard segment fractions were phase-separated. VE-cadherin (for cell–cell adherence) and vinculin (for cell–matrix focal adhesion) localized at the tip of interconnecting cells in the tube structures indicated synchronized cell–cell and cell–matrix interactions.

Because streptavidin is a homotetramer, it can bind multiple biotinylated ligands and cause target aggregation. To allow biotin detection without clustering, we previously engineered monomeric streptavidin (mSA) that is structurally similar to a single streptavidin subunit. Introducing the S25H mutation near the binding site increases the biotin dissociation half-life t_1/2 to 83 minutes. The slowly dissociating mutant, mSA2, is useful in imaging studies because it allows stable labeling of biotinylated targets. We show that mSA2 conjugated with Alexa 488 binds biotinylated receptors on HEK293 with high specificity, and bound mSA2–Alexa488 does not dissociate significantly during an imaging study lasting 50 minutes. As a structural monomer, mSA2 can be fused to other proteins to create bifunctional molecules. We tested the use of mSA2 in proximity dependent biotinylation, in which mSA2 is fused to a peptide or a protein that binds a protein of interest (POI) and is used to recruit photoactivatable biotin (PA-biotin) to the target molecule. Once the resulting cluster of interacting proteins is subjected to UV-initiated distance-dependent biotinylation, subsequent affinity purification of biotinylated proteins on streptavidin beads can identify protein molecules that interact with POI. In addition to proteins that directly interact with the mSA2 fusion, mSA2 also induces biotinylation of other proteins that are associated through a series of noncovalent interactions. We show that mSA2 fused to an antibody recognition domain can be recruited to the kinase Erk-2 using a commercially available antibody and induce biotinylation of a known Erk-2 substrate, GST-Elk-1. Therefore, mSA2 can be used to implement proximity dependent biotinylation and detect transient enzyme-substrate interactions.

Tissue engineering is an innovative field of research applied to treat intestinal diseases. Engineered smooth muscle requires dense smooth muscle tissue and robust vascularization to support contraction. The purpose of this study was to use heparan sulfate (HS) and collagen coatings to increase the attachment of smooth muscle cells (SMCs) to scaffolds and improve their survival after implantation. SMCs grown on biologically coated scaffolds were evaluated for maturity and cell numbers after 2, 4 and 6 weeks in vitro and both 2 and 6 weeks in vivo. Implants were also assessed for vascularization. Collagen-coated scaffolds increased attachment, growth and maturity of SMCs in culture. HS-coated implants increased angiogenesis after 2 weeks, contributing to an increase in SMC survival and growth compared to HS-coated scaffolds grown in vitro. The angiogenic effects of HS may be useful for engineering intestinal smooth muscle.

Major exhaust emissions from diesel engines are CO, CO₂, PM, UHC and NO_x, of which NO_x is one of the most harmful. A number of techniques have been utilized to control NO_x emissions and exhaust gas recirculation (EGR) is one of the widely used techniques that show outstanding results in NO_x reduction in both light and heavy duty diesel engines. In the present study, the experiment has been conducted on a four-stroke, single-cylinder water cooled diesel engine. Here, a long-route EGR system was used in both hot (insulated) and partially cooled (without insulation) conditions. EGR rate was varied from 0 to 24% in steps of 6% and the engine ran at various load conditions. The research objective was to investigate the effects of varying EGR ratios and temperatures on engine performance parameters and determine the effective EGR rate where the engine gives high performance, low fuel consumption and produces low emissions.

Aberrant cellular signaling networks are implicated in major diseases including cancer, but are difficult to reliably quantitate, as many signaling proteins are expressed at low abundance and further reduced following specimen collection. MTIP is an integrated tissue-imaging platform that leverages bright, fluorescent reporters and sensitive spectral instrumentation, along with automated staining, image acquisition and non-parametric image analysis, to attain reproducible, multiplexed quantitation of signaling proteins in tissue. MTIP captured the phosphoactivity of six key PI3K/MAPK proteins (pAKTS473, pAKT308, pPRAS40, pS6, peIF4G and pERK1/2) at high precision (coefficient of variation, CV <10%), four-log dynamic range and subcellular resolution. We demonstrated the MTIP platform's capability to capture a diversity of PI3K/MAPK networks present in breast tumors. These protein networks are heterogeneously distributed across the tumor tissue and associated with subgroups of cells and underscore the importance of accessing information about signaling networks in spatially intact tissue. Analysis of PI3K/MAPK networks by hierarchical clustering showed that PI3K/MAPK networks do not strictly correlate with PI3K pathway mutations, also pointing to the value of functional signaling network profiling together with genomic information. MTIP's reliable quantitative capability can be applied to guide therapeutic development and selection in precision medicine.

An antibody-based sensor (immunosensor) has been developed that features an all-in-one extraction and detection of the enteric pathogen E. coli O57 : H7 using the same redox-active polyaniline-coated iron oxide nanoparticles (PIONs). Capture efficiency for E. coli O57 : H7 is shown to be 81–99% in various food matrices with varying properties. The immunosensor's detection range is 10⁰ to 10⁵ CFU/mL with a detection limit of 5 CFU. Furthermore, magnetic separation is showing great promise as an alternative to existing conventional sample processing systems. Given the bacteria's low contamination level in food and water, this biosensor technology has great potential in monitoring enteric microbial contaminants in the food supply chain where simplicity, sensitivity, and ease of use are important.

Therapeutic mesenchymal stromal cells (MSCs) are attractive in part due to their immunomodulatory properties, achieved by their paracrine secretion of factors including prostaglandin E2 (PGE2). Despite promising pre-clinical data, demonstrating clinical efficacy has proven difficult. The current studies were designed to develop approaches to pre-induce desired functions from naïve MSCs and examine MSC donor variability, two factors contributing to this disconnect. MSCs from six human donors were pre-activated with interleukin 1 beta (IL-1β) at a concentration and duration identified as optimal or interferon gamma (IFN-γ) as a comparator. Their secretion of PGE2 after pre-activation and secondary exposure to pro-inflammatory molecules was measured. Modulation of tumor necrosis factor alpha (TNF-α) secretion from M1 pro-inflammatory macrophages by co-cultured pre-activated MSCs was also measured. Our results indicated that pre-activation of MSCs with IL-1β resulted in upregulated PGE2 secretion post exposure. Pre-activation with IL-1β or IFN-γ resulted in higher sensitivity to induction by secondary stimuli compared to no pre-activation. While IL-1β pre-activation led to enhanced MSC-mediated attenuation of macrophage TNF-α secretion, IFN-γ pre-activation resulted in enhanced TNF-α secretion. Donor variability was noted in PGE2 secretion and upregulation and the level of improved or impaired macrophage modulation.