Subunit‐Specific Labeling of Ubiquitin Chains by Using Sortase: Insights into the Selectivity of Deubiquitinases

Information embedded in different ubiquitin chains is transduced by proteins with ubiquitin‐binding domains (UBDs) and erased by a set of hydrolytic enzymes referred to as deubiquitinases (DUBs). Understanding the selectivity of UBDs and DUBs is necessary for decoding the functions of different ubiquitin chains. Critical to these efforts is the access to chemically defined ubiquitin chains bearing site‐specific fluorescent labels. One approach toward constructing such molecules involves peptide ligation by sortase (SrtA), a bacterial transpeptidase responsible for covalently attaching cell surface proteins to the cell wall. Here, we demonstrate the utility of SrtA in modifying individual subunits of ubiquitin chains. Using ubiquitin derivatives in which an N‐terminal glycine is unveiled after protease‐mediated digestion, we synthesized ubiquitin dimers, trimers, and tetramers with different isopeptide linkages. SrtA was then used in combination with fluorescent depsipeptide substrates to effect the modification of each subunit in a chain. By constructing branched ubiquitin chains with individual subunits tagged with a fluorophore, we provide evidence that the ubiquitin‐specific protease USP15 prefers ubiquitin trimers but has little preference for a particular isopeptide linkage. Our results emphasize the importance of subunit‐specific labeling of ubiquitin chains when studying how DUBs process these chains.     

Nebulizer-spray chamber apparatus and method for production of multi-component particle types and their use in affecting cellular responses

A particle generation apparatus and methodology is described for its application to prepare heterogeneous particles of defined chemical composition and their subsequent delivery to human lung cell cultures. A concentric type nebulizer was used together with a single pass spray chamber that was operated at two different temperature settings, either room temperature or to produce intact particles, at nonuniform temperature with the central axis measuring 54°C. Silica (SiO₂) particles in the micrometer size range were produced from the aggregation of nanoparticles. Tumor necrosis factor (TNF)-α, a well-characterized cytokine, was incorporated as a secondary soluble component within host silica particles for application as a proof-of-concept heterogeneous particle type. The overall particle deposition and wetting efficiency into media was 2.0 ± 0.4%, and the average size of particles that impacted and sank in the growth medium was 3.4 µm. The particle dose, reported as particle-to-cell ratio, spanned 0.1 to ～2.5. Co-cultures of A549 and differentiated human monocytic cells (THP-1*) exposed to silica particles caused IL-6 upregulation, but not IL-8. For the silica plus TNF-α particle type, ICAM-1 signal increased in A549 mono-cultures as the particle-to-cell ratio increased, yet an estimated 90% of the TNF-α was denatured or possibly bound to the silica particle host, and was therefore bio-unavailable. Method quantitation using particles having two different chemical compositions has been described, illustrating capability for subsequent systematic investigation of the role of particles having different chemical composition in the human health aspects of the particulate air pollution issue.

Copyright © 2016 American Association for Aerosol Research     

Surface-tailoring chlorine resistant materials and strategies for polyamide thin film composite reverse osmosis membranes

Polyamide thin film composite membranes have dominated current reverse osmosis market on account of their excellent separation performances compared to the integrally skinned counterparts. Despite their very promising separation performance, chlorine-induced degradation resulted from the susceptibility of polyamide toward chlorine attack has been regarded as the Achilles’s heel of polyamide thin film composite. The free chlorine species present during chlorine treatment can impair membrane performance through chlorination and depolymerization of the polyamide selective layer. From material point of view, a chemically stable membrane is crucial for the sustainable application of membrane separation process as it warrants a longer membrane lifespan and reduces the cost involved in membrane replacement. Various strategies, particularly those involved membrane material optimization and surface modifications, have been established to address this issue. This review discusses membrane degradation by free chlorine attack and its correlation with the surface chemistry of polyamide. The advancement in the development of chlorine resistant polyamide thin film composite membranes is reviewed based on the state-of-the-art surface modifications and tailoring approaches which include the in situ and post-fabrication membrane modifications using a broad range of functional materials. The challenges and future directions in this field are also highlighted.     

Effects of 3‐D microwell culture on initial fate specification in human embryonic stem cells

Several studies have demonstrated that three‐dimensional (3‐D) culture systems influence human embryonic stem cell (hESC) phenotypes and fate choices. However, the effect that these microenvironmental changes have on signaling pathways governing hESC behaviors is not well understood. Here, a 3‐D microwell array was used to investigate differences in activation of developmental pathways between 2‐D and 3‐D cultures of both undifferentiated hESCs and hESCs undergoing initial differentiation in embryoid bodies (EBs). An increased induction into mesoderm and endoderm and differences in expression of genes from multiple signaling pathways that regulate development, including Wnt/β‐catenin, TGF‐β superfamily, Notch, and FGF during EB‐mediated differentiation were observed in 3‐D microwells as compared with the 2‐D substrates. In undifferentiated hESCs, differences in epithelial‐mesenchymal transition phenotypes and the TGFβ/BMP pathway between cultures in 3‐D and 2‐D were also observed. These results illustrate that 3‐D culture influences multiple pathways that may regulate the differentiation trajectories of hESCs. © 2014 American Institute of Chemical Engineers AIChE J, 60: 1225–1235, 2014     

RAFT polymerization of temperature- and salt-responsive block copolymers as reversible hydrogels

Reversible-addition fragmentation chain transfer (RAFT) polymerization enabled the synthesis of novel, stimuli-responsive, AB and ABA block copolymers. The B block contained oligo(ethylene glycol) methyl ether methacrylate (OEG) and was permanently hydrophilic in the conditions examined. The A block consisted of diethylene glycol methyl ether methacrylate (DEG) and [2-(methacryloyloxy)ethyl]trimethylammonium chloride (TMA). The A block displayed both salt- and temperature-response with lower critical solution temperatures (LCSTs) dependent on the molar content of TMA and the presence of salt. Higher TMA content in the AB diblock copolymers increased the critical micelle temperatures (CMT) in HPLC-grade water due to an increased hydrophilicity of the A block. Upon addition of 0.9 wt% NaCl, the CMTs of poly(OEG-b-DEG₉₅TMA₅) decreased from 50 °C to 36 °C due to screening of electrostatic repulsion between the TMA units. ABA triblock copolymers displayed excellent hydrogel properties with salt- and temperature-dependent gel points. TMA incorporation in the A block increased the gel points for all triblock copolymers, and salt-response increased with higher TMA composition in the A block. For example, poly(DEG₉₈TMA₂-b-OEG-b-DEG₉₈TMA₂) formed a hydrogel at 40 °C in HPLC-grade water and 26 °C in 0.9 wt% NaCl aqueous solution. These salt- and temperature-responsive AB diblock and ABA triblock copolymers find applications as drug delivery vehicles, adhesives, and hydrogels.     

Material properties and fire test results

Five material properties commonly used to describe the fire behavior of solids were evaluated as sole explanatory variables for four small‐scale fire tests with pass/fail outcomes by using a physically based probabilistic (phlogistic) burning model. The phlogistic model describes the likelihood of passing vertical Bunsen burner tests and a regulatory heat release rate test reasonably well over a wide range of material properties, as deduced from the correlation coefficient and mean deviation of the predicted and measured values. Of the thermal, combustion, and fire properties examined, the best predictors of the likelihood of passing the fire tests of this study were the heat of combustion of the sample, the heat release capacity, and the heat release parameter. The relative merits and drawbacks of qualitative (threshold) and quantitative (probabilistic) approaches to predicting fire test results using thermal and combustion properties are discussed. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

A yeast surface display platform for plant hormone receptors: Toward directed evolution of new biosensors

Small-molecule biosensors have major applications in biotechnology and medicine but remain difficult to engineer. Plant hormone receptors represent an attractive platform for engineering such biosensors because their chemically induced dimerization architectures naturally decouple small-molecule sensing and sensor actuation. Rapid biosensor engineering will require quantitative high-throughput screening methods. Here we develop a yeast surface display (YSD) platform for the PYR1/HAB1 abscisic acid sensor of Arabidopsis thaliana. We extensively optimized PYR1 surface display, HAB1 purification, and binding reaction conditions. Our system reproduces previous results with wild-type and engineered receptors, and a mathematical analysis of the PYR1/HAB1 system allows us to infer all binding constants. Critically, we find that a previously engineered PYR1 receptor with altered ligand specificity binds HAB1 with identical affinity, suggesting that substantial reengineering of the PYR1 binding pocket does not compromise sensor actuation. This YSD platform for A. thaliana PYR1/HAB1 will facilitate future biosensor engineering efforts.     

Characteristics of Bi-metallic Interfaces Formed During Direct Energy Deposition Additive Manufacturing Processing

Metallurgical and Materials Transactions B - Various additive manufacturing processes are being evaluated to reduce the time and cost for fabrication of low volume, complex, and multifunctional...     

Reaction Engineering Approach (REA) to model the drying kinetics of droplets with different initial sizes—experiments and analyses

Droplets with different initial sizes, which are typical in conventional liquid atomization for spray drying applications, will result in varying drying and crust formation histories. It is essential for any droplet drying model to accurately capture such fundamental phenomena. This study used a newly constructed glass-filament single droplet rig to evaluate the applicability of the Reaction Engineering Approach (REA) in describing such effect. For the three initial sizes (1, 2 and 3 μL) tested, the glass filament gravimetric method clearly distinguished the different drying kinetics and the crust formation phenomenon, delineated by the drying behavior. Analysis from the drying kinetics revealed that the main premise of the REA, which utilizes a material-specific master activation energy curve, is applicable to droplets of different initial sizes at all the three air temperatures tested. This allowed the REA to accurately predict the different temperature and moisture histories given by droplets with different initial sizes. The result supports the REA as a good modeling approach for a wide range of initial droplet conditions. A new master curve approach was proposed to predict the diameter change of droplets with different initial concentrations. Validation with the current and past experimental data revealed that this approach has strong potential to account for the different feed concentrations typically found in spray drying applications.