Reduced type-A carbohydrate-binding module interactions to cellulose I leads to improved endocellulase activity.

Dissociation of nonproductively bound cellulolytic enzymes from cellulose is hypothesized to be a key rate-limiting factor impeding cost-effective biomass conversion to fermentable sugars. However, the role of carbohydrate-binding modules (CBMs) in enabling nonproductive enzyme binding is not well understood. Here, we examine the subtle interplay of CBM binding and cellulose hydrolysis activity for three models type-A CBMs (Families 1, 3a, and 64) tethered to multifunctional endoglucanase (CelE) on two distinct cellulose allomorphs (i.e., cellulose I and III). We generated a small library of mutant CBMs with varying cellulose affinity, as determined by equilibrium binding assays, followed by monitoring cellulose hydrolysis activity of CelE-CBM fusion constructs. Finally, kinetic binding assays using quartz crystal microbalance with dissipation were employed to measure CBM adsorption and desorption rate constants kon and koff , respectively, towards nanocrystalline cellulose derived from both allomorphs. Overall, our results indicate that reduced CBM equilibrium binding affinity towards cellulose I alone, resulting from increased desorption rates ( koff ) and reduced effective adsorption rates ( nkon ), is correlated to overall improved endocellulase activity. Future studies could employ similar approaches to unravel the role of CBMs in nonproductive enzyme binding and develop improved cellulolytic enzymes for industrial applications.

Biofabrication Strategies and Engineered In Vitro Systems for Vascular Mechanobiology.

The vascular system is integral for maintaining organ-specific functions and homeostasis. Dysregulation in vascular architecture and function can lead to various chronic or acute disorders. Investigation of the role of the vascular system in health and disease has been accelerated through the development of tissue-engineered constructs and microphysiological on-chip platforms. These in vitro systems permit studies of biochemical regulation of vascular networks and parenchymal tissue and provide mechanistic insights into the biophysical and hemodynamic forces acting in organ-specific niches. Detailed understanding of these forces and the mechanotransductory pathways involved is necessary to develop preventative and therapeutic strategies targeting the vascular system. This review describes vascular structure and function, the role of hemodynamic forces in maintaining vascular homeostasis, and measurement approaches for cell and tissue level mechanical properties influencing vascular phenomena. State-of-the-art techniques for fabricating in vitro microvascular systems, with varying degrees of biological and engineering complexity, are summarized. Finally, the role of vascular mechanobiology in organ-specific niches and pathophysiological states, and efforts to recapitulate these events using in vitro microphysiological systems, are explored. It is hoped that this review will help readers appreciate the important, but understudied, role of vascular-parenchymal mechanotransduction in health and disease toward developing mechanotherapeutics for treatment strategies.

The Influence of Matrix-Induced Dormancy on Metastatic Breast Cancer Chemoresistance.

Metastasis remains the leading cause of cancer-associated death worldwide. Disseminated tumor cells can undergo dormancy upon infiltration of secondary organs, and chemotherapeutics fail to effectively eliminate dormant populations. Mechanistic understanding of dormancy-associated chemoresistance could lead to development of targeted therapeutic strategies. Toward this goal, we implemented three poly(ethylene glycol) (PEG)-based hydrogel formulations fabricated from proteolytically degradable PEG (PEG-PQ), integrin ligating PEG-RGDS, and the non-degradable cross-linker N-vinylpyrrolidone (NVP) to induce three distinct phenotypes in triple negative MDA-MB-231 breast cancer cells. With constant 5% w/v PEG-PQ, PEG-RGDS and NVP concentrations were tuned to induce (i) a growth state characterized by high proliferation, high metabolic activity, significant temporally increased cell density, and an invasive morphology; (ii) a balanced dormancy state characterized by a temporal balance (~1:1 ratio) in new live and dead cell density and a non-invasive morphology; and (iii) a cellular dormancy state characterized by rounded, solitary quiescent cells with low viability, proliferation, and metabolic activity. The cellular responses to doxorubicin (DOX), paclitaxel (PAC), and 5-fluorouracil (5-FU) in the three phenotypic states were quantified. Under DOX treatment, cells in dormant states demonstrated increased chemoresistance with a 1.4- to 1.8-fold increase in half maximal effective concentration (EC50) and 1.3- to 1.8-fold increase in half maximal inhibitory concentration (IC50) compared to cells in the growth state. PAC and 5-FU treatment led to similar results. To mechanistically investigate the role of dormancy in conferring DOX resistance, cytoplasmic and nuclear accumulation of DOX was measured. The results indicated comparable DOX accumulation between all three phenotypic states; however, the intracellular to intranuclear distribution indicated a ~1.5 fold increase in DOX nuclear accumulation in cells in the growth state compared to the two dormant states. These results further validate the utility of implementing engineered hydrogels as in vitro platforms of breast cancer dormancy for the development of anti-dormancy therapeutic strategies.

Soft Robotic Manipulation and Locomotion with a 3D Printed Electroactive Hydrogel.

Electroactive hydrogels (EAH) that exhibit large deformation in response to an electric field have received great attention as a potential actuating material for soft robots and artificial muscle. However, their application has been limited due to the use of traditional two-dimensional (2D) fabrication methods. Here we present soft robotic manipulation and locomotion with 3D printed EAH microstructures. Through 3D design and precise dimensional control enabled by a digital light processing (DLP) based micro 3D printing technique, complex 3D actuations of EAH are achieved. We demonstrate soft robotic actuations including gripping and transporting an object and a bidirectional locomotion.

Engineered In Vitro Models of Tumor Dormancy and Reactivation.

Metastatic recurrence is a major hurdle to overcome for successful control of cancer-associated death. Residual tumor cells in the primary site, or disseminated tumor cells in secondary sites, can lie in a dormant state for long time periods, years to decades, before being reactivated into a proliferative growth state. The microenvironmental signals and biological mechanisms that mediate the fate of disseminated cancer cells with respect to cell death, single cell dormancy, tumor mass dormancy and metastatic growth, as well as the factors that induce reactivation, are discussed in this review. Emphasis is placed on engineered, in vitro, biomaterial-based approaches to model tumor dormancy and subsequent reactivation, with a focus on the roles of extracellular matrix, secondary cell types, biochemical signaling and drug treatment. A brief perspective of molecular targets and treatment approaches for dormant tumors is also presented. Advances in tissue-engineered platforms to induce, model, and monitor tumor dormancy and reactivation may provide much needed insight into the regulation of these processes and serve as drug discovery and testing platforms.