Designing cell-targeted therapeutic proteins reveals the interplay between domain connectivity and cell binding.

The therapeutic efficacy of cytokines is often hampered by severe side effects due to their undesired binding to healthy cells. One strategy for overcoming this obstacle is to tether cytokines to antibodies or antibody fragments for targeted cell delivery. However, how to modulate the geometric configuration and relative binding affinity of the two domains for optimal activity remains an outstanding question.

Unique nucleotide sequence-guided assembly of repetitive DNA parts for synthetic biology applications.

Recombination-based DNA construction methods, such as Gibson assembly, have made it possible to easily and simultaneously assemble multiple DNA parts, and they hold promise for the development and optimization of metabolic pathways and functional genetic circuits. Over time, however, these pathways and circuits have become more complex, and the increasing need for standardization and insulation of genetic parts has resulted in sequence redundancies--for example, repeated terminator and insulator sequences--that complicate recombination-based assembly. We and others have recently developed DNA assembly methods, which we refer to collectively as unique nucleotide sequence (UNS)-guided assembly, in which individual DNA parts are flanked with UNSs to facilitate the ordered, recombination-based assembly of repetitive sequences.

Rapid construction of insulated genetic circuits via synthetic sequence-guided isothermal assembly.

In vitro recombination methods have enabled one-step construction of large DNA sequences from multiple parts. Although synthetic biological circuits can in principle be assembled in the same fashion, they typically contain repeated sequence elements such as standard promoters and terminators that interfere with homologous recombination. Here we use a computational approach to design synthetic, biologically inactive unique nucleotide sequences (UNSes) that facilitate accurate ordered assembly.

Two- and three-input TALE-based AND logic computation in embryonic stem cells.

Biological computing circuits can enhance our ability to control cellular functions and have potential applications in tissue engineering and medical treatments. Transcriptional activator-like effectors (TALEs) represent attractive components of synthetic gene regulatory circuits, as they can be designed de novo to target a given DNA sequence. We here demonstrate that TALEs can perform Boolean logic computation in mammalian cells.

The elastic properties of valve interstitial cells undergoing pathological differentiation.

Increasing evidence indicates that the progression of calcific aortic valve disease (CAVD) is influenced by the mechanical forces experienced by valvular interstitial cells (VICs) embedded within the valve matrix. The ability of VICs to sense and respond to tissue-level mechanical stimuli depends in part on cellular-level biomechanical properties, which may change with disease. In this study, we used micropipette aspiration to measure the instantaneous elastic modulus of normal VICs and of VICs induced to undergo pathological differentiation in vitro to osteoblast or myofibroblast lineages on compliant and stiff collagen gels, respectively.

Cell-matrix interactions in the pathobiology of calcific aortic valve disease: critical roles for matricellular, matricrine, and matrix mechanics cues.

The hallmarks of calcific aortic valve disease (CAVD) are the significant changes that occur in the organization, composition, and mechanical properties of the extracellular matrix (ECM), ultimately resulting in stiffened stenotic leaflets that obstruct flow and compromise cardiac function. Increasing evidence suggests that ECM maladaptations are not simply a result of valve cell dysfunction; they also contribute to CAVD progression by altering cellular and molecular signaling. In this review, we summarize the ECM changes that occur in CAVD.

β-catenin mediates mechanically regulated, transforming growth factor-β1-induced myofibroblast differentiation of aortic valve interstitial cells.

Objective: In calcific aortic valve disease, myofibroblasts and activation of the transforming growth factor-β1 (TGF-β1) and Wnt/β-catenin pathways are observed in the fibrosa, the stiffer layer of the leaflet, but their association is unknown. We elucidated the roles of β-catenin and extracellular matrix stiffness in TGF-β1-induced myofibroblast differentiation of valve interstitial cells (VICs).

Methods And Results: TGF-β1 induced rapid β-catenin nuclear translocation in primary porcine aortic VICs in vitro through TGF-β receptor I kinase.

Microfabricated arrays for high-throughput screening of cellular response to cyclic substrate deformation.

Mechanical forces play an important role in regulating cellular function and have been shown to modulate cellular response to other factors in the cellular microenvironment. Presently, no technique exists to rapidly screen for the effects of a range of uniform mechanical forces on cellular function. In this work, we developed and characterized a novel microfabricated array capable of simultaneously applying cyclic equibiaxial substrate strains ranging in magnitude from 2 to 15% to small populations of adherent cells.

Boning up on Wolff's Law: mechanical regulation of the cells that make and maintain bone.

Bone tissue forms and is remodeled in response to the mechanical forces that it experiences, a phenomenon described by Wolff's Law. Mechanically induced formation and adaptation of bone tissue is mediated by bone cells that sense and respond to local mechanical cues. In this review, the forces experienced by bone cells, the mechanotransduction pathways involved, and the responses elicited are considered.

Calcification by valve interstitial cells is regulated by the stiffness of the extracellular matrix.

Objective: Extensive remodeling of the valve ECM in calcific aortic valve sclerosis alters its mechanical properties, but little is known about the impact of matrix mechanics on the cells within the valve interstitium. In this study, the influence of matrix stiffness in modulating calcification by valve interstitial cells (VICs), and their differentiation to pathological phenotypes was assessed.

Methods And Results: Primary porcine aortic VICs were cultured in standard media or calcifying media on constrained type I fibrillar collagen gels.

Identification and characterization of aortic valve mesenchymal progenitor cells with robust osteogenic calcification potential.

Advanced valvular lesions often contain ectopic mesenchymal tissues, which may be elaborated by an unidentified multipotent progenitor subpopulation within the valve interstitium. The identity, frequency, and differentiation potential of the putative progenitor subpopulation are unknown. The objectives of this study were to determine whether valve interstitial cells (VICs) contain a subpopulation of multipotent mesenchymal progenitor cells, to measure the frequencies of the mesenchymal progenitors and osteoprogenitors, and to characterize the osteoprogenitor subpopulation because of its potential role in calcific aortic valve disease.