The mechanics of the microenvironment continuously modulates cell functions like growth, survival, apoptosis, differentiation, and morphogenesis via cytoskeletal remodeling and actomyosin contractility 1 – 3 . Although all these processes consume energy 4 , 5 , it is unknown if and how cells adapt their metabolic activity to variable mechanical cues. Here, we report that transfer of human bronchial epithelial cells (HBECs) from stiff to soft substrates causes downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by stress fiber disassembly, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small cell lung cancer cells (NSCLCs), which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress fiber population that is insensitive to substrate stiffness. In sum, our data unveil a mechanism by which glycolysis responds to architectural features of the actomyosin cytoskeleton, thus coupling cell metabolism to the mechanical properties of the surrounding tissue. These processes enable normal cells to attune energy production in variable microenvironments, while the resistance of the cytoskeleton to respond to mechanical cues allows high glycolytic rates to persist in cancer cells despite constant alterations of the tumor tissue.

Resilin, the highly elastomeric protein found in specialized compartments of most arthropods, possesses superior resilience and excellent high-frequency responsiveness. Enabled by biosynthetic strategies, we have designed and produced a modular, recombinant resilin-like polypeptide bearing both mechanically active and biologically active domains in order to create novel biomaterial microenvironments for engineering mechanically active tissues such as blood vessels, cardiovascular tissues and vocal folds. Preliminary studies revealed that these recombinant materials exhibit promising mechanical properties and support the adhesion of NIH 3T3 fibroblasts. In this report, we detail the characterization of the dynamic mechanical properties of these materials, as assessed via dynamic oscillatory shear rheology at various protein concentrations and cross-linking ratios. Simply by varying the polypeptide concentration and cross-linker ratios, the storage modulus G′ can be easily tuned within the range of 500Pa to 10kPa. Strain-stress cycles and resilience measurements were probed via standard tensile testing methods and indicated the excellent resilience (>90%) of these materials, even when the mechanically active domains are intercepted by non-mechanically active biological cassettes. Further evaluation, at high frequencies, of the mechanical properties of these materials were assessed by a custom-designed torsional wave apparatus (TWA) at frequencies close to human phonation, indicated elastic modulus values from 200-2500Pa, which is within the range of experimental data collected on excised porcine and human vocal fold tissues. The results validate the outstanding mechanical properties of the engineered materials, which are highly comparable to the mechanical properties of targeted vocal fold tissues. The ease of production of these biologically active materials, coupled with their outstanding mechanical properties over a range of compositions, suggests their potential in tissue regeneration applications.

Natural resilin, the rubber-like protein that exists in specialized compartments of most arthropods, possesses excellent mechanical properties such as low stiffness, high resilience and effective energy storage. Recombinantly-engineered resilin-like polypeptides (RLPs) that possess the favorable attributes of native resilin would be attractive candidates for the modular design of biomaterials for engineering mechanically active tissues. Based on our previous success in creating a novel RLP-based hydrogel and demonstrating useful mechanical and cell-adhesive properties, we have produced a suite of new RLP-based constructs, each equipped with 12 repeats of the putative resilin consensus sequence and a single, distinct biologically active domain. This approach allows independent control over the concentrations of cell-binding, MMP-sensitive, and polysaccharide-sequestration domains in hydrogels comprising mixtures of the various RLPs. The high purity, molecular weight and correct compositions of each new polypeptide have been confirmed via high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and amino acid analysis. These RLP-based polypeptides exhibit largely random-coil conformation, both in solution and in the cross-linked hydrogels, as indicated by circular dichroic and infrared spectroscopic analyses. Hydrogels of various compositions, with a range of elastic moduli (1kPa to 25kPa) can be produced from these polypeptides, and the activity of the cell-binding and matrix metalloproteinase (MMP) sensitive domains was confirmed. Tris(hydroxymethyl phosphine) cross-linked RLP hydrogels were able to maintain their mechanical integrity as well as the viability of encapsulated primary human mesenchymal stem cells (MSCs). These results validate the promising properties of these RLP-based elastomeric biomaterials.

Delay alternating with nutation for tailored excitation (DANTE) pulse trains are well appreciated as frequency-selective excitation methods in Fourier transform NMR and for spatial tagging in MRI. In this study, nonselective DANTE pulse trains are used in combination with gradient pulses and short repetition times as motion-sensitive preparation modules. We show that while the longitudinal magnetization of static tissue is mostly preserved, flowing spins are largely (or fully) attenuated as they fail to establish transverse steady state due to a spoiling effect caused by flow along the applied gradient. The attenuation of flowing spins is effectively insensitive to spin velocity (above a low threshold) and can be approximately quantified with a simple T₁ longitudinal magnetization decay model. The relevant analytical equations for moving spins and static spins during DANTE module application are derived for both transient and steady state epochs. The equations are validated by comparing analytical solutions and numerical Bloch equation simulations against experimental observations in phantoms and in vivo. Based on this contrast mechanism, the application of the DANTE preparation to black blood vessel imaging is proposed. A simple demonstration of DANTE black blood imaging modules shows that it provides excellent blood signal suppression and static tissue signal preservation.