Mechanical regulation of glycolysis via cytoskeleton architecture.

The mechanics of the cellular microenvironment continuously modulates cell functions such as growth, survival, apoptosis, differentiation and morphogenesis via cytoskeletal remodelling and actomyosin contractility1-3. Although all of these processes consume energy4,5, it is unknown whether and how cells adapt their metabolic activity to variable mechanical cues. Here we report that the transfer of human bronchial epithelial cells from stiff to soft substrates causes a downregulation of glycolysis via proteasomal degradation of the rate-limiting metabolic enzyme phosphofructokinase (PFK). PFK degradation is triggered by the disassembly of stress fibres, which releases the PFK-targeting E3 ubiquitin ligase tripartite motif (TRIM)-containing protein 21 (TRIM21). Transformed non-small-cell lung cancer cells, which maintain high glycolytic rates regardless of changing environmental mechanics, retain PFK expression by downregulating TRIM21, and by sequestering residual TRIM21 on a stress-fibre subset that is insensitive to substrate stiffness. Our data reveal 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 tune energy production in variable microenvironments, whereas the resistance of the cytoskeleton in response to mechanical cues enables the persistence of high glycolytic rates in cancer cells despite constant alterations of the tumour tissue.

A Protein-Adsorbent Hydrogel with Tunable Stiffness for Tissue Culture Demonstrates Matrix-Dependent Stiffness Responses.

Although tissue culture plastic has been widely employed for cell culture, the rigidity of plastic is not physiologic. Softer hydrogels used to culture cells have not been widely adopted in part because coupling chemistries are required to covalently capture extracellular matrix (ECM) proteins and support cell adhesion. To create an in vitro system with tunable stiffnesses that readily adsorbs ECM proteins for cell culture, we present a novel hydrophobic hydrogel system via chemically converting hydroxyl residues on the dextran backbone to methacrylate groups, thereby transforming non-protein adhesive, hydrophilic dextran to highly protein adsorbent substrates. Increasing methacrylate functionality increases the hydrophobicity in the resulting hydrogels and enhances ECM protein adsorption without additional chemical reactions. These hydrophobic hydrogels permit facile and tunable modulation of substrate stiffness independent of hydrophobicity or ECM coatings. Using this approach, we show that substrate stiffness and ECM adsorption work together to affect cell morphology and proliferation, but the strengths of these effects vary in different cell types. Furthermore, we reveal that stiffness mediated differentiation of dermal fibroblasts into myofibroblasts is modulated by the substrate ECM. Our material system demonstrates remarkable simplicity and flexibility to tune ECM coatings and substrate stiffness and study their effects on cell function.

Nonlinear relationship between untraditional lipid parameters and the risk of prediabetes: a large retrospective study based on Chinese adults.

BACKGROUND: Abnormal lipid metabolism poses a risk for prediabetes. However, research on lipid parameters used to predict the risk of prediabetes is scarce, and the significance of traditional and untraditional lipid parameters remains unexplored in prediabetes. This study aimed to comprehensively evaluate the association between 12 lipid parameters and prediabetes and their diagnostic value. METHODS: This cross-sectional study included data from 100,309 Chinese adults with normal baseline blood glucose levels. New onset of prediabetes was the outcome of concern. Untraditional lipid parameters were derived from traditional lipid parameters. Multivariate logistic regression and smooth curve fitting were used to examine the nonlinear relationship between lipid parameters and prediabetes. A two-piecewise linear regression model was used to identify the critical points of lipid parameters influencing the risk of prediabetes. The areas under the receiver operating characteristic curve estimated the predictive value of the lipid parameters. RESULTS: A total of 12,352 participants (12.31%) were newly diagnosed with prediabetes. Following adjustments for confounding covariables, high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol were negatively correlated with prediabetes risk. Conversely, total cholesterol, triglyceride (TG), lipoprotein combine index (LCI), atherogenic index of plasma (AIP), non-HDL-C, atherogenic coefficient, Castelli's index-I, remnant cholesterol (RC), and RC/HDL-C ratio displayed positive correlations. In younger adults, females, individuals with a family history of diabetes, and non-obese individuals, LCI, TG, and AIP exhibited higher predictive values for the onset of prediabetes compared to other lipid profiles. CONCLUSION: Nonlinear associations were observed between untraditional lipid parameters and the risk of prediabetes. The predictive value of untraditional lipid parameters for prediabetes surpassed that of traditional lipid parameters, with LCI emerging as the most effective predictor for prediabetes.

Production of succinate with two CO2 fixation reactions from fatty acids in Cupriavidus necator H16.

BACKGROUND: Biotransformation of CO2 into high-value-added carbon-based products is a promising process for reducing greenhouse gas emissions. To realize the green transformation of CO2, we use fatty acids as carbon source to drive CO2 fixation to produce succinate through a portion of the 3-hydroxypropionate (3HP) cycle in Cupriavidus necator H16. RESULTS: This work can achieve the production of a single succinate molecule from one acetyl-CoA molecule and two CO2 molecules. It was verified using an isotope labeling experiment utilizing NaH13CO3. This implies that 50% of the carbon atoms present in succinate are derived from CO2, resulting in a twofold increase in efficiency compared to prior methods of succinate biosynthesis that relied on the carboxylation of phosphoenolpyruvate or pyruvate. Meanwhile, using fatty acid as a carbon source has a higher theoretical yield than other feedstocks and also avoids carbon loss during acetyl-CoA and succinate production. To further optimize succinate production, different approaches including the optimization of ATP and NADPH supply, optimization of metabolic burden, and optimization of carbon sources were used. The resulting strain was capable of producing succinate to a level of 3.6 g/L, an increase of 159% from the starting strain. CONCLUSIONS: This investigation established a new method for the production of succinate by the implementation of two CO2 fixation reactions and demonstrated the feasibility of ATP, NADPH, and metabolic burden regulation strategies in biological carbon fixation.

Upregulation of the key biomarker kinesin family member 20A (KIF20A) is associated with pulmonary artery hypertension.

OBJECTIVE: Pulmonary artery hypertension (PAH) is a complex, fatal disease with limited treatments. This study aimed to investigate possible key targets in PAH through bioinformatics. METHODS: GSE144274 were obtained from Gene Expression Omnibus (GEO) database. Then, differentially expressed genes (DEGs) between idiopathic pulmonary hypertension (IPAH) and healthy subjects were identified and analyzed. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) were analyzed, and a protein-protein interaction (PPI) network was constructed using STRING. The hub genes were identified by MCODE method. The expression levels of hub genes were validated in vitro and in vivo models. Finally, the ROC analysis was performed based on the level of hub genes in clinical plasma samples. RESULTS: A total of 363 DEGs were identified. GO analysis on these DEGs were mainly enriched in cell division, inflammatory response, among others. In the KEGG pathways analysis, DEGs mainly involved in cytokine-cytokine receptor interaction, rheumatoid arthritis, and IL-17 signaling pathways were enriched. The DEGs were analyzed with the STRING for PPI network analysis, and 62 hub genes were identified by MCODE. Finally, 6 central genes, KIF18B, SPC25, DLGAP5, KIF20A, CEP55 and ANLN, were screened out due to their novelty role in PAH. The expression of KIF20A was validated to be significantly upregulated both in the lung tissue of hypoxia-induced pulmonary hypertension (HPH) mice and proliferative PASMCs. Additionally, KIF20A levels is evelated in PAH plasma and the area under the curve (AUC) to identify PAH was 0.8591 for KIF20A. CONCLUSION: The level of KIF20A elevates during the progression of PAH, which suggestes it could be a potential diagnostic and therapeutic target for the PAH.

Visceral adiposity index is positively associated with fasting plasma glucose: a cross-sectional study from National Health and Nutrition Examination Survey 2017-2020.

BACKGROUND: Visceral adiposity index (VAI) has been recognized as a reliable indicator for visceral adiposity. However, it remains largely unexplored on its association with fasting plasma glucose (FPG). The current study aims to explore the association between VAI and FPG using a representative dataset. METHODS: A cross-sectional study was carried out based on the dataset from National Health and Nutrition Examination Survey (NHANES) 2017-2020. Univariate and Multiple linear regression analysis were performed to explore the relationship between VAI and FPG. Generalized additive model (GAM) and smooth curve fitting analysis were performed to explore the nonlinear relationship between VAI and FPG. Receiver operating characteristic (ROC) analysis was used to evaluate the predictive value of VAI for FPG elevation. RESULTS: A total of 4437 participants with complete data were finally included in the research. Individuals were divided into 4 quartiles according to the calculated VAI value: Q1 (VAI<0.69), Q2 (0.69 ≤ VAI < 1.18), Q3 (1.18 ≤ VAI < 2.02) and Q4 (VAI ≥ 2.02). FPG significantly increased with the increasing VAI quartile. Multiple linear regression analysis showed VAI was independently positively associated with FPG after adjusting confounding factors. As a continuous variable, an increase of one unit in VAI was correlated with 0.52 mmol/L (95% CI: 0.41-0.63, p < 0.0001) higher FPG level. As a categorical variable, 4th VAI quartile group was related to 0.71 mmol/L (95% CI: 0.47-0.95, p < 0.001) higher FPG level compared with 1st VAI group. GAM and smooth curve fitting analysis identified the non-linear relationship between VAI and FPG, and 4.02 was identified as the inflection point using two-piecewise linear regression. The positive association between VAI and FPG existed when VAI was lower (ß = 0.73, p < 0.0001) and higher than 4.02 (ß = 0.23, p = 0.0063). ROC analysis indicated VAI has a good predictive value for FPG elevation (AUC = 0.7169, 95% CI: 0.6948-0.7389), and the best threshold of VAI was 1.4315. CONCLUSION: VAI was an independently risk indicator for FPG, and VAI was nonlinearly positively associated with FPG. VAI had a good predictive value for elevated FPG. VAI might become a useful indicator for risk assessment and treatment of hyperglycemia in clinical practice.

Quantification of cerebral blood volume changes caused by visual stimulation at 3 T using DANTE-prepared dual-echo EPI.

PURPOSE: We investigate the influence of moving blood-attenuation effects when using "delay alternating with nutation for tailored excitation" (DANTE) pulses in conjunction with blood oxygen level dependent (BOLD) of functional MRI (fMRI) at 3 T. Based on the effects of including DANTE pulses, we propose quantification of cerebral blood volume (CBV) changes following functional stimulation. METHODS: Eighteen volunteers in total underwent fMRI scans at 3 T. Seven volunteers were scanned to investigate the effects of DANTE pulses on the fMRI signal. CBV changes in response to visual stimulation were quantified in 11 volunteers using a DANTE-prepared dual-echo EPI sequence. RESULTS: The inflow effects from flowing blood in arteries and draining vein effects from flowing blood in large veins can be suppressed by use of a DANTE preparation module. Using DANTE-prepared dual-echo EPI, we quantitatively measured intravascular-weighted microvascular CBV changes of 25.4%, 29.8%, and 32.6% evoked by 1, 5, and 10 Hz visual stimulation, respectively. The extravascular fraction (∆S/S)extra at TE = 30 ms in total BOLD signal was determined to be 64.8 ± 3.4%, which is in line with previous extravascular component estimation at 3 T. Results show that the microvascular CBV changes are linearly dependent on total BOLD changes at TE = 30 ms with a slope of 0.113, and this relation is independent of stimulation frequency and subject. CONCLUSION: The DANTE preparation pulses can be incorporated into a standard EPI fMRI sequence for the purpose of minimizing inflow effects and reducing draining veins effects in large vessels. Additionally, the DANTE-prepared dual-echo EPI sequence is a promising fast imaging tool for quantification of intravascular-weighted CBV change in the microvascular space at 3 T.

Magnetization transfer weighted EPI facilitates cortical depth determination in native fMRI space.

The increased availability of ultra-high field scanners provides an opportunity to perform fMRI at sub-millimeter spatial scales and enables in vivo probing of laminar function in the human brain. In most previous studies, the definition of cortical layers, or depths, is based on an anatomical reference image that is collected by a different acquisition sequence and exhibits different geometric distortion compared to the functional images. Here, we propose to generate the anatomical image with the fMRI acquisition technique by incorporating magnetization transfer (MT) weighted imaging. Small flip angle binomial pulse trains are used as MT preparation, with a flexible duration (several to tens of milliseconds), which can be applied before each EPI segment without constraining the acquisition length (segment or slice number). The method's feasibility was demonstrated at 7T for coverage of either a small slab or the near-whole brain at 0.8 mm isotropic resolution. Tissue contrast was found to be similar to that obtained with a state-of-art anatomical reference based on MP2RAGE. This MT-weighted EPI image allows an automatic reconstruction of the cortical surface to support laminar analysis in native fMRI space, obviating the need for distortion correction and registration.

The role of JrPPOs in the browning of walnut explants.

BACKGROUND: Tissue culture is an effective method for the rapid breeding of seedlings and improving production efficiency, but explant browning is a key limiting factor of walnut tissue culture. Specifically, the polymerization of PPO-derived quinones that cause explant browning of walnut is not well understood. This study investigated explants of 'Zanmei' walnut shoot apices cultured in agar (A) or vermiculite (V) media, and the survival percentage, changes in phenolic content, POD and PPO activity, and JrPPO expression in explants were studied to determine the role of PPO in the browning of walnut explants. RESULTS: The results showed that the V media greatly reduced the death rate of explants, and 89.9 and 38.7% of the explants cultured in V media and A media survived, respectively. Compared with that of explants at 0 h, the PPO of explants cultured in A was highly active throughout the culture, but activity in those cultured in V remained low. The phenolic level of explants cultured in A increased significantly at 72 h but subsequently declined, and the content in the explants cultured in V increased to a high level only at 144 h. The POD in explants cultured in V showed high activity that did not cause browning. Gene expression assays showed that the expression of JrPPO1 was downregulated in explants cultured in both A and V. However, the expression of JrPPO2 was upregulated in explants cultured in A throughout the culture and upregulated in V at 144 h. JrPPO expression analyses in different tissues showed that JrPPO1 was highly expressed in stems, young leaves, mature leaves, catkins, pistils, and hulls, and JrPPO2 was highly expressed in mature leaves and pistils. Moreover, browning assays showed that both explants in A and leaf tissue exhibited high JrPPO2 activity. CONCLUSION: The rapid increase in phenolic content caused the browning and death of explants. V media delayed the rapid accumulation of phenolic compounds in walnut explants in the short term, which significantly decreased explants mortality. The results suggest that JrPPO2 plays a key role in the oxidation of phenols in explants after branch injury.

Integrated VASO and perfusion contrast: A new tool for laminar functional MRI.

Earlier research in cats has shown that both cerebral blood volume (CBV) and cerebral blood flow (CBF) can be used to identify layer-dependent fMRI activation with spatial specificity superior to gradient-echo blood-oxygen-level-dependent (BOLD) contrast (Jin and Kim, 2008a). CBF contrast of perfusion fMRI at ultra-high field has not been widely applied in humans to measure laminar activity due to its low sensitivity, while CBV contrast for fMRI using vascular space occupancy (VASO) has been successfully used. However, VASO can be compromised by interference of blood in-flow effects and a temporally limited acquisition window around the blood-nulling time point. Here, we proposed to use DANTE (Delay Alternating with Nutation for Tailored Excitation) pulse trains combined with 3D-EPI to acquire an integrated VASO and perfusion (VAPER) contrast. The signal origin of the VAPER contrast was theoretically evaluated with respect to its CBV and CBF contributions using a four-compartment simulation model. The feasibility of VAPER to measure layer-dependent activity was empirically investigated in human primary motor cortex at 7 â€‹T. We demonstrated this new tool, with its highly specified functional layer profile, robust reproducibility, and improved sensitivity, to allow investigation of layer-specific cortical functions.

Transient Support from Fibroblasts is Sufficient to Drive Functional Vascularization in Engineered Tissues.

Formation of capillary blood vasculature is a critical requirement for native as well as engineered organs and can be induced in vitro by co-culturing endothelial cells with fibroblasts. However, whether these fibroblasts are required only in the initial morphogenesis of endothelial cells or needed throughout is unknown, and the ability to remove these stromal cells after assembly could be useful for clinical translation. In this study, we introduce a technique termed CAMEO (Controlled Apoptosis in Multicellular Tissues for Engineered Organogenesis), whereby fibroblasts are selectively ablated on demand, and utilize it to probe the dispensability of fibroblasts in vascular morphogenesis. The presence of fibroblasts is shown to be necessary only during the first few days of endothelial cell morphogenesis, after which they can be ablated without significantly affecting the structural and functional features of the developed vasculature. Furthermore, we demonstrate the use of CAMEO to vascularize a construct containing primary human hepatocytes that improved tissue function. In conclusion, this study suggests that transient, initial support from fibroblasts is sufficient to drive vascular morphogenesis in engineered tissues, and this strategy of engineering-via-elimination may provide a new general approach for achieving desired functions and cell compositions in engineered organs.

Navigator-based reacquisition and estimation of motion-corrupted data: Application to multi-echo spin echo for carotid wall MRI.

PURPOSE: To assess whether artifacts in multi-slice multi-echo spin echo neck imaging, thought to be caused by brief motion events such as swallowing, can be corrected by reacquiring corrupted central k-space data and estimating the remainder with parallel imaging. METHODS: A single phase-encode line (ky = 0, phase-encode direction anteroposterior) navigator echo was used to identify motion-corrupted data and guide the online reacquisition. If motion corruption was detected in the 7 central k-space lines, they were replaced with reacquired data. Subsequently, GRAPPA reconstruction was trained on the updated central portion of k-space and then used to estimate the remaining motion-corrupted k-space data from surrounding uncorrupted data. Similar compressed sensing-based approaches have been used previously to compensate for respiration in cardiac imaging. The g-factor noise amplification was calculated for the parallel imaging reconstruction of data acquired with a 10-channel neck coil. The method was assessed in scans with 9 volunteers and 12 patients. RESULTS: The g-factor analysis showed that GRAPPA reconstruction of 2 adjacent motion-corrupted lines causes high noise amplification; therefore, the number of 2-line estimations should be limited. In volunteer scans, median ghosting reduction of 24% was achieved with 2 adjacent motion-corrupted lines correction, and image quality was improved in 2 patient scans that had motion corruption close to the center of k-space. CONCLUSION: Motion-corrupted echo-trains can be identified with a navigator echo. Combined reacquisition and parallel imaging estimation reduced motion artifacts in multi-slice MESE when there were brief motion events, especially when motion corruption was close to the center of k-space.

Recovery of Tractions Exerted by Single Cells in Three-Dimensional Nonlinear Matrices.

Cell-generated tractions play an important role in various physiological and pathological processes such as stem-cell differentiation, cell migration, wound healing, and cancer metastasis. Traction force microscopy (TFM) is a technique for quantifying cellular tractions during cell-matrix interactions. Most applications of this technique have heretofore assumed that the matrix surrounding the cells is linear elastic and undergoes infinitesimal strains, but recent experiments have shown that the traction-induced strains can be large (e.g., more than 50%). In this paper, we propose a novel three-dimensional (3D) TFM approach that consistently accounts for both the geometric nonlinearity introduced by large strains in the matrix, and the material nonlinearity due to strain-stiffening of the matrix. In particular, we pose the TFM problem as a nonlinear inverse hyperelasticity problem in the stressed configuration of the matrix, with the objective of determining the cellular tractions that are consistent with the measured displacement field in the matrix. We formulate the inverse problem as a constrained minimization problem and develop an efficient adjoint-based minimization procedure to solve it. We first validate our approach using simulated data, and quantify its sensitivity to noise. We then employ the new approach to recover tractions exerted by NIH 3T3 cells fully encapsulated in hydrogel matrices of varying stiffness. We find that neglecting nonlinear effects can induce significant errors in traction reconstructions. We also find that cellular tractions roughly increase with gel stiffness, while the strain energy appears to saturate.

The role of microRNAs in the involvement of vascular smooth muscle cells in the development of atherosclerosis.

MicroRNAs (miRNAs) are a class of nonprotein-encoding RNAs of ~22 nucleotides in length that bind to or complement each other with a target gene messenger RNA (mRNA) to promote mRNA degradation or inhibit translation of the target mRNA. The protein required [such as Toll-like receptor (TLR) proteins] is controlled at an optimal level. By affecting protein translation, miRNAs have become powerful regulators of biological processes, including development, differentiation, cell proliferation, and apoptosis. MiRNAs are involved in the regulation of proliferation, migration, and apoptosis of vascular smooth muscle cells (VSMCs), thereby affecting the formation of atherosclerosis (AS). In recent years, the role and mechanism of miRNAs involved in AS development in VSMCs have been studied extensively. In the current study, the results and progress in miRNA research are reviewed.

Chrysin Alleviates Chronic Hypoxia-Induced Pulmonary Hypertension by Reducing Intracellular Calcium Concentration in Pulmonary Arterial Smooth Muscle Cells.

Chrysin (CH), the main ingredient of many medicinal plants, has been reported to be a very potent flavonoid possessing a large number of pharmacological activities. Recent studies have shown that CH significantly improves hemodynamic parameters such as right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodeling in a rat model of chronic hypoxia-induced pulmonary hypertension (CHPH). These improvements are through the inhibition of NOX4 expression, reactive oxygen species and malondialdehyde production, pulmonary arterial smooth muscle cell (PASMC) proliferation, and collagen accumulation. In this study, we investigated another mechanism by which CH alleviates CHPH by regulating intracellular calcium concentrations ([Ca]i) in PASMCs, as well as the underlying signaling pathway. The results show that (1) in CHPH model rats, CH substantially attenuated elevated right ventricular pressure, right ventricular hypertrophy, and pulmonary vascular remodeling; (2) in cultured rat distal PASMCs, CH inhibited the hypoxia-triggered promotion of cell proliferation, store-operated Ca entry and [Ca]i; and (3) CH significantly suppressed the hypoxia-upregulated HIF-1α, BMP4, TRPC1, and TRPC6 expression in distal pulmonary arteries (PAs) and cultured rat distal PASMCs. These results indicate that CH likely exerts its CHPH protective activity by regulating [Ca]i, which may result from the downregulation of HIF-1α, BMP4, TRPC1, and TRPC in PASMCs.

A novel approach to probing in vivo metabolite relaxation: Linear quantification of spatially modulated magnetization.

PURPOSE: Conventional sequences for metabolite transverse relaxation quantification all generally measure signal changes at different echo times (TEs). However, quantification results obtained via these conventional methods can be very different and are highly dependent on the type of sequence being applied. TE-dependent effects such as diffusion, macromolecule baseline, and J-coupling modulation contribute significantly to these differences. Here, we propose a novel technique-multiple flip angle pulse-driven ratio of longitudinal steady states (MARzss)-for preparing magnetization with T2 /T1 weighting. Using premeasured T1 values, T2 values for metabolites can thereby be determined. The measurement procedure does not require varying TE and is TE independent; T2 , diffusion, and J-coupling effects induced by the readout sequence are cancelled. METHOD: Longitudinal steady states at different flip angles were prepared with trains of radio frequency pulses interspersed with field gradients. The resulting spatially modulated longitudinal magnetization was acquired with a PRESS readout module. A new linear equation for quantification of MARzss was derived from Bloch equations. RESULTS: By implementing this readout-independent method, T2 measurement of brain metabolites at 7T was demonstrated through Bloch simulations, phantom, and in vivo experiments. CONCLUSIONS: The proposed MARzss technique can be used to largely avoid multi-TE associated interference, including diffusion, macromolecules, and J modulation. This MARzss technology, which is uniquely insensitive to readout sequence type and TE, is a promising technique for more accurately probing in vivo metabolite relaxation. Magn Reson Med 79:2491-2499, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Micromechanical characterization of soft, biopolymeric hydrogels: stiffness, resilience, and failure.

Detailed understanding of the local structure-property relationships in soft biopolymeric hydrogels can be instrumental for applications in regenerative tissue engineering. Resilin-like polypeptide (RLP) hydrogels have been previously demonstrated as useful biomaterials with a unique combination of low elastic moduli, excellent resilience, and cell-adhesive properties. However, comprehensive mechanical characterization of RLP hydrogels under both low-strain and high-strain conditions has not yet been conducted, despite the unique information such measurements can provide about the local structure and macromolecular behavior underpinning mechanical properties. In this study, mechanical properties (elastic modulus, resilience, and fracture initiation toughness) of equilibrium swollen resilin-based hydrogels were characterized via oscillatory shear rheology, small-strain microindentation, and large-strain puncture tests as a function of polypeptide concentration. These methods allowed characterization, for the first time, of the resilience and failure in hydrogels with low polypeptide concentrations (<20 wt%), as the employed methods obviate the handling difficulties inherent in the characterization of such soft materials via standard mechanical techniques, allowing characterization without any special sample preparation and requiring minimal volumes (as low as 50 µL). Elastic moduli measured from small-strain microindentation showed good correlation with elastic storage moduli obtained from oscillatory shear rheology at a comparable applied strain rate, and evaluation of multiple loading-unloading cycles revealed decreased resilience values at lower hydrogel concentrations. In addition, large-strain indentation-to-failure (or puncture) tests were performed to measure large-strain mechanical response and fracture toughness on length scales similar to biological cells (∼10-50 µm) at various polypeptide concentrations, indicating very high fracture initiation toughness for high-concentration hydrogels. Our results establish the utility of employing microscale mechanical methods for the characterization of the local mechanical properties of biopolymeric hydrogels of low concentrations (<20 wt%), and show how the combination of small and large-strain measurements can provide unique insight into structure-property relationships for biopolymeric elastomers. Overall, this study provides new insight into the effects on local mechanical properties of polypeptide concentration near the overlap polymer concentration c* for resilin-based hydrogels, confirming their unique elastomeric features for applications in regenerative medicine.

T2-Weighted intracranial vessel wall imaging at 7 Tesla using a DANTE-prepared variable flip angle turbo spin echo readout (DANTE-SPACE).

PURPOSE: To optimize intracranial vessel wall imaging (VWI) at 7T for sharp wall depiction and high boundary contrast. METHODS: A variable flip angle turbo spin echo scheme (SPACE) was optimized for VWI. SPACE provides black-blood contrast, but has less crushing effect on cerebrospinal fluid (CSF). However, a delay alternating with nutation for tailored excitation (DANTE) preparation suppresses the signal from slowly moving spins of a few mm per second. Therefore, we optimized a DANTE-preparation module for 7T. Signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), and signal ratio for vessel wall, CSF, and lumen were calculated for SPACE and DANTE-SPACE in 11 volunteers at the middle cerebral artery (MCA). An exemplar MCA stenosis patient was scanned with DANTE-SPACE. RESULTS: The 7T-optimized SPACE sequence improved the vessel wall point-spread function by 17%. The CNR between the wall and CSF was doubled (12.2 versus 5.6) for the DANTE-SPACE scans compared with the unprepared SPACE. This increase was significant in the right hemisphere (P = 0.016), but not in the left (P = 0.090). The CNR between wall and lumen was halved, but remained at a high value (24.9 versus 56.5). CONCLUSION: The optimized SPACE sequence improves VWI at 7T. Additional DANTE preparation increases the contrast between the wall and CSF. Increased outer boundary contrast comes at the cost of reduced inner boundary contrast. Magn Reson Med 77:655-663, 2017. © 2016 The Authors Magnetic Resonance in Medicine published by Wiley Periodicals, Inc. on behalf of International Society for Magnetic Resonance in Medicine. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

Structural imaging of the cervical spinal cord with suppressed CSF signal using DANTE pulse trains.

PURPOSE: We propose DANTE (Delays Alternating with Nutation for Tailored Excitation) moving fluid attenuation preparation pulse trains, in conjunction with T1 , T2 , and proton-density-weighted fast spin-echo (T1w-TSE, T2w-TSE and PDw-TSE) imaging readout, and three-dimensional fast low flip angle shots (3D-FLASH) T1 -weighted imaging readout to achieve CSF-suppressed high-spatial resolution multicontrast cervical spinal cord images. METHODS: DANTE pulse trains, consisting of a rapid series of low flip angle radiofrequency pulses interspersed with gradients, were used to substantially attenuate the longitudinal magnetization of flowing spins relative to static tissue/fluid, whose longitudinal magnetization is mostly preserved. We hypothesized that the contrast between spinal cord and cerebrospinal fluid (CSF) could be maximized due to moving CSF signal suppression. RESULTS: We demonstrate that metrics of contrast-to-noise ratio between spinal cord, nerve root, and CSF regions (CNRcord-CSF and CNRnerve-CSF ) are improved by at least a factor of 2 when compared with images acquired with non-prepared approaches and with 2D multiple-echo data image combination (MEDIC) imaging. In addition, we find that sagittal image quality can be significantly improved due to flow suppression effects from the DANTE preparation pluses. CONCLUSION: DANTE prepared imaging techniques for moving CSF signal attenuation are promising tools for cervical spinal cord imaging.

Temperature-triggered phase separation of a hydrophilic resilin-like polypeptide.

Temperature-triggered phase separation of recombinant proteins has offered substantial opportunities in the design of nanoparticles for a variety of applications. Herein, the temperature-triggered phase separation behavior of a recombinant hydrophilic resilin-like polypeptide (RLP) is described. The transition temperature and sizes of RLP-based nanoparticles can be modulated based on variations in polypeptide concentration, salt identity, ionic strength, pH, and denaturing agents, as indicated via UV-Vis spectroscopy and dynamic light scattering (DLS). The irreversible particle formation is coupled with secondary conformational changes from a random coil conformation to a more ordered ß-sheet structure. These RLP-based nanoparticles could find potential use as mechanically-responsive components in drug delivery, nanospring, nanotransducer, and biosensor applications.