The protective roles of integrin α4ß7 and Amphiregulin-expressing innate lymphoid cells in lupus nephritis.

Type 2 innate lymphoid cells (ILC2s) have emerged as key regulators of the immune response in renal inflammatory diseases such as lupus nephritis. However, the mechanisms underlying ILC2 adhesion and migration in the kidney remain poorly understood. Here, we revealed the critical role of integrin α4ß7 in mediating renal ILC2 adhesion and function. We found that integrin α4ß7 enables the retention of ILC2s in the kidney by binding to VCAM-1, E-cadherin, or fibronectin on structural cells. Moreover, integrin α4ß7 knockdown reduced the production of the reparative cytokine amphiregulin (Areg) by ILC2s. In lupus nephritis, TLR7/9 signaling within the kidney microenvironment downregulates integrin α4ß7 expression, leading to decreased Areg production and promoting the egress of ILC2s. Notably, IL-33 treatment upregulated integrin α4ß7 and Areg expression in ILC2s, thereby enhancing survival and reducing inflammation in lupus nephritis. Together, these findings highlight the potential of targeting ILC2 adhesion as a therapeutic strategy for autoimmune kidney diseases.

Efficient Combination Chemo-Sonodynamic Cancer Therapy Using Mitochondria-Targeting Sonosensitizer-Loaded Polysorbate-Based Micelles.

Sonodynamic therapy (SDT), utilizing ultrasound (US) and sonosensitizers, holds immense potential as a noninvasive and targeted treatment for a variety of deep-seated tumors. However, the clinical translation of SDT is hampered by several key limitations in sonosensitizers, especially their low aqueous stability and poor cellular uptake. In this study, non-ionic polysorbate (Tween 80, T80) was adopted to formulate effective nanocarriers for the safe and efficient delivery of sonosensitizers to cancer cells. Mitochondria-targeting triphenylphosphonium (TPP)-conjugated chlorin e6 (Ce6) sonosensitizer was loaded into T80-based micelles for efficient SDT. Pro-oxidant piperlongumine (PL) was co-encapsulated with TPP-conjugated Ce6 (T-Ce6) in T80 micelles to enable combination chemo-SDT. T80 micelles substantially enhanced the cellular internalization of T-Ce6. As a result, T80 micelles loaded with T-Ce6 and PL [T80(T-Ce6/PL)] significantly elevated intracellular reactive oxygen species (ROS) generation in MCF-7 human breast cancer cells upon US exposure. Moreover, T-Ce6 exhibited selective accumulation within the mitochondria, leading to efficient cell death under US irradiation. Importantly, T80(T-Ce6/PL) micelles caused cancer-specific cell death by selectively triggering apoptosis in cancer cells through PL. This study demonstrated the feasibility of using T80(T-Ce6/PL) micelles for efficient and cancer-specific combination chemo-SDT.

Improvement of transdermal absorption rate by nonthermal biocompatible atmospheric pressure plasma.

Nonthermal biocompatible plasma (NBP) is a promising option for improving medication absorption into the human skin. Currently, most plasma devices for cosmetics employ a floating-electrode plasma source for treating the skin. Human skin serves as the ground electrode in the floating-electrode plasma discharge, and discharge occurs between the skin and electrodes of the device. In this in vitro study, we aimed to evaluate the effect of NBP on the skin permeation of niacinamide. We have quantified the transdermal absorption rates of niacinamide in both untreated skin and skin treated with NBP for a duration of 10 s. The absorption of niacinamide for both without and with NBP treatment was observed until 12 h incubation time. Without plasma treatment, the human skin exhibited stable and low transdermal absorption of niacinamide up to 12 h. However, the NBP treatment significantly increased the transdermal absorption of niacinamide from 0.5 h to 6 h and continuously increased skin penetration over a duration of more than 12 h incubation period. The obtained results suggest that NBP-treated human skin showed a 60-fold higher penetration rate than non-treated skin. The increased penetration rate of niacinamide can be mainly attributed to plasmaporation subsequent to NBP treatment. The findings of this study demonstrate that NBP treatment results in remarkable skin permeability, making it a promising candidate for both cosmetic and pharmaceutical delivery applications.

In Situ Simultaneous Detection of Surface Protein and microRNA in Clustered Extracellular Vesicles from Cancer Cell Lines Using Flow Cytometry.

Extracellular vesicles (EVs) are becoming increasingly important in liquid biopsy for cancer because they contain multiple biomarkers, including proteins and RNAs, and circulate throughout the body. Cancer cell-derived EVs are highly heterogeneous, and multiplexed biomarker detection techniques are required to improve the accuracy of diagnosis. In addition, in situ EV biomarker detection increases the efficiency of the detection process because EVs are difficult to handle. In this study, in situ simultaneous detection of EV surface proteins, programmed cell death-ligand 1 (PD-L1), and internal miRNA-21 (miR-21) analyzed by conventional flow cytometry was developed for a breast cancer liquid biopsy. However, the majority of EVs were not recognized by flow cytometry for biomarker detection because the size of EVs was below the detectable size range of the flow cytometer. To solve this problem, the formation of EV clusters was induced by 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-polyethylene glycol-DSPE during biomarker detection. Consequently, both PD-L1 and miR-21 detection signals from cancer cell-derived EVs were drastically increased, making them distinguishable from normal cell-derived EVs. The in situ simultaneous cancer biomarker detection from EV clusters analyzed by flow cytometry contributes to an increase in the sensitivity and accuracy of the EV-based liquid biopsy for cancer.

Defining Integrin Tension Required for Chemotaxis of Metastatic Breast Cancer Cells in Confinement.

Cancer metastasis is affected by chemical factors and physical cues. From cell adhesion to migration, mechanical tension applied to integrin expresses on the cell membrane and physical confinement significantly regulates cancer cell behaviors. Despite the physical interplay between integrins in cells and ligands in the tumor microenvironment, quantitative analysis of integrin tension during cancer cell migration in microconfined spaces remains elusive owing to the limited experimental tools. Herein, a platform termed microconfinement tension gauge tether to monitor spatial integrin tension with single-molecule precision by analyzing the epithelial-growth-factor-induced chemotaxis of metastatic human breast cancer cells in microfluidic channels is developed. The results reveal that the metastatic cancer cells exert the strongest integrin tension in the range of 54-100 pN at the leading edges of cells during chemokinetic migration on a planar surface, while the cells exert the strongest integrin tension exceeding 100 pN at the cell rear when entering microconfinement. Further analysis demonstrates that cells undergo mesenchymal migration under high integrin tension and less confinement, which is converted to amoeboid migration under low integrin tension or high confinement. In summary, the results identify a basic mechanism underlying the mechanical interactions between integrin tension and microenvironment that determines cancer invasion and metastasis.

Characterization of the role of Kunitz-type protease inhibitor domain in dimerization of amyloid precursor protein.

A major difference between amyloid precursor protein (APP) isoforms (APP695 and APP751) is the existence of a Kunitz type protease inhibitor (KPI) domain which has a significant impact on the homo- and hetero-dimerization of APP isoforms. However, the exact molecular mechanisms of dimer formation remain elusive. To characterize the role of the KPI domain in APP dimerization, we performed a single molecule pull down (SiMPull) assay where homo-dimerization between tethered APP molecules and soluble APP molecules was highly preferred regardless of the type of APP isoforms, while hetero-dimerization between tethered APP751 molecules and soluble APP695 molecules was limited. We further investigated the domain level APP-APP interactions using coarse-grained models with the Martini force field. Though the model initial ternary complexes (KPI-E1, KPI-KPI, KPI-E2, E1-E1, E2-E2, and E1-E2) generated using HADDOCK (HD) and AlphaFold2 (AF2), the binding free energy profiles and the binding affinities of the domain combinations were investigated via the umbrella sampling with Martini force field. Additionally, membrane-bound microenvironments at the domain level were modeled. As a result, it was revealed that the KPI domain has a stronger attractive interaction with itself than the E1 and E2 domains, as reported elsewhere. Thus, the KPI domain of APP751 may form additional attractive interactions with E1, E2 and the KPI domain itself, whereas it is absent in APP695. In conclusion, we found that the APP751 homo-dimer formation is predominant than the homodimerization in APP695, which is facilitated by the presence of the KPI domain.

Senescent stroma induces nuclear deformations in cancer cells via the inhibition of RhoA/ROCK/myosin II-based cytoskeletal tension.

The presence of senescent cells within tissues has been functionally linked to malignant transformations. Here, using tension-gauge tethers technology, particle-tracking microrheology, and quantitative microscopy, we demonstrate that senescent-associated secretory phenotype (SASP) derived from senescent fibroblasts impose nuclear lobulations and volume shrinkage on malignant cells, which stems from the loss of RhoA/ROCK/myosin II-based cortical tension. This loss in cytoskeletal tension induces decreased cellular contractility, adhesion, and increased mechanical compliance. These SASP-induced morphological changes are, in part, mediated by Lamin A/C. These findings suggest that SASP induces defective outside-in mechanotransduction from actomyosin fibers in the cytoplasm to the nuclear lamina, thereby triggering a cascade of biophysical and biomolecular changes in cells that associate with malignant transformations.

Characterization of Integrin Molecular Tension of Human Breast Cancer Cells on Anisotropic Nanopatterns.

Physical interactions between cells and micro/nanometer-sized architecture presented in an extracellular matrix (ECM) environment significantly influence cell adhesion and morphology, often facilitating the incidence of diseases, such as cancer invasion and metastasis. Sensing and responding to the topographical cues are deeply associated with a physical interplay between integrins, ligands, and mechanical force transmission, ultimately determining diverse cell behavior. Thus, how the tension applied to the integrin-ligand bonds controls cells' response to the topographical cues needs to be elucidated through quantitative analysis. Here, in this brief research report, we reported a novel platform, termed "topo-tension gauge tether (TGT)," to visualize single-molecule force applied to the integrin-ligand on the aligned anisotropic nanopatterns. Using the topo-TGT assay, first, topography-induced adhesion and morphology of cancerous and normal cells were compared with the pre-defined peak integrin tension. Next, spatial integrin tensions underneath cells were identified using reconstructed integrin tension maps. As a result, we characterized each cell's capability to comply with nanotopographies and the magnitude of the spatial integrin tension. Altogether, the quantitative information on integrin tension will be a valuable basis for understanding the biophysical mechanisms underlying the force balance influencing adhesion to the topographical cues.

Pro-oxidant drug-loaded porphyrinic zirconium metal-organic-frameworks for cancer-specific sonodynamic therapy.

Sonodynamic therapy, which utilizes ultrasound (US) to produce cytotoxic reactive oxygen species (ROS), can overcome the critical drawbacks of photodynamic therapy, such as limited tissue penetration depth. However, the development of sonosensitizers having superior sonodynamic effects and desirable biocompatibility remains a major challenge. In this study, nanoscale zirconium-based porphyrinic metal organic frameworks (MOFs) (PCN-222) were developed as safe and effective nanosonosensitizers. Polyethylene glycol (PEG)-coated PCN-222 (PEG-PCN) was loaded with a pro-oxidant drug, piperlongumine (PL), to enable tumor-specific chemo-photodynamic combination therapy. Both PEG-PCN and PL-incorporated PEG-PCN (PL-PEG-PCN) showed high colloidal stability in biological media. In addition, nanoscale PL-PEG-PCN was efficiently internalized by breast cancer cells, leading to substantially increased ROS generation under US exposure. The effective intracellular delivery of PL by PEG-PCN further elevated the level of intracellular ROS in breast cancer cells owing to the pro-oxidative activity of PL. Therefore, PL-PEG-PCN revealed significantly higher sonotoxicity than free PL and PEG-PCN. Owing to the cancer-specific apoptosis triggered by PL, PL-PEG-PCN showed cancer-selective cell death in breast cancer cells compared with normal fibroblast cells. This study demonstrates that pro-oxidant drug-loaded porphyrinic MOFs are biocompatible and effective sonosensitizers for cancer-targeted chemo-sonodynamic combination therapy.

Molecular Nanomechanical Mapping of Histamine-Induced Smooth Muscle Cell Contraction and Shortening.

Mechanical response to external stimuli is a conserved feature of many cell types. For example, neurotransmitters (e.g., histamine) trigger calcium signals that induce actomyosin-regulated contraction of airway smooth muscle (ASM); the resulting cell shortening causes airway narrowing, the excess of which can cause asthma. Despite intensive studies, however, it remains unclear how physical forces are propagated through focal adhesion (FA)-the major force-transmission machinery of the cell-during ASM shortening. We provide a nanomechanical platform to directly image single molecule forces during ASM cell shortening by repurposing DNA tension sensors. Surprisingly, cell shortening and FA disassembly that immediately precedes it occurred long after histamine-evoked increases in intracellular calcium levels ([Ca2+]i). Our mathematical model that fully integrates cell edge protrusion and retraction with contractile forces acting on FA predicted that (1) stabilization of FA impedes cell shortening and (2) the disruption of FAs is preceded by their strengthening through actomyosin-activated molecular tension. We confirmed these predictions via real-time imaging and molecular force measurements. Together, our work highlights a key role of FA dynamics in regulating ASM contraction induced by an allergen with potential therapeutic implications.

Circulating mitochondrial N-formyl peptides contribute to secondary nosocomial infection in patients with septic shock.

Secondary infections typically worsen outcomes of patients recovering from septic shock. Neutrophil [polymorphonuclear leukocytes (PMNs)] migration to secondarily inoculated sites may play a key role in inhibiting progression from local bacterial inoculation to secondary infection. Mitochondrial N-formyl peptide (mtFP) occupancy of formyl peptide receptor-1 (FPR1) has been shown to suppress PMN chemotaxis. Therefore, we studied the association between circulating mtFPs and the development of secondary infection in patients with septic shock. We collected clinical data and plasma samples from patients with septic shock admitted to the intensive care unit for longer than 72 h. Impacts of circulating nicotinamide adenine dinucleotide dehydrogenase subunit-6 (ND6) upon clinical outcomes were analyzed. Next, the role of ND6 in PMN chemotaxis was investigated using isolated human PMNs. Studying plasma samples from 97 patients with septic shock, we found that circulating ND6 levels at admission were independently and highly associated with the development of secondary infection (odds ratio = 30.317, 95% CI: 2.904 to 316.407, P = 0.004) and increased 90-d mortality (odds ratio = 1.572, 95% CI: 1.002 to 2.465, P = 0.049). In ex vivo experiments, ND6 pretreatment suppressed FPR1-mediated PMN chemotactic responses to bacterial peptides in the presence of multiple cytokines and chemokines, despite increased nondirectional PMN movements. Circulating mtFPs appear to contribute to the development of secondary infection and increased mortality in patients with septic shock who survive their early hyperinflammatory phase. The increased susceptibility to secondary infection is probably partly mediated by the suppression of FPR1-mediated PMN chemotaxis to secondary infected sites.

Mechanical stress determines the configuration of TGFß activation in articular cartilage.

Our incomplete understanding of osteoarthritis (OA) pathogenesis has significantly hindered the development of disease-modifying therapy. The functional relationship between subchondral bone (SB) and articular cartilage (AC) is unclear. Here, we found that the changes of SB architecture altered the distribution of mechanical stress on AC. Importantly, the latter is well aligned with the pattern of transforming growth factor beta (TGFß) activity in AC, which is essential in the regulation of AC homeostasis. Specifically, TGFß activity is concentrated in the areas of AC with high mechanical stress. A high level of TGFß disrupts the cartilage homeostasis and impairs the metabolic activity of chondrocytes. Mechanical stress stimulates talin-centered cytoskeletal reorganization and the consequent increase of cell contractile forces and cell stiffness of chondrocytes, which triggers αV integrin-mediated TGFß activation. Knockout of αV integrin in chondrocytes reversed the alteration of TGFß activation and subsequent metabolic abnormalities in AC and attenuated cartilage degeneration in an OA mouse model. Thus, SB structure determines the patterns of mechanical stress and the configuration of TGFß activation in AC, which subsequently regulates chondrocyte metabolism and AC homeostasis.

Novel fusion peptide-mediated siRNA delivery using self-assembled nanocomplex.

BACKGROUND: Gene silencing using siRNA can be a new potent strategy to treat many incurable diseases at the genetic level, including cancer and viral infections. Treatments using siRNA essentially requires an efficient and safe method of delivering siRNA into cells while maintaining its stability. Thus, we designed novel synergistic fusion peptides, i.e., SPACE and oligoarginine. RESULTS: Among the novel fusion peptides and siRNAs, nanocomplexes have enhanced cellular uptake and gene silencing effect in vitro and improved retention and gene silencing effects of siRNAs in vivo. Oligoarginine could attract siRNAs electrostatically to form stable and self-assembled nanocomplexes, and the SPACE peptide could interact with the cellular membrane via hydrogen bonding. Therefore, nanocomplexes using fusion peptides showed improved and evident cellular uptake and gene silencing of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) via the lipid raft-mediated endocytosis pathway, especially to the HDFn cells of the skin, and all of the fusion peptides were biocompatible. Also, intratumorally injected nanocomplexes had increased retention time of siRNAs at the site of the tumor. Finally, nanocomplexes demonstrated significant in vivo gene silencing effect without overt tissue damage and immune cell infiltration. CONCLUSIONS: The new nanocomplex strategy could become a safe and efficient platform for the delivery of siRNAs into cells and tissues to treat various target diseases through gene silencing.

Force-dependent trans-endocytosis by breast cancer cells depletes costimulatory receptor CD80 and attenuates T cell activation.

In this study, we investigated the biophysical interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) and CD80. CTLA-4 is a key molecule in immunosuppression, and CD80 is a costimulatory receptor promoting T cell activation. We observed that after cell-cell contact was established between breast cancer cells and antigen presenting cells (APCs), CTLA-4 expressed on the breast cancer cells bind to CD80 expressed on the APCs, and underwent trans-endocytosis to deplete CD80. Force measurement and live cell imaging revealed that upon binding to CD80, forces generated by breast cancer cells and transmitted via CTLA-4 were sufficiently strong to displace CD80 from the surface of APCs to be internalized by breast cancer cells. We further demonstrated that because of the force-dependent trans-endocytosis of CD80, the capacity of APCs to activate T cells was significantly attenuated. Furthermore, inhibiting force generation in cancer cells would increase the T cell activating capacity of APCs. Our results provide a possible mechanism behind the immunosuppression commonly seen in breast cancer patients, and may lead to a new strategy to restore anti-tumor immunity by inhibiting pathways of force-generation.

Differential interleukin-1ß induction by uropathogenic Escherichia coli correlates with its phylotype and serum C-reactive protein levels in Korean infants.

Urinary tract infection (UTI) is one of the most common bacterial infections in infants less than age 1 year. UTIs frequently recur and result in long-term effects include sepsis and renal scarring. Uropathogenic Escherichia coli (UPEC), the most prevalent organism found in UTIs, can cause host inflammation via various virulence factors including hemolysin and cytotoxic necrotizing factors by inducing inflammatory cytokines such as interleukin (IL)-1ß. However, the ability of each UPEC organism to induce IL-1ß production may differ by strain. Furthermore, the correlation between differential IL-1ß induction and its relevance in pathology has not been well studied. In this study, we isolated UPEC from children under age 24 months and infected bone-marrow derived macrophages with the isolates to investigate secretion of IL-1ß. We found that children with higher concentrations of C-reactive protein (CRP) were more likely to harbor phylotype B2 UPEC strains that induced more IL-1ß production than phylotype D. We also observed a significant correlation between serum CRP level and in vitro IL-1ß induction by phylotype B2 UPEC bacteria. Our results highlight the diversity of UPEC in terms of IL-1ß induction capacity in macrophages and suggest a potential pathogenic role in UTIs by inducing inflammation in infants.

Rapid translocation of pluripotency-related transcription factors by external uniaxial forces.

Human embryonic stem cells subjected to a one-time uniaxial stretch for as short as 30-min on a flexible substrate coated with Matrigel experienced rapid and irreversible nuclear-to-cytoplasmic translocation of NANOG and OCT4, but not Sox2. Translocations were directed by intracellular transmission of biophysical signals from cell surface integrins to nuclear CRM1 and were independent of exogenous soluble factors. On E-CADHERIN-coated substrates, presumably with minimal integrin engagement, mechanical strain-induced rapid nuclear-to-cytoplasmic translocation of the three transcription factors. These findings might provide fundamental insights into early developmental processes and may facilitate mechanotransduction-mediated bioengineering approaches to influencing stem cell fate determination.

TGFß1 reinforces arterial aging in the vascular smooth muscle cell through a long-range regulation of the cytoskeletal stiffness.

Here we report exquisitely distinct material properties of primary vascular smooth muscle (VSM) cells isolated from the thoracic aorta of adult (8 months) vs. aged (30 months) F344XBN rats. Individual VSM cells derived from the aged animals showed a tense internal network of the actin cytoskeleton (CSK), exhibiting increased stiffness (elastic) and frictional (loss) moduli than those derived from the adult animals over a wide frequency range of the imposed oscillatory deformation. This discrete mechanical response was long-lived in culture and persistent across a physiological range of matrix rigidity. Strikingly, the pro-fibrotic transforming growth factor ß1 (TGFß1) emerged as a specific modifier of age-associated VSM stiffening in vitro. TGFß1 reinforced the mechanical phenotype of arterial aging in VSM cells on multiple time and length scales through clustering of mechanosensitive α5ß1 and αvß3 integrins. Taken together, these studies identify a novel nodal point for the long-range regulation of VSM stiffness and serve as a proof-of-concept that the broad-based inhibition of TGFß1 expression, or TGFß1 signal transduction in VSM, may be a useful therapeutic approach to mitigate the pathologic progression of central arterial wall stiffening associated with aging.

Sphingolipids facilitate age asymmetry of membrane proteins in dividing yeast cells.

One proposed mechanism of cellular aging is the gradual loss of certain cellular components that are insufficiently renewed. In an earlier study, multidrug resistance transporters (MDRs) were postulated to be such aging determinants during the yeast replicative life span (RLS). Aged MDR proteins were asymmetrically retained by the aging mother cell and did not diffuse freely into the bud, whereas newly synthesized MDR proteins were thought to be deposited mostly in the bud before cytokinesis. In this study, we further demonstrate the proposed age asymmetry of MDR proteins in dividing yeast cells and investigate the mechanism that controls diffusive properties of MDR proteins to maintain this asymmetry. We found that long-chain sphingolipids, but not the septin/endoplasmic reticulum-based membrane diffusion barrier, are important for restricting MDR diffusion. Depletion of sphingolipids or shortening of their long acyl chains resulted in an increase in the lateral mobility of MDR proteins, causing aged MDR protein in the mother cell to enter the bud. We used a mathematical model to understand the effect of diminished MDR age asymmetry on yeast cell aging, the result of which was qualitatively consistent with the observed RLS shortening in sphingolipid mutants.

Notch-Jagged complex structure implicates a catch bond in tuning ligand sensitivity.

Notch receptor activation initiates cell fate decisions and is distinctive in its reliance on mechanical force and protein glycosylation. The 2.5-angstrom-resolution crystal structure of the extracellular interacting region of Notch1 complexed with an engineered, high-affinity variant of Jagged1 (Jag1) reveals a binding interface that extends ~120 angstroms along five consecutive domains of each protein. O-Linked fucose modifications on Notch1 epidermal growth factor-like (EGF) domains 8 and 12 engage the EGF3 and C2 domains of Jag1, respectively, and different Notch1 domains are favored in binding to Jag1 than those that bind to the Delta-like 4 ligand. Jag1 undergoes conformational changes upon Notch binding, exhibiting catch bond behavior that prolongs interactions in the range of forces required for Notch activation. This mechanism enables cellular forces to regulate binding, discriminate among Notch ligands, and potentiate Notch signaling.

Dynamic simulations show repeated narrowing maximizes DNA linearization in elastomeric nanochannels.

This paper uses computer simulations to reveal unprecedented details about linearization of deoxyribonucleic acid (DNA) inside dynamic nanochannels that can be repeatedly widened and narrowed. We first analyze the effect of rate of channel narrowing on DNA linearization dynamics. Quick (∼0.1 s) narrowing of nanoscale channels results in rapid overstretching of the semi-flexible chain followed by a slower (∼0.1-10 s) relaxation to an equilibrium extension. Two phenomena that induce linearization during channel narrowing, namely, elongational-flow and confinement, occur simultaneously, regardless of narrowing speed. Interestingly, although elongational flow is a minimum at the mid-point of the channel and increases towards the two ends, neither the linearization dynamics nor the degree of DNA extension varies significantly with the center-of-mass of the polymer projected on the channel axis. We also noticed that there was a significant difference in time to reach the equilibrium length, as well as the degree of DNA linearization at short times, depending on the initial conformation of the biopolymer. Based on these observations, we tested a novel linearization protocol where the channels are narrowed and widened repeatedly, allowing DNA to explore multiple conformations. Repeated narrowing and widening, something uniquely enabled by the elastomeric nanochannels, significantly decrease the time to reach the equilibrium-level of stretch when performed within periods comparable to the chain relaxation time and more effectively untangle chains into more linearized biopolymers.