Biology subjects

Patten, J.

Publications and source records attributed to Patten, J..

5 recordsLinked to original sources

bioRxiv2025

The interplay between stiffness and hyperglycemia on diabetic foot ulcer wound closure

IntroductionDiabetic foot ulcers are open wounds with impaired wound closure at the bottom of the foot. Although diabetic plantar skin is stiffer, which should enhance fibroblast mechanotransduction, fibroblasts still fail to migrate effectively. This suggests impaired wound closure is driven by another factor; hyperglycemia ([≥]11.1 mM glucose), which alters fibroblast mechanotransduction. PurposeTo mimic diabetic foot ulcers by developing a 2D circular in vitro wound closure model system to investigate fibroblast mechanoresponse under diabetic plantar skin stiffness and hyperglycemia. MethodsPolydimethylsiloxane was used as substrate, fabricated at 57 kPa and 90 kPa for normal and diabetic plantar skin stiffnesses, respectively. Cell culture media contained a 5.5 mM glucose concentration simulating normal blood glucose or an altered 11.1 mM glucose concentration simulating hyperglycemia. ResultsTime-lapse fluorescent imaging of wound assays reveals a restrictive effect of higher stiffness on migrating fibroblasts under normal glucose conditions, and a biphasic response to hyperglycemic conditions. Fibroblasts migrating on softer substrates mimicking normal plantar skin stiffness and under hyperglycemia have decreased velocity as predicted. Whereas cells migrating on stiffer substrates mimicking diabetic plantar skin stiffness and under hyperglycemia demonstrate increased cell velocity, overcoming the higher stiffnesss restrictive effect. Despite faster cell velocities on higher stiffness, wounds under normal glucose conditions still close faster than those under hyperglycemic conditions. ConclusionThis research establishes a wound closure model demonstrating significantly slower wound closure in diabetic plantar skin with higher stiffness and hyperglycemic glucose levels compared to normal plantar skin with softer stiffness and normal glucose levels.

bioengineering↗

bioRxiv2025

FAK Differentially Mechanoregulates Cell Migration During Wound Closure

Cell migration is an essential step in wound healing. Mechanical input from the local microenvironment controls much of cell velocity and directionality during migration, which is translated into biochemical cues by focal adhesion kinase (FAK) inside the cell. FAK induces both regeneration and fibrosis. The mechanisms by which FAK decide wound fate (regenerative or fibrotic repair) in soft, normal wounds or stiff, fibrotic wounds remains unclear. Here we show that FAK differentially mechanoregulates wound behavior on soft substrates mimicking normal wounds and stiff substrates mimicking fibrotic wounds by converting mechanical substrate stimuli into variable cell velocity, directionality, and angle during wound healing. Cells on soft substrates migrate slower and less persistently; cells on stiff substrates migrate faster and more persistently with the same angle as the cells on normal wound substrates. Inhibition of FAK results in substantially slower, less persistent, and less correctly angled cell migration, which leads to slowed wound closure. Moreover, FAK inhibition impairs fibroblast ability to respond to substrate stiffness when migrating. Here we show FAK is an essential mechanoregulator of wound migration in fibroblast wound closure and is responsible for controlling cell migration dynamics in response to substrate stiffnesses mimicking normal or fibrotic wounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/646098v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b93ab5org.highwire.dtl.DTLVardef@bd4693org.highwire.dtl.DTLVardef@646059org.highwire.dtl.DTLVardef@1f1aec1_HPS_FORMAT_FIGEXP M_FIG TOC Graphic: Cell migration dynamics regulated by substrate stiffness via focal adhesion kinase (FAK). Cells on softer substrates mimicking normal wounds display slower, more random migration that is impaired by FAK inhibition. Cells migrate faster and more persistently on stiffer substrates mimicking fibrotic wounds that is dysregulated by FAK inhibition. C_FIG

bioengineering↗

bioRxiv2024

EDA Fibronectin Microarchitecture and YAP Translocation During Wound Closure

Fibronectin (Fn) is an extracellular matrix glycoprotein with mechanosensitive structure-function. EDA Fn, a Fn isoform, is not present in adult tissue but is required for tissue repair. Curiously, EDA Fn is linked to both regenerative and fibrotic tissue repair. Given that Fn mechanoregulates cell behavior, Fn EDA organization during wound closure might play a role in mediating these differing responses. One mechanism by which cells sense and respond to their microenvironment is by activating a transcriptional co-activator, Yes-associated protein (YAP). Interestingly, YAP activity is not only required for wound closure, but similarly linked to both regenerative and fibrotic repair. Therefore, this study aims to evaluate how, during normal and fibrotic wound closure, EDA Fn organization might modulate YAP translocation by culturing human dermal fibroblasts on polydimethylsiloxane (PDMS) substrates mimicking normal (soft: 18 kPa) and fibrotic (stiff: 146 kPa) wounded skin. On stiffer substrates mimicking fibrotic wounds, fibroblasts assembled an aligned EDA Fn matrix comprising thinner fibers, suggesting increased microenvironmental tension. To evaluate if cell binding to the EDA domain of Fn was essential to overall matrix organization, fibroblasts were treated with Irigenin, which inhibits binding to the EDA domain within Fn. Blocking adhesion to EDA led to randomly organized EDA Fn matrices with thicker fibers, suggesting reduced microenvironmental tension even during fibrotic wound closure. To evaluate if YAP signaling plays a role in EDA Fn organization, fibroblasts were treated with CA3, which suppresses YAP activity in a dose-dependent manner. Treatment with CA3 also led to randomly organized EDA Fn matrices with thicker fibers, suggesting a potential connected mechanism of reducing tension during fibrotic wound closure. Next, YAP activity was assessed to evaluate the impact of EDA Fn organization. Interestingly, fibroblasts migrating on softer substrates mimicking normal wounds increased YAP activity but on stiffer substrates, decreased YAP activity. When fibroblasts on stiffer substrates were treated with Irigenin or CA3, fibroblasts increased YAP activity. These results suggest there may be disrupted signaling between EDA Fn organization and YAP translocation during fibrotic wound closure that could be restored when reestablishing normal EDA Fn matrix organization to instead drive regenerative wound repair.

bioengineering↗

bioRxiv2022

CAPG is required for Ebola virus infection by controlling virus egress from infected cells.

Replication of Ebola virus (EBOV) is dependent upon actin functionality, especially at cell entry through macropinocytosis and at release of virus from cells. Previously, major actin-regulatory factors such as Rac1 and ARP2/3, involved in actin nucleation were shown important in both steps. However, downstream of nucleation, many other cell factors, are needed to control actin dynamics. How these regulate EBOV infection remains largely unknown. Here, we identified the actin-regulating protein, CAPG, as important for EBOV replication. Notably, knockdown (KD) of CAPG specifically inhibited viral infectivity and yield of infectious particles. Mechanistic analysis revealed a requirement of CAPG for virus production from infected cells. Proximity ligation and split GFP reconstitution assays revealed strong association of CAPG with VP40 that was mediated through the S1 domain of CAPG. Overall, CAPG is a novel host factor regulating EBOV infection through connecting actin filament stabilization to viral egress from cells.

microbiology↗

bioRxiv2021

Multidose evaluation of 6,710 drug repurposing library identifies potent SARS-CoV-2 infection inhibitors In Vitro and In Vivo.

Identification of host factors contributing to replication of viruses and resulting disease progression remains a promising approach for development of new therapeutics. Here, we evaluated 6710 clinical and preclinical compounds targeting 2183 host proteins by immunocytofluorescence-based screening to identify SARS-CoV-2 infection inhibitors. Computationally integrating relationships between small molecule structure, dose-response antiviral activity, host target and cell interactome networking produced cellular networks important for infection. This analysis revealed 389 small molecules, >12 scaffold classes and 813 host targets with micromolar to low nanomolar activities. From these classes, representatives were extensively evaluated for mechanism of action in stable and primary human cell models, and additionally against Beta and Delta SARS-CoV-2 variants and MERS-CoV. One promising candidate, obatoclax, significantly reduced SARS-CoV-2 viral lung load in mice. Ultimately, this work establishes a rigorous approach for future pharmacological and computational identification of novel host factor dependencies and treatments for viral diseases.

microbiology↗