The umbrella cell keratin network: organization as a tile-like mesh, formation of a girded layer in response to bladder filling, and dependence on the plectin cytolinker.

The keratin cytoskeleton and associated desmosomes contribute to the mechanical stability of epithelial tissues, but their organization in bladder umbrella cells and their responses to bladder filling are poorly understood. Using super-resolution confocal microscopy, along with 3D image reconstruction and platinum replica electron microscopy, we observed that the apical keratin network of umbrella cells was organized as a dense tile-like mesh comprised of tesserae bordered on their edges by cortical actin filaments, filled with woven keratin filaments, and crosslinked by plectin. A band of keratin was also observed at the cell periphery that was linked to the junction-associated actin ring by plectin. During bladder filling, the junction-localized desmosomal necklace expanded, and a subjacent girded layer was formed that linked the keratin network to desmosomes, including those at the umbrella cell-intermediate cell interface. Disruption of plectin led to focal keratin network dissolution, loss of the junction-associated band of keratin, perturbation of tight junction continuity, and loss of cell-cell cohesion. Our studies reveal a novel tile-like organization of the umbrella cell keratin cytoskeleton that is dependent on plectin, that reorganizes in response to bladder filling, and that likely serves to maintain umbrella cell continuity in the face of mechanical distension.

Antinociceptive effect of the calcitonin gene-related peptide receptor antagonist BIBN4096BS in mice with bacterial cystitis.

Patients with urinary tract infections (UTIs) suffer from urinary frequency, urgency, dysuria, and suprapubic pain, but the mechanisms by which bladder afferents sense the presence of uropathogens and encode this information is not well understood. Calcitonin gene-related peptide (CGRP) is a 37-mer neuropeptide found in a subset of bladder afferents that terminate primarily in the lamina propria. Here, we report that the CGRP receptor antagonist BIBN4096BS lessens lower urinary tract symptoms and prevents the development of pelvic allodynia in mice inoculated with uropathogenic Escherichia coli (UPEC) without altering urine bacterial loads or the host immune response to the infection. These findings indicate that CGRP facilitates the processing of noxious/inflammatory stimuli during UPEC infection. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria, a region where afferent fibers containing CGRP terminate, that expresses the canonical CGRP receptor components Calcrl and Ramp1. We propose that these fibroblasts, in conjunction with CGRP+ afferents, form a circuit that senses substances released during the infection and transmit this noxious information to the central nervous system.NEW & NOTEWORTHY Afferent C fibers release neuropeptides including calcitonin gene-related peptide (CGRP). Here, we show that the specific CGRP receptor antagonist, BIBN409BS, ameliorates lower urinary tract symptoms and pelvic allodynia in mice inoculated with uropathogenic E. coli. Using fluorescent in situ hybridization, we identified a population of suburothelial fibroblasts in the lamina propria that expresses the canonical CGRP receptor. Our findings indicate that CGRP contributes to the transmission of nociceptive information arising from the bladder.

Accelerated in vitro oxidative degradation testing of polypropylene surgical mesh.

Polypropylene (PP) surgical mesh had reasonable success in repair of hernia and treatment of stress urinary incontinence (SUI); however, their use for the repair of pelvic organ prolapse (POP) resulted in highly variable results with lifelong complications in some patients. One of several factors that could be associated with mesh-related POP complications is changes in the properties of the implanted surgical mesh due to oxidative degradation of PP in vivo. Currently, there are no standardized in vitro bench testing methods available for assessing the susceptibility to oxidative degradation and estimating long-term in vivo stability of surgical mesh. In this work, we adapted a previously reported automated reactive accelerated aging (aRAA) system, which uses elevated temperatures and high concentrations of hydrogen peroxide (H2 O2 ), for accelerated bench-top oxidative degradation testing of PP surgical mesh. Since H2 O2 is highly unstable at elevated temperatures and for prolonged periods, the aRAA system involves a feedback loop based on electrochemical detection methods to maintain consistent H2 O2 concentration in test solutions. Four PP mesh samples with varying mesh knit designs, filament diameter, weight, and % porosity, were selected for testing using aRAA up to 4 weeks and characterized using thermal analysis, Fourier-transform infrared spectroscopy-attenuated total reflectance (FTIR-ATR) and scanning electron microscopy (SEM). Additionally, the oxidation index (OI) values were calculated based on the FTIR-ATR spectra to estimate the oxidative degradation and oxidation reaction kinetics of PP surgical mesh. The OI values and surface damage in the form of surface flaking, peeling, and formation of transverse cracks increased with aRAA aging time. The aRAA test method introduced here could be used to standardize the assessment of long-term stability of surgical mesh and may also be adopted for accelerated oxidative degradation testing of other polymer-based medical devices.

GGA-MLP: A Greedy Genetic Algorithm to Optimize Weights and Biases in Multilayer Perceptron.

The task of designing an Artificial Neural Network (ANN) can be thought of as an optimization problem that involves many parameters whose optimal value needs to be computed in order to improve the classification accuracy of an ANN. Two of the major parameters that need to be determined during the design of an ANN are weights and biases. Various gradient-based optimization algorithms have been proposed by researchers in the past to generate an optimal set of weights and biases. However, due to the tendency of gradient-based algorithms to get trapped in local minima, researchers have started exploring metaheuristic algorithms as an alternative to the conventional techniques. In this paper, we propose the GGA-MLP (Greedy Genetic Algorithm-Multilayer Perceptron) approach, a learning algorithm, to generate an optimal set of weights and biases in multilayer perceptron (MLP) using a greedy genetic algorithm. The proposed approach increases the performance of the traditional genetic algorithm (GA) by using a greedy approach to generate the initial population as well as to perform crossover and mutation. To evaluate the performance of GGA-MLP in classifying nonlinear input patterns, we perform experiments on datasets of varying complexities taken from the University of California, Irvine (UCI) repository. The experimental results of GGA-MLP are compared with the existing state-of-the-art techniques in terms of classification accuracy. The results show that the performance of GGA-MLP is better than or comparable to the existing state-of-the-art techniques.

Thread Size and Polymer Composition of 3D Printed and Electrospun Wound Dressings Affect Wound Healing Outcomes in an Excisional Wound Rat Model.

Thread size and polymer composition are critical properties to consider for achieving a positive healing outcome with a wound dressing. Three-dimensional (3D) printed scaffolds and electrospun mats both offer distinct advantages as replaceable wound dressings. This research aims to determine if the thread size and polymer compositions of the scaffolds affect skin wound healing outcomes, an aspect that has not been adequately explored. Using a modular polymer platform, four polyester direct-write 3D printed scaffolds and electrospun mats were fabricated into wound dressings. The dressings were applied to splinted, full thickness skin wounds in an excisional wound rat model and evaluated against control wounds to which no dressing was applied. Wound closure rates and reduction of the wound bed width were not affected by the thread size or polymer composition. However, epidermal thickness was larger in wounds treated with electrospun dressings and was slightly affected by the polymer composition. Two of the four tested polymer compositions lead to delayed reorganization of granulation tissues. Moreover, enhanced angiogenesis was seen in wounds treated with 3D printed dressings compared to those treated with electrospun dressings. The results from this study can be used to inform the choice of dressing architecture and polymer compositions to achieve positive wound healing outcomes.

Effect of Dexamethasone on Room Temperature Three-Dimensional Printing, Rheology, and Degradation of a Low Modulus Polyester for Soft Tissue Engineering.

Three-dimensional (3D) printing has enabled benchtop fabrication of customized bioengineered constructs with intricate architectures. Various approaches are being explored to enable optimum integration of such constructs into the physiological environment including addition of bioactive fillers. In this work, we incorporated a corticosteroid drug, dexamethasone (Dex), in a low modulus polyester (SC5050) and examined the effect of Dex incorporation on solvent-, initiator-, and monomer-free pneumatic extrusion-based 3D printing of the polymer. Dex-SC5050 interactions were characterized by plotting thermodynamic binary phase diagrams based on the Flory-Huggins theory. The effect of Dex composition on the 3D printability of the SC5050 polyester was examined by rheological characterization and by image analysis of each layer of the 3D printed scaffolds. The drug release and the degradation of the polymer from the 3D printed scaffolds was used to analyze the effect of Dex composition on the performance of the 3D printed scaffolds. We found that Dex was insoluble in SC5050 polyester at relevant 3D printing temperatures and the insoluble drug particles physically reinforced the polymer, increasing the viscosity and the shear modulus of the base polymer. In addition, the reinforcing effect improved the shape fidelity of the printed filaments and the overall quality of the scaffolds. The Dex particles demonstrated a two-phase release, with an initial burst release and a slower sustained release of drug under in vitro conditions. To investigate preliminary host response of the 3D printed SC5050 scaffolds for tissue engineering applications, the printed scaffolds were implanted subcutaneously in Sprague-Dawley rats for 6 weeks and examined for fibrous tissue formation, infiltration of cells, and vascularization into the pores of the scaffolds.

Hypoxia inducible factor as a therapeutic target for atherosclerosis.

Atherosclerosis is a highly prevalent disease that can significantly increase the risk of major vascular events, such as myocardial or cerebral infarctions. The anoxemia theory states that a disparity between oxygen supply and demand contributes to atherosclerosis. Hypoxia inducible factor-1 (HIF-1) is a heterodimeric protein, part of the basic helix-loop-helix family and one of the main regulators of cellular responses in a low­oxygen environment. It plays a key role in the development of atherosclerosis through cell-specific responses, acting on endothelial cells, vascular smooth muscle cells (SMCs) and macrophages. Through the upregulation of VEGF, NO, ROS and PDGF, HIF-1 is able to cause endothelial cell dysfunction, proliferation, angiogenesis and inflammation. Activation of the NF-kB pathway in endothelial cells is an important contributor to inflammation and positively feedbacks to HIF-1. HIF-1 also plays a significant role in both the proliferation and migration of smooth muscle cells - two important features of atherosclerosis, while the formation of foam cells (lipid-laden macrophages) is also a critical step in atherosclerosis and mediated by HIF-1 through various mechanisms such as dysfunctional efflux pathways in macrophages. Overall, HIF-1 exerts its effect on the pathogenesis of atherosclerosis via a variety of molecular and cellular events in the process. In this review article, we examine the effects HIF-1 on vascular cells and macrophages in the development of atherosclerosis, highlighting the environmental cues and signalling pathways that control HIF-1 expression/activation within the vasculature. We will highlight the potential of HIF-1 as a therapeutic target on the disease development.

An Osteoconductive Antibiotic Bone Eluting Putty with a Custom Polymer Matrix.

With the rising tide of antibiotic resistant bacteria, extending the longevity of the current antibiotic arsenal is becoming a necessity. Developing local, controlled release antibiotic strategies, particularly for difficult to penetrate tissues such as bone, may prove to be a better alternative. Previous efforts to develop an osteoconductive local antibiotic release device for bone were created as solid molded composites; however, intimate contact with host bone was found to be critical to support host bone regrowth; thus, an osteocondconductive antibiotic releasing bone void filling putty was developed. Furthermore, a controlled releasing polymer matrix was refined using pendant-functionalized diols to provide tailorable pharmacokinetics. In vitro pharmacokinetic and bioactivity profiles were compared for a putty formulation with an analogous composition as its molded counterpart as well as four new pendant-functionalized polymers. A best-fit analysis of polymer composition in either small cylindrical disks or larger spheres revealed that the new pendant-functionalized polymers appear to release vancomycin via both diffusion and erosion regardless of the geometry of the putty. In silico simulations, a valuable technique for diffusion mediated controlled release models, will be used to confirm and optimize this property.