A deep learning approach to identify and segment alpha‑smooth muscle actin stress fiber positive cells

Cardiac fibrosis is a pathological process characterized by excessive tissue deposition, matrix remodeling, and tissue stiffening, which eventually leads to organ failure. On a cellular level, the development of fibrosis is associated with the activation of cardiac fibroblasts into myofibroblasts, a highly contractile and secretory phenotype. Myofibroblasts are commonly identified in vitro by the de novo assembly of alpha‑smooth muscle actin stress fibers; however, there are few methods to automate stress fiber identification, which can lead to subjectivity and tedium in the process. To address this limitation, we present a computer vision model to classify and segment cells containing alpha‑smooth muscle actin stress fibers into 2 classes (α‑SMA SF + and α‑SMA SF‑), with a high degree of accuracy (cell accuracy: 77%, F1 score 0.79). The model combines standard image processing methods with deep learning techniques to achieve semantic segmentation of the different cell phenotypes. We apply this model to cardiac fibroblasts cultured on hyaluronic acid‑based hydrogels of various moduli to induce alpha‑smooth muscle actin stress fiber formation. The model successfully predicts the same trends in stress fiber identification as obtained with a manual analysis. Taken together, this work demonstrates a process to automate stress fiber identification in in vitro fibrotic models, thereby increasing reproducibility in fibroblast phenotypic characterization.This research was supported by the Burroughs Wellcome Fund (CASI-1015895, A.M.R) and by the National Sci- ence Foundation (NSF MRSEC DMR-1720595 A.M.R). The authors acknowledge the use of shared research facili- ties supported in part by the Texas Materials Institute, the Center for Dynamics and Control of Materials: an NSF MRSEC (DMR-1720595), and the NSF National Nanotechnology Coordinated Infrastructure (ECCS-1542159). We also acknowledge the use of shared facilities in the UT Proteomics Facility (CPRIT RP110782). Finally, we also acknowledge the Texas Advanced Computing Center for their high performance computing resources.Center for Dynamics and Control of Material

5-HT 3 receptors mediate the time-dependent vagal afferent modulation of nociception during chronic food allergen-sensitized visceral hyperalgesia in rats

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/72949/1/j.1365-2982.2009.01335.x.pd

Genetic Control of Radical Crosslinking in a Semi-Synthetic Hydrogel

Enhancing materials with the qualities of living systems, including sensing, computation, and adaptation, is an important challenge in designing next-generation technologies. Living materials address this challenge by incorporating live cells as actuating components that control material function. For abiotic materials, this requires new methods that couple genetic and metabolic processes to material properties. Toward this goal, we demonstrate that extracellular electron transfer (EET) from Shewanella oneidensis can be leveraged to control radical cross-linking of a methacrylate-functionalized hyaluronic acid hydrogel. Cross-linking rates and hydrogel mechanics, specifically storage modulus, were dependent on various chemical and biological factors, including S. oneidensis genotype. Bacteria remained viable and metabolically active in the networks for a least 1 week, while cell tracking revealed that EET genes also encode control over hydrogel microstructure. Moreover, construction of an inducible gene circuit allowed transcriptional control of storage modulus and cross-linking rate via the tailored expression of a key electron transfer protein, MtrC. Finally, we quantitatively modeled hydrogel stiffness as a function of steady-state mtrC expression and generalized this result by demonstrating the strong relationship between relative gene expression and material properties. This general mechanism for radical cross-linking provides a foundation for programming the form and function of synthetic materials through genetic control over extracellular electron transfer.Center for Dynamics and Control of Material

Increased 5-HT3-mediated signalling in pelvic afferent neurons from mice deficient in P2X2 and/or P2X3 receptor subunits

Extracellular ATP and 5-hydroxytryptamine (5-HT) are both involved in visceral sensory pathways by interacting with P2X and 5-HT3 receptors, respectively. We have investigated the changes in P2X and 5-HT3-mediated signalling in pelvic afferent neurons in mice deficient in P2X2 and/or P2X3 subunits by whole-cell recording of L6–S2 dorsal root ganglion (DRG) neurons and by multi-unit recording of pelvic afferents of the colorectum. In wildtype DRG neurons, ATP evoked transient, sustained or mixed (biphasic) inward currents. Transient currents were absent in P2X3−/− neurons, whereas sustained currents were absent in P2X2−/− DRG neurons. Neither transient nor sustained currents were observed following application of ATP or α,β-methylene ATP (α,β-meATP) in P2X2/P2X3Dbl−/− DRG neurons. 5-HT was found to induce a fast inward current in 63% of DRG neurons from wildtype mice, which was blocked by tropisetron, a 5-HT3 receptor antagonist. The percentage of DRG neurons responding to 5-HT was significantly increased in P2X 2−/−, P2X3−/− and P2X2/P2X3Dbl−/− mice, and the amplitude of 5-HT response was significantly increased in P2X2/P2X3Dbl−/− mice. The pelvic afferent response to colorectal distension was attenuated in P2X2/P2X3Dbl−/− mice, but the response to serosal application of 5-HT was enhanced. Furthermore, tropisetron resulted in a greater reduction in pelvic afferent responses to colorectal distension in the P2X2/P2X3Dbl−/− preparations. These data suggest that P2X receptors containing the P2X2 and/or P2X3 subunits mediate purinergic activation of colorectal afferents and that 5-HT signalling in pelvic afferent neurons is up-regulated in mice lacking P2X2 or P2X3 receptor genes. This effect is more pronounced when both subunits are absent

Serotonin and cholecystokinin synergistically stimulate rat vagal primary afferent neurones

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/65669/1/jphysiol.2004.064816.pd

Mechanical Induction of PGE2 in Osteocytes Blocks Glucocorticoid-Induced Apoptosis Through Both the β-Catenin and PKA Pathways

Glucocorticoids are known to induce osteocyte apoptosis, whereas mechanical loading has been shown to sustain osteocyte viability. Here we show that mechanical loading in the form of fluid-flow shear stress blocks dexamethasone-induced apoptosis of osteocyte-like cells (MLO-Y4). Prostaglandin E2 (PGE2), a rapidly induced signaling molecule produced by osteocytes, was shown to be protective against dexamethasone-induced apoptosis, whereas indomethacin reversed the antiapoptotic effects of shear stress. This protective effect of shear stress was mediated through EP2 and EP4 receptors, leading to activation of the cAMP/protein kinase A signaling pathway. Activation of phosphatidylinositol 3-kinase, an inhibitor of glycogen synthesis kinase 3, also occurred, leading to the nuclear translocation of β-catenin, an important signal transducer of the Wnt signaling pathway. Both shear stress and prostaglandin increased the phosphorylation of glycogen synthesis kinase 3 α/β. Lithium chloride, an activator of the Wnt pathway, also was protective against glucocorticoid-induced apoptosis. Whereas it is known that mechanical loading increases cyclooxygenase-2 and EP2 receptor expression and prostaglandin production, dexamethasone was shown to inhibit expression of these components of the prostaglandin pathway and to reduce β-catenin protein expression. β-catenin siRNA knockdown experiments abrogated the protective effects of PGE2, confirming the central role of β-catenin in mediating the protection against dexamethasone-induced cell death. Our data support a central role for PGE2 acting through the cAMP/PKA and β-catenin signaling pathways in the protection of osteocyte apoptosis by fluid-flow shear stress. © 2010 American Society for Bone and Mineral Research

Microfluidic Enhancement of Intramedullary Pressure Increases Interstitial Fluid Flow and Inhibits Bone Loss in Hindlimb Suspended Mice

Interstitial fluid flow (IFF) has been widely hypothesized to mediate skeletal adaptation to mechanical loading. Although a large body of in vitro evidence has demonstrated that fluid flow stimulates osteogenic and antiresorptive responses in bone cells, there is much less in vivo evidence that IFF mediates loading-induced skeletal adaptation. This is due in large part to the challenges associated with decoupling IFF from matrix strain. In this study we describe a novel microfluidic system for generating dynamic intramedullary pressure (ImP) and IFF within the femurs of alert mice. By quantifying fluorescence recovery after photobleaching (FRAP) within individual lacunae, we show that microfluidic generation of dynamic ImP significantly increases IFF within the lacunocanalicular system. In addition, we demonstrate that dynamic pressure loading of the intramedullary compartment for 3 minutes per day significantly eliminates losses in trabecular and cortical bone mineral density in hindlimb suspended mice, enhances trabecular and cortical structural integrity, and increases endosteal bone formation rate. Unlike previously developed modalities for enhancing IFF in vivo, this is the first model that allows direct and dynamic modulation of ImP and skeletal IFF within mice. Given the large number of genetic tools for manipulating the mouse genome, this model is expected to serve as a powerful investigative tool in elucidating the role of IFF in skeletal adaptation to mechanical loading and molecular mechanisms mediating this process. © 2010 American Society for Bone and Mineral Research

Endocannabinoids-related compounds in gastrointestinal diseases

The endocannabinoid system (ECS) is an endogenous signalling pathway involved in the control of several gastrointestinal (GI) functions at both peripheral and central levels. In recent years, it has become apparent that the ECS is pivotal in the regulation of GI motility, secretion and sensitivity, but endocannabinoids (ECs) are also involved in the regulation of intestinal inflammation and mucosal barrier permeability, suggesting their role in the pathophysiology of both functional and organic GI disorders. Genetic studies in patients with irritable bowel syndrome (IBS) or inflammatory bowel disease have indeed shown significant associations with polymorphisms or mutation in genes encoding for cannabinoid receptor or enzyme responsible for their catabolism, respectively. Furthermore, ongoing clinical trials are testing EC agonists/antagonists in the achievement of symptomatic relief from a number of GI symptoms. Despite this evidence, there is a lack of supportive RCTs and relevant data in human beings, and hence, the possible therapeutic application of these compounds is raising ethical, political and economic concerns. More recently, the identification of several EC-like compounds able to modulate ECS function without the typical central side effects of cannabino-mimetics has paved the way for emerging peripherally acting drugs. This review summarizes the possible mechanisms linking the ECS to GI disorders and describes the most recent advances in the manipulation of the ECS in the treatment of GI diseases

Antisense-Mediated Knockdown of NaV1.8, but Not NaV1.9, Generates Inhibitory Effects on Complete Freund's Adjuvant-Induced Inflammatory Pain in Rat

Tetrodotoxin-resistant (TTX-R) sodium channels NaV1.8 and NaV1.9 in sensory neurons were known as key pain modulators. Comparing with the widely reported NaV1.8, roles of NaV1.9 on inflammatory pain are poorly studied by antisense-induced specific gene knockdown. Here, we used molecular, electrophysiological and behavioral methods to examine the effects of antisense oligodeoxynucleotide (AS ODN) targeting NaV1.8 and NaV1.9 on inflammatory pain. Following complete Freund's adjuvant (CFA) inflammation treatment, NaV1.8 and NaV1.9 in rat dorsal root ganglion (DRG) up-regulated mRNA and protein expressions and increased sodium current densities. Immunohistochemical data demonstrated that NaV1.8 mainly localized in medium and small-sized DRG neurons, whereas NaV1.9 only expressed in small-sized DRG neurons. Intrathecal (i.t.) delivery of AS ODN was used to down-regulate NaV1.8 or NaV1.9 expressions confirmed by immunohistochemistry and western blot. Unexpectedly, behavioral tests showed that only NaV1.8 AS ODN, but not NaV1.9 AS ODN could reverse CFA-induced heat and mechanical hypersensitivity. Our data indicated that TTX-R sodium channels NaV1.8 and NaV1.9 in primary sensory neurons played distinct roles in CFA-induced inflammatory pain and suggested that antisense oligodeoxynucleotide-mediated blocking of key pain modulator might point toward a potential treatment strategy against certain types of inflammatory pain

Effect of rehabilitation exercise durations on the dynamic bone repair process by coupling polymer scaffold degradation and bone formation

Implantation of biodegradable scaffold is considered as a promising method to treat bone disorders, but knowledge of the dynamic bone repair process is extremely limited. In this study, based on the representative volume cell of a periodic scaffold, the influence of rehabilitation exercise duration per day on the bone repair was investigated by a computational framework. The framework coupled scaffold degradation and bone remodeling. The scaffold degradation was described by a function of stochastic hydrolysis independent of mechanical stimulation, and the bone formation was remodeled by a function of the mechanical stimulation, i.e., strain energy density. Then, numerical simulations were performed to study the dynamic bone repair process. The results showed that the scaffold degradation and the bone formation in the process were competitive. An optimal exercise duration per day emerged. All exercise durations promoted the bone maturation with a final Young's modulus of 1.9 ± 0.3 GPa. The present study connects clinical rehabilitation and fundamental research, and is helpful to understand the bone repair process and further design bone scaffold for bone tissue engineering