ABSTRACT
PURPOSE: Utilising non-invasive imaging parameters to assess human oocyte fertilisation, development and implantation; and their influence on transcriptomic profiles. METHODS: A ranking tool was designed using imaging data from 957 metaphase II stage oocytes retrieved from 102 patients undergoing ART. Hoffman modulation contrast microscopy was conducted with an Olympus IX53 microscope. Images were acquired prior to ICSI and processed using ImageJ for optical density and grey-level co-occurrence matrices texture analysis. Single-cell RNA sequencing of twenty-three mature oocytes classified according to their competence was performed. RESULT(S): Overall fertilisation, blastulation and implantation rates were 73.0%, 62.6% and 50.8%, respectively. Three different algorithms were produced using binary logistic regression methods based on "optimal" quartiles, resulting in an accuracy of prediction of 76.6%, 67% and 80.7% for fertilisation, blastulation and implantation. Optical density, gradient, inverse difference moment (homogeneity) and entropy (structural complexity) were the parameters with highest predictive properties. The ranking tool showed high sensitivity (68.9-90.8%) but with limited specificity (26.5-62.5%) for outcome prediction. Furthermore, five differentially expressed genes were identified when comparing "good" versus "poor" competent oocytes. CONCLUSION(S): Imaging properties can be used as a tool to assess differences in the ooplasm and predict laboratory and clinical outcomes. Transcriptomic analysis suggested that oocytes with lower competence may have compromised cell cycle either by non-reparable DNA damage or insufficient ooplasmic maturation. Further development of algorithms based on image parameters is encouraged, with an increased balanced cohort and validated prospectively in multicentric studies.
Subject(s)
Oocytes , Transcriptome , Humans , Transcriptome/genetics , Oogenesis/genetics , Embryo Implantation , Gene Expression ProfilingABSTRACT
OBJECTIVE: The effects of mechanical stimulation in preterm amniotic membrane (AM) defects were explored. METHODS: Preterm AM was collected from women undergoing planned preterm caesarean section (CS) due to fetal growth restriction or emergency CS after spontaneous preterm prelabour rupture of the membranes (sPPROM). AM explants near the cervix or placenta were subjected to trauma and/or mechanical stimulation with the Cx43 antisense. Markers for nuclear morphology (DAPI), myofibroblasts (αSMA), migration (Cx43), inflammation (PGE2 ) and repair (collagen, elastin and transforming growth factor ß [TGFß1 ]) were examined by confocal microscopy, second harmonic generation, qPCR and biochemical assays. RESULTS: In preterm AM defects, myofibroblast nuclei were highly deformed and contractile and expressed αSMA and Cx43. Mechanical stimulation increased collagen fibre polarisation and the effects on matrix markers were dependent on tissue region, disease state, gestational age and the number of fetuses. PGE2 levels were broadly similar but reduced after co-treatment with Cx43 antisense in late sPPROM AM defects. TGFß1 and Cx43 gene expression were significantly increased after trauma and mechanical stimulation but this response dependent on gestational age. CONCLUSION: Mechanical stimulation affects Cx43 signalling and cell/collagen mechanics in preterm AM defects. Establishing how Cx43 regulates mechanosignalling could be an approach to repair tissue integrity after trauma.
Subject(s)
Amnion , Fetal Membranes, Premature Rupture , Pregnancy , Infant, Newborn , Humans , Female , Connexin 43 , Cesarean Section , Mechanotransduction, CellularABSTRACT
Endocytosis, an important macromolecule uptake process in cells, is known to be dysregulated in cancer. Clathrin and caveolin-1 proteins play a major role in receptor-mediated endocytosis. We have used a quantitative, unbiased and semi-automated method to measure in situ protein expression of clathrin and caveolin-1 in cancerous and paired normal (cancer adjacent, non-cancerous) human prostate tissue. There was a significant (p < 0.0001) increase in the expression of clathrin in prostate cancer samples (N = 29, n = 91) compared to normal tissue (N = 29, n = 67) (N = number of patients, n = number of cores in tissue arrays). Conversely, there was a significant (p < 0.0001) decrease in expression of caveolin-1 in prostate cancer tissue compared to normal prostate tissue. The opposite change in expression of the two proteins was highly correlated to increasing cancer aggressiveness. There was also a concurrent increase in the expression of epidermal growth factor receptor (EGFR), a key receptor in carcinogenesis, with clathrin in prostate cancer tissue, indicating recycling of EGFR through clathrin-mediated endocytosis (CME). These results indicate that in prostate cancer, caveolin-1-mediated endocytosis (CavME) may be acting as a brake and increase in CME may facilitate tumorigenicity and aggressiveness of prostate cancer through recycling of EGFR. Changes in the expression of these proteins can also potentially be used as a biomarker for prostate cancer to aid in diagnosis and prognosis and clinical decision-making.
Subject(s)
Caveolin 1 , Prostatic Neoplasms , Male , Humans , Caveolin 1/metabolism , Clathrin/metabolism , Prostate , ErbB Receptors/metabolism , EndocytosisABSTRACT
The wound healing capacity of the fetal membranes after spontaneous or iatrogenic membrane rupture is unclear. We examined the healing mechanisms in amniotic membrane (AM) defects after trauma. Traumatised human AM defects were cultured for 4 days. Markers for nuclear (DAPI), cell type (vimentin, αSMA) and healing (Cx43, TGFß1, collagen) were examined by immunofluorescence (IMF) confocal microscopy, Second Harmonic Generation (SHG) imaging and RT-qPCR. After trauma, AMCs and myofibroblasts migrated to the AM wound edge. Within four days, αSMA expressing myofibroblasts showed abundant Cx43 localized in the cytoplasmic processes. The highly contractile spindle-shaped myofibroblasts were present in the defect site and released collagen. In contrast, AMCs expressed vimentin and formed Cx43 plaques between cells found in the outer edges of the wound. Whilst AMCs were absent in the defect site, αSMA expressing myofibroblasts continued to elongate and polarize the collagen fibres. Both TGFß1 and Cx43 gene expression were significantly increased after trauma. Cx43 has differential effects on AM cell populations that increase cellularity, contraction and potentially migration to the wound edge resulting in collagen polarisation in the AM defect site. Establishing how Cx43 regulates AM cell function could be an approach to repair defects in the membranes after trauma.
Subject(s)
Amnion/metabolism , Collagen/metabolism , Connexin 43/metabolism , Myofibroblasts/metabolism , Extraembryonic Membranes/metabolism , Female , Fetal Membranes, Premature Rupture/metabolism , Humans , Pregnancy , Vimentin/metabolism , Wound Healing/physiologyABSTRACT
Coenzyme A (CoA) is an essential cofactor present in all living cells. Under physiological conditions, CoA mainly functions to generate metabolically active CoA thioesters, which are indispensable for cellular metabolism, the regulation of gene expression, and the biosynthesis of neurotransmitters. When cells are exposed to oxidative or metabolic stress, CoA acts as an important cellular antioxidant that protects protein thiols from overoxidation, and this function is mediated by protein CoAlation. CoA and its derivatives are strictly maintained at levels controlled by nutrients, hormones, metabolites, and cellular stresses. Dysregulation of their biosynthesis and homeostasis has deleterious consequences and has been noted in a range of pathological conditions, including cancer, diabetes, Reye's syndrome, cardiac hypertrophy, and neurodegeneration. The biochemistry of CoA biosynthesis, which involves five enzymatic steps, has been extensively studied. However, the existence of a CoA biosynthetic complex and the mode of its regulation in mammalian cells are unknown. In this study, we report the assembly of all five enzymes that drive CoA biosynthesis, in HEK293/Pank1ß and A549 cells, using the in situ proximity ligation assay. Furthermore, we show that the association of CoA biosynthetic enzymes is strongly upregulated in response to serum starvation and oxidative stress, whereas insulin and growth factor signaling downregulate their assembly.
Subject(s)
Biosynthetic Pathways/genetics , Coenzyme A/metabolism , Gene Expression Regulation , Oxidative Stress , A549 Cells , Coenzyme A/biosynthesis , HEK293 Cells , Humans , Insulin/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Signal TransductionABSTRACT
INTRODUCTION: Posterior urethral valves (PUV) is the most common cause of congenital bladder outflow obstruction with persistent lower urinary tract and renal morbidities. There is a spectrum of functional bladder disorders ranging from hypertonia to bladder underactivity, but the aetiology of these clinical conditions remains unclear. AIMS AND OBJECTIVES: We tested the hypothesis that replacement of detrusor muscle with non-muscle cells and excessive deposition of connective tissue is an important factor in bladder dysfunction with PUV. We used isolated detrusor samples from children with PUV and undergoing primary or secondary procedures in comparison to age-matched data from children with functionally normal bladders. In vitro contractile properties, as well as passive stiffness, were measured and matched to histological assessment of muscle and connective tissue. We examined if a major pathway for fibrosis was altered in PUV tissue samples. METHODS: Isometric contractions were measured in vitro in response to either stimulation of motor nerves to detrusor or exposure to cholinergic and purinergic receptor agonists. Passive mechanical stiffness was measured by rapid stretching of the tissue and recording changes to muscle tension. Histology measured the relative amounts of detrusor muscle and connective tissue. Multiplex quantitative immunofluorescence labelling using five epitope markers was designed to determine cellular pathways, in particular the Wnt-signalling pathway, responsible for any changes to excessive deposition of connective tissue. RESULTS AND DISCUSSION: PUV tissue showed equally reduced contractile function to efferent nerve stimulation or exposure to contractile agonists. Passive muscle stiffness was increased in PUV tissue samples. The smooth muscle:connective tissue ratio was also diminished and mirrored the reduction of contractile function and the increase of passive stiffness. Immunofluorescence labelling showed in PUV samples increased expression of the matrix metalloproteinase, MMP-7; as well as cyclin-D1 expression suggesting cellular remodelling. However, elements of a fibrosis pathway associated with Wnt-signalling were either reduced (ß-catenin) or unchanged (c-Myc). The accumulation of extracellular matrix, containing collagen, will contribute to the reduced contractile performance of the bladder wall. It will also increase tissue stiffness that in vivo would lead to reduced filling compliance. CONCLUSIONS: Replacement of smooth muscle with fibrosis is a major contributory factor in contractile dysfunction in the hypertonic PUV bladder. This suggests that a potential strategy to restore normal contractile and filling properties is development of the effective use of antifibrotic agents.
Subject(s)
Muscle, Smooth , Urinary Bladder Diseases , Child , Fibrosis , Humans , Muscle ContractionABSTRACT
OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.
Subject(s)
Antisense Elements (Genetics)/administration & dosage , Connexin 43/antagonists & inhibitors , Extraembryonic Membranes/injuries , Peptides/administration & dosage , Wound Healing/drug effects , Adult , Amniotic Fluid/chemistry , Coculture Techniques , Drug Evaluation, Preclinical , Extraembryonic Membranes/ultrastructure , Female , Fetoscopy/adverse effects , Humans , Peptides/chemistry , PregnancyABSTRACT
HYPOTHESIS: Developing oral formulations to enable effective release of poorly water-soluble drugs like progesterone is a major challenge in pharmaceutics. Coaxial electrospray can generate drug-loaded nanoparticles of strategic compositions and configurations to enhance physiological dissolution and bioavailability of poorly water-soluble drug progesterone. EXPERIMENTS: Six formulations comprising nanoparticles encapsulating progesterone in different poly(lactide-co-glycolide) (PLGA) matrix configurations and compositions were fabricated and characterized in terms of morphology, molecular crystallinity, drug encapsulation efficiency and release behavior. FINDINGS: A protocol of fabrication conditions to achieve 100% drug encapsulation efficiency in nanoparticles was developed. Scanning electron microscopy shows smooth and spherical morphology of 472.1±54.8 to 588.0±92.1 nm in diameter. Multiphoton Airyscan super-resolution confocal microscopy revealed core-shell nanoparticle configuration. Fourier transform infrared spectroscopy confirmed presence of PLGA and progesterone in all formulations. Diffractometry indicated amorphous state of the encapsulated drug. UV-vis spectroscopy showed drug release increased with hydrophilic copolymer glycolide ratio while core-shell formulations with progesterone co-dissolved in PLGA core exhibited enhanced release over five hours at 79.9±1.4% and 70.7±3.5% for LA:GA 50:50 and 75:25 in comparison with pure progesterone without polymer matrix in the core at 67.0±1.7% and 57.5±2.8%, respectively. Computational modeling showed good agreement with the experimental drug release behavior in vitro.
Subject(s)
Nanoparticles/chemistry , Polylactic Acid-Polyglycolic Acid Copolymer/chemistry , Progesterone/administration & dosage , Progesterone/pharmacokinetics , Biological Availability , Drug Carriers/chemistry , Drug Carriers/pharmacokinetics , Drug Compounding/methods , Drug Delivery Systems , Drug Liberation , Microscopy, Electron, Scanning , Nanoparticles/administration & dosage , Particle Size , Solubility , Spectrophotometry, Ultraviolet , Spectroscopy, Fourier Transform Infrared , Water/chemistry , X-Ray DiffractionABSTRACT
Equine penile squamous cell carcinoma (EpSCC) is a relatively common cutaneous neoplasm with a poor prognosis. In this study, we aimed to determine the protein expression and colocalisation of FRA1, c-Myc, Cyclin D1, and MMP7 in normal (NT), tumour (T), hyperplastic epidermis and/or squamous papilloma (Hyp/Pap), poorly-differentiated (PDSCC), or well-differentiated (WDSCC) EpSCC using a tissue array approach. Further objectives were to correlate protein expression to (i) levels of inflammation, using a convolutional neural network (ii) equine papillomavirus 2 (EcPV2) infection, detected using PCR amplification. We found an increase in expression of FRA1 in EpSCC compared to NT samples. c-Myc expression was higher in Hyp/Pap and WDSCC but not PDSCC whereas MMP7 was reduced in WDSCC compared with NT. There was a significant increase in the global intersection coefficient (GIC) of FRA1 with MMP7, c-Myc, and Cyclin D1 in EpSCC. Conversely, GIC for MMP7 with c-Myc was reduced in EpSCC tissue. Inflammation was positively associated with EcPV2 infection in both NT and EpSCC but not Hyp/Pap. Changes in protein expression could be correlated with EcPV2 for Cyclin D1 and c-Myc. Our results evaluate novel biomarkers of EpSCC and a putative correlation between the expression of biomarkers, EcPV2 infection and inflammation.
Subject(s)
Biomarkers, Tumor/genetics , Carcinoma, Squamous Cell/genetics , Gene Expression Regulation, Neoplastic , Papillomaviridae/isolation & purification , Papillomavirus Infections/genetics , Penile Neoplasms/genetics , Animals , Biomarkers, Tumor/metabolism , Carcinoma, Squamous Cell/diagnosis , Carcinoma, Squamous Cell/virology , Cyclin D1/genetics , Cyclin D1/metabolism , Horses , Male , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 7/metabolism , Papillomaviridae/physiology , Papillomavirus Infections/diagnosis , Papillomavirus Infections/virology , Penile Neoplasms/diagnosis , Penile Neoplasms/virology , Protein Binding , Proto-Oncogene Proteins c-fos/genetics , Proto-Oncogene Proteins c-fos/metabolism , Proto-Oncogene Proteins c-myc/genetics , Proto-Oncogene Proteins c-myc/metabolism , ROC Curve , Tissue Array Analysis/methodsABSTRACT
KEY POINTS: Wnt ligands belonging to both canonical and non-canonical Wnt pathways regulate membrane potential signifying a very early event in the signal transduction. Wnts activate K+ currents by elevating intracellular Ca2+ and trigger Ca2+ release from intracellular stores. Control of potential by Wnt ligands has significant implications for gene transcription and opens up a novel avenue to interfere with this critical pathway. ABSTRACT: The Wnt signalling network determines gene transcription with free intracellular Ca2+ ( Cai2+ ) and ß-catenin as major intracellular signal transducers. Despite its critical importance during development and disease, many of the basic mechanisms of Wnt signal activation remain unclear. Here we show by single cell recording and simultaneous Cai2+ imaging in mammalian prostate cancer cells that an early step in the signal cascade is direct action on the cell membrane potential. We show that Wnt ligands 5A, 9B and 10B rapidly hyperpolarized the cells by activating K+ current by Ca2+ release from intracellular stores. Medium-throughput multi-well recordings showed responses to Wnts at concentrations of 2 nm. We identify a putative target for early events as a TRPM channel. Wnts thus act as ligands for ion channel activation in mammalian cells and membrane potential is an early indicator of control of transcription.
Subject(s)
Calcium Signaling , Membrane Potentials , Wnt Signaling Pathway , Cell Membrane/drug effects , Cell Membrane/physiology , Humans , MCF-7 Cells , Potassium Channels/metabolism , TRPM Cation Channels/metabolism , Wnt Proteins/metabolism , Wnt Proteins/pharmacologyABSTRACT
Mechanical and inflammatory signals in the fetal membrane play an important role in extracellular matrix (ECM) remodelling in order to dictate the timing of birth. We developed a mechanical model that mimics repetitive stretching of the amniotic membrane (AM) isolated from regions over the placenta (PAM) or cervix (CAM) and examined the effect of cyclic tensile strain (CTS) on mediators involved in mechanotransduction (Cx43, AKT), tissue remodelling (GAGs, elastin, collagen) and inflammation (PGE2, MMPs). In CAM and PAM specimens, the application of CTS increased GAG synthesis, PGE2 release and MMP activity, with concomitant reduction in collagen and elastin content. Co-stimulation with CTS and pharmacological agents that inhibit either Cx43 or AKT, differentially influenced collagen, GAG and elastin in a tissue-dependent manner. SHG confocal imaging of collagen fibres revealed a reduction in SHG intensity after CTS, with regions of disorganisation dependent on tissue location. CTS increased Cx43 and AKT protein and gene expression and the response could be reversed with either CTS, the Cx43 antisense or AKT inhibitor. We demonstrate that targeting Cx43 and AKT prevents strain-induced ECM damage and promotes tissue remodelling mechanisms in the AM. We speculate that a combination of inflammatory and mechanical factors could perturb typical mechanotransduction processes mediated by Cx43 signalling. Cx43 could therefore be a potential therapeutic target to prevent inflammation and preterm premature rupture of the fetal membranes.
Subject(s)
Amnion/metabolism , Mechanotransduction, Cellular/physiology , Amnion/physiology , Cervix Uteri/metabolism , Collagen/metabolism , Connexin 43/genetics , Connexin 43/metabolism , Dinoprostone/metabolism , Elastin/metabolism , Female , Gene Expression Regulation, Developmental , Humans , Inflammation/metabolism , Matrix Metalloproteinases/metabolism , Placenta/metabolism , Pregnancy , Proto-Oncogene Proteins c-akt/genetics , Proto-Oncogene Proteins c-akt/metabolismABSTRACT
The availability of intracellular, stabilized ß-catenin, a transcription factor coactivator, is tightly regulated; ß-catenin is translocated into the nucleus in response to Wnt ligand binding to its cell membrane receptors. Here we show that Wnt signal activation in mammalian cells activates intracellular mobilization of connexin 43 (Cx43), which belongs to a gap junction protein family, a new target protein in response to extracellular Wnt signal activation. Transmission electron microscopy showed that the nuclear localization of Cx43 was increased by 8- to 10-fold in Wnt5A- and 9B-treated cells compared with controls; this Wnt-induced increase was negated in the cells where Cx43 and ß-catenin were knocked down using shRNA. There was a significant (P < 0.001) and concomitant depletion of the cell membrane and cytosolic signal of Cx43 in Wnt-treated cells with an increase in the nuclear signal for Cx43; this was more obvious in cells where ß-catenin was knocked down using shRNA. Conversely, Cx43 knockdown resulted in increased ß-catenin in the nucleus in the absence of Wnt activation. Coimmunoprecipitation of Cx43 and ß-catenin proteins with a casein kinase (CKIδ) antibody showed that Cx43 interacts with ß-catenin and may form part of the so-called destruction complex. Functionally, Wnt activation increased the rate of wound reepithelization in rat skin in vivo.
Subject(s)
Connexin 43/metabolism , Gap Junctions/metabolism , Wnt Signaling Pathway/physiology , Cell Nucleus/metabolism , Cells, Cultured , Gap Junctions/genetics , Humans , Male , Wnt Signaling Pathway/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
Class II antiarrhythmics or ß-blockers are antisympathetic nervous system agents that act by blocking ß-adrenoceptors. Despite their common clinical use, little is known about the effects of ß-blockers on free intracellular calcium (Ca2+ i), an important cytosolic second messenger and a key regulator of cell function. We investigated the role of four chemical analogs, commonly prescribed ß-blockers (atenolol, metoprolol, propranolol, and sotalol), on Ca2+ i release and whole-cell currents in mammalian cancer cells (PC3 prostate cancer and MCF7 breast cancer cell lines). We discovered that only propranolol activated free Ca2+ i release with distinct kinetics, whereas atenolol, metoprolol, and sotalol did not. The propranolol-induced Ca2+ i release was significantly inhibited by the chelation of extracellular calcium with ethylene glycol tetraacetic acid (EGTA) and by dantrolene, an inhibitor of the endoplasmic reticulum (ER) ryanodine receptor channels, and it was completely abolished by 2-aminoethoxydiphenyl borate, an inhibitor of the ER inositol-1,4,5-trisphosphate (IP3) receptor channels. Exhaustion of ER stores with 4-chloro-m-cresol, a ryanodine receptor activator, or thapsigargin, a sarco/ER Ca2+ ATPase inhibitor, precluded the propranolol-induced Ca2+ i release. Finally, preincubation of cells with sotalol or timolol, nonselective blockers of ß-adrenoceptors, also reduced the Ca2+ i release activated by propranolol. Our results show that different ß-blockers have differential effects on whole-cell currents and free Ca2+ i release and that propranolol activates store-operated Ca2+ i release via a mechanism that involves calcium-induced calcium release and putative downstream transducers such as IP3 The differential action of class II antiarrhythmics on Ca2+ i release may have implications on the pharmacology of these drugs.
Subject(s)
Anti-Arrhythmia Agents/pharmacology , Calcium/metabolism , Intracellular Space/drug effects , Intracellular Space/metabolism , Adrenergic beta-Antagonists/pharmacology , Humans , Kinetics , MCF-7 Cells , PC-3 Cells , Propranolol/pharmacology , Receptors, Adrenergic, beta/metabolismABSTRACT
Polymeric scaffolds comprising two size scales of microfibers and submicron fibers can better support three-dimensional (3D) cell growth in tissue engineering, making them an important class of healthcare material. However, a major manufacturing barrier hampers their translation into wider practical use: scalability. Traditional production of two-scale scaffolds by electrospinning is slow and costly. For day-to-day cell cultures, the scaffolds need to be affordable, made in high yield to drive down cost. Combining expertise from academia and industry from the United Kingdom and United States, this study uses a new series of high-yield, low-cost scaffolds made by shear spinning for tissue engineering. The scaffolds comprise interwoven submicron fibers and microfibers throughout as observed under scanning electron microscopy and demonstrate good capability to support cell culturing for tumor modeling. Three model human cancer cell lines (HEK293, A549 and MCF-7) with stable expression of GFP were cultured in the scaffolds and found to exhibit efficient cell attachment and sustained 3D growth and proliferation for 30 days. Cryosection and multiphoton fluorescence microscopy confirmed the formation of compact 3D cell clusters throughout the scaffolds. In addition, comparative growth curves of 2D and 3D cultures show significant cell-type-dependent differences. This work applies high-yield shear-spun scaffolds in mammalian tissue engineering and brings practical, affordable applications of multiscale scaffolds closer to reality. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2750, 2019.
Subject(s)
Shear Strength , Cell Proliferation , Cell Survival , Humans , Microscopy, Fluorescence , Tissue Engineering , Tissue Scaffolds , Tumor Cells, CulturedABSTRACT
Prostate cancer is the third highest cause of male mortality in the developed world, with the burden of the disease increasing dramatically with demographic change. There are significant limitations to the current diagnostic regimens and no established effective screening modality. To this end, research has discovered hundreds of potential 'biomarkers' that may one day be of use in screening, diagnosis or prognostication. However, the barriers to bringing biomarkers to clinical evaluation and eventually into clinical usage have yet to be realised. This is an operational challenge that requires some new thinking and development of paradigms to increase the efficiency of the laboratory process and add 'value' to the clinician. Value comes in various forms, whether it be a process that is seamlessly integrated into the hospital laboratory environment or one that can provide additional 'information' for the clinical pathologist in terms of risk profiling. We describe, herein, an efficient and tissue-conserving pipeline that uses Tissue Microarrays in a semi-automated process that could, one day, be integrated into the hospital laboratory domain, using seven putative prostate cancer biomarkers for illustration.
ABSTRACT
PURPOSE: Despite posterior vitreous detachment being a common ocular event affecting most individuals in an aging population, there is little consensus regarding its precise anatomic definition. We investigated the morphologic appearance and molecular composition of the posterior hyaloid membrane to determine whether the structure clinically observed enveloping the posterior vitreous surface after posterior vitreous detachment is a true basement membrane and to postulate its origin. Understanding the relationship between the vitreous (in both its attached and detached state) and the internal limiting membrane of the retina is essential to understanding the cause of rhegmatogenous retinal detachment and vitreoretinal interface disorders, as well as potential future prophylactic and treatment strategies. DESIGN: Clinicohistologic correlation study. PARTICIPANTS: Thirty-six human donor globes. METHODS: Vitreous bodies identified to have posterior vitreous detachment were examined with phase-contrast microscopy and confocal microscopy after immunohistochemically staining for collagen IV basement membrane markers, in addition to extracellular proteins that characterize the vitreoretinal junction (fibronectin, laminin) and vitreous gel (opticin) markers. The posterior retina similarly was stained to evaluate the internal limiting membrane. Findings were correlated to the clinical appearance of the posterior hyaloid membrane observed during slit-lamp biomicroscopy after posterior vitreous detachment and compared with previously published studies. MAIN OUTCOME MEASURES: Morphologic appearance and molecular composition of the posterior hyaloid membrane. RESULTS: Phase-contrast microscopy consistently identified a creased and distinct glassy membranous sheet enveloping the posterior vitreous surface, correlating closely with the posterior hyaloid membrane observed during slit-lamp biomicroscopy in patients with posterior vitreous detachment. Immunofluorescent confocal micrographs demonstrated the enveloping membranous structure identified on phase-contrast microscopy to show positive stain results for type IV collagen. Immunofluorescence of the residual intact internal limiting membrane on the retinal surface also showed positive stain results for type IV collagen. CONCLUSIONS: The results of this study provide immunohistochemical evidence that the posterior hyaloid membrane is a true basement membrane enveloping the posterior hyaloid surface. Because this membranous structure is observed only after posterior vitreous detachment, the results of this study indicate that it forms part of the internal limiting membrane when the vitreous is in its attached state.
Subject(s)
Basement Membrane/diagnostic imaging , Collagen/metabolism , Vitreous Body/pathology , Vitreous Detachment/diagnosis , Adult , Aged , Aged, 80 and over , Basement Membrane/chemistry , Female , Humans , Imaging, Three-Dimensional , Immunohistochemistry , Male , Microscopy, Acoustic , Microscopy, Confocal , Middle Aged , Prospective Studies , Vitrectomy , Vitreous Body/surgery , Vitreous Detachment/surgeryABSTRACT
Few quantifiable tissue biomarkers for the diagnosis and prognosis of prostate cancer exist. Using an unbiased, quantitative approach, this study evaluates the potential of three proteins of the 40S ribosomal protein complex as putative biomarkers of malignancy in prostate cancer. Prostate tissue arrays, constructed from 82 patient samples (245 tissue cores, stage pT3a or pT3b), were stained for antibodies against three ribosomal proteins, RPS19, RPS21 and RPS24. Semi-automated Ox-DAB signal quantification using ImageJ software revealed a significant change in expression of RPS19, RPS21 and RPS24 in malignant vs non-malignant tissue (p<0.0001). Receiver operating characteristics curves were calculated to evaluate the potential of each protein as a biomarker of malignancy in prostate cancer. Positive likelihood ratios for RPS19, RPS21 and RPS24 were calculated as 2.99, 4.21, and 2.56 respectively, indicating that the overexpression of the protein is correlated with the presence of disease. Triple-labelled, quantitative, immunofluorescence (with RPS19, RPS21 and RPS24) showed significant changes (p<0.01) in the global intersection coefficient, a measure of how often two fluorophore signals intersect, for RPS19 and RPS24 only. No change was observed in the co-localization of any other permutations of the three proteins. Our results show that RPS19, RPS21 or RPS24 are upregulated in malignant tissue and may serve as putative biomarkers for prostate cancer.
Subject(s)
Biomarkers, Tumor/genetics , Gene Expression Regulation, Neoplastic , Prostatic Neoplasms/diagnosis , Ribosomal Proteins/genetics , Aged , Biomarkers, Tumor/metabolism , Fluorescent Antibody Technique , Humans , Image Interpretation, Computer-Assisted , Male , Middle Aged , Neoplasm Staging , Prostate/metabolism , Prostate/pathology , Prostatic Neoplasms/genetics , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , ROC Curve , Retrospective Studies , Ribosomal Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Ribosomes/pathology , Tissue Array AnalysisABSTRACT
Chronic wounds are a growing problem worldwide with no effective therapeutic treatments available. Our objective was to understand the composition of the dermal tissue surrounding venous leg ulcers and diabetic foot ulcers (DFU). We used novel 2-photon imaging techniques alongside classical histology to examine biopsies from the edges of two common types of chronic wound, venous leg ulcers and DFU. Compared to normal intact skin, we found that collagen levels are significantly reduced throughout the dermis of venous leg ulcer biopsies and DFU, with a reduction in both fibril thickness and abundance. Both wound types showed a significant reduction in elastin in the upper dermis, but in DFU, the loss was throughout the dermis. Loss of extracellular matrix correlated with high levels of CD68- and CD18-positive leukocytes. 2-photon imaging of the extracellular matrix in the intact tissue surrounding a chronic wound with a hand-held device may provide a useful clinical indicator on the healing progression or deterioration of these wounds.
Subject(s)
Dermis/diagnostic imaging , Diabetic Foot/diagnostic imaging , Elasticity Imaging Techniques/methods , Extracellular Matrix/ultrastructure , Varicose Ulcer/diagnostic imaging , Wound Healing/physiology , Wounds and Injuries/diagnostic imaging , Aged , Aged, 80 and over , Chronic Disease , Humans , Male , Middle Aged , Wounds and Injuries/physiopathologyABSTRACT
OBJECTIVE: We developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects. METHOD: Term human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining. RESULTS: After trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture. CONCLUSION: Cx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.
Subject(s)
Connexin 43/analysis , Extraembryonic Membranes/injuries , Amnion/chemistry , Amnion/pathology , Cell Survival , Collagen/chemistry , Collagen/ultrastructure , Epithelial Cells/chemistry , Extraembryonic Membranes/pathology , Female , Fetal Membranes, Premature Rupture/pathology , Fluorescent Antibody Technique , Humans , Mesenchymal Stem Cells/chemistry , Microscopy, Confocal , Pregnancy , Wounds and Injuries/metabolismABSTRACT
Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.