Intermediate Filament Expression Research Articles

The expression of intermediate filaments in the abomasum of ruminants: A comparative study.

Intermediate filaments (IFs) are key molecular factors of the cell and have been reported to play an important role in maintaining the structural integrity and functionality of the abomasum. This study was designed to determine the regional distribution, cellular localization and expression of several IFs, including CK8, CK18, CK19, vimentin, desmin, peripherin and nestin, as well as the connective tissue component laminin, in the bovine, ovine and caprine abomasa. Immunohistochemical analyses demonstrated varying levels of expression of CK8, CK18, CK19, vimentin, desmin, nestin, peripherin and laminin in the bovine, ovine and caprine abomasa. CK8 immunoreactions were particularly evident in the luminal and glandular epithelia of the glands found in the abomasal cardia, fundus and pylorus in all three species. In the bovine abomasum, CK18 immunoreactions were stronger in the parietal cells, compared to the chief cells. In the abomasum of all three species, the smooth muscle as well as the smooth muscle cells of the vascular media in the cardiac, fundic and pyloric regions showed strong immunoreactivity. In all three species, the cardiac, fundic and pyloric regions of the abomasum showed strong peripherin and nestin immunoreactions in the luminal and glandular epithelial cells, stromal and smooth muscle cells, nervous plexuses and blood vessels. The expression patterns of IFs and laminin in the ruminant abomasum suggest that these proteins play a structural role in the cytoskeleton and are effective in maintaining abomasal tissue integrity and stability.

Expression of oestrogen and progesterone receptor and intermediate filament proteins in the oviduct of mares with endometrosis

Equine endometrosis is one of the major causes of equine infertility, in the course of which periglandular fibrosis signs occur in the endometrium. As an oviduct is a key place of early embryo development, we hypothesized that loss of contact between mucosal cells and perturbation in hormonal action may coexist in the uterus and oviducts. Thus, this study aimed to investigate the expression of the intermediate filaments (vimentin and desmin) as well as the estrogen receptor alpha (ERα) and progesterone receptor A (PRA) in the stroma and epithelia of endometrium and oviduct in healthy mares and these with endometrosis. Out of 56 culled-in slaughterhouse mare endometrium samples were annotated in two groups (i) healthy endometrium (n=26) and endometrium with signs of moderate and severe endometrosis (n=20). The expression of vimentin, desmin, ERα, and PRA was investigated immunohistochemically and compared between the above groups as well as between the phase of the estrous cycle. The expression of both vimentin and desmin was higher in the endometrial stroma of endometrosis group compared to healthy endometrium. The expression of ERα was higher in the healthy endometrial stroma and luminal epithelium during the follicular phase than luteal, however, no differences were noted in endometrium of mares with endometrosis. Moreover, PRA expression was significantly higher in the stoma oviduct during the follicular phase than luteal. It seems possible that mucosal cells through their altered interactions may support the occurrence of moderate and severe endometrosis. In addition, the oviduct appears to be less sensitive to hormonal changes than the uterus. The changed expression of intermediate filaments and steroid hormones may be some of the leading factors which can contribute to the oviduct functional status in endometrosis.

Retinal gliosis and phenotypic diversity of intermediate filament induction and remodeling upon acoustic blast overpressure (ABO) exposure to the rat eye

Traumatic brain injury (TBI) caused by acoustic blast overpressure (ABO) is frequently associated with chronic visual deficits in military personnel and civilians. In this study, we characterized retinal gliotic response in adult male rats following a single ABO exposure directed to one side of the head. Expression of gliosis markers and intermediate filaments was assessed at 48 h and 1 wk post-ABO exposure, in comparison to age-matched non-exposed control retina. In response to a single ABO exposure, type III IF, glial fibrillary acidic protein (GFAP) was variably induced in a subpopulation of retinal Müller glia in ipsilateral eyes. ABO-exposed eyes exhibited radial Müller glial GFAP filament extension through the inner plexiform layer (IPL) and the inner nuclear layer (INL) through the retina in both the nasal quadrant and juxta-optic nerve head (jONH) eye regions at 1 wk post-ABO. We observed an ∼6-fold increase (p ≤ 0.05) in radial glial GFAP immunolabeling in the IPL in both eye regions, in comparison to regionally matched controls. Similarly, GFAP extension through the INL into the outer retina was elevated ∼3-fold, p ≤ 0.05 in the nasal retina, but exhibited wider variability in the jONH retina. In contrast, constitutive type III IF vimentin exhibited greater remodeling in retinal Müller glia through the jONH retina compared to the nasal retina in response to ABO. We observed areas of lateral vimentin remodeling through the Müller glial end-feet, and greater mid-outer retinal radial vimentin IF extension in a subpopulation of glia at 1 wk post-ABO. We also observed a significant increase in total retinal levels of the type III IF desmin in ABO-exposed retina vs. controls (∼1.6-fold, p ≤ 0.01). In addition, ABO-exposure elicited varied glial induction of developmentally regulated type VI family IFs (nestin and synemin) in subpopulations of Müller cells at 48 h and 1 wk post-ABO. We demonstrate that multiple glial phenotypes emerge in the rat retina following a single ABO exposure, rather than a global homogeneous retinal glial response, involving less well characterized IF protein forms which warrant further investigation in the context of ABO-induced retinal gliosis.

Dynamic changes in whole genome DNA methylation, chromatin and gene expression during mouse lens differentiation

BackgroundCellular differentiation is marked by temporally and spatially coordinated gene expression regulated at multiple levels. DNA methylation represents a universal mechanism to control chromatin organization and its accessibility. Cytosine methylation of CpG dinucleotides regulates binding of methylation-sensitive DNA-binding transcription factors within regulatory regions of transcription, including promoters and distal enhancers. Ocular lens differentiation represents an advantageous model system to examine these processes as lens comprises only two cell types, the proliferating lens epithelium and postmitotic lens fiber cells all originating from the epithelium.ResultsUsing whole genome bisulfite sequencing (WGBS) and microdissected lenses, we investigated dynamics of DNA methylation and chromatin changes during mouse lens fiber and epithelium differentiation between embryos (E14.5) and newborns (P0.5). Histone H3.3 variant chromatin landscapes were also generated for both P0.5 lens epithelium and fibers by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Tissue-specific features of DNA methylation patterns are demonstrated via comparative studies with embryonic stem (ES) cells and neural progenitor cells (NPCs) at Nanog, Pou5f1, Sox2, Pax6 and Six3 loci. Comparisons with ATAC-seq and RNA-seq data demonstrate that reduced methylation is associated with increased expression of fiber cell abundant genes, including crystallins, intermediate filament (Bfsp1 and Bfsp2) and gap junction proteins (Gja3 and Gja8), marked by high levels of histone H3.3 within their transcribed regions. Interestingly, Pax6-binding sites exhibited predominantly DNA hypomethylation in lens chromatin. In vitro binding of Pax6 proteins showed Pax6’s ability to interact with sites containing one or two methylated CpG dinucleotides.ConclusionsOur study has generated the first data on methylation changes between two different stages of mammalian lens development and linked these data with chromatin accessibility maps, presence of histone H3.3 and gene expression. Reduced DNA methylation correlates with expression of important genes involved in lens morphogenesis and lens fiber cell differentiation.

Expression of Intermediate Filaments in the Developing Testis and Testicular Germ Cell Cancer.

Cytokeratin and desmin expression have been associated with Sertoli cell maturity and the development of testicular germ cell cancer (TGCC). Thus, the present study aimed to characterize the expression of these intermediate filaments in normal testis development and TGCC. Cytokeratin and desmin were determined by immunohistochemistry and immunofluorescence in human fetal, and adult testis and tissue from patients with pre-invasive germ cell neoplasia in-situ (GCNIS) or invasive TGCC. Desmin was expressed in Sertoli cells of the human fetal testis, and the proportion of desmin expressing Sertoli cells was significantly reduced in the second trimester, compared with the first trimester (31.14% vs. 6.74%, p = 0.0016). Additionally, Desmin was expressed in the majority of Sertoli cells in the adult testis and TGCC samples. Cytokeratin was detected in Sertoli cells of human fetal testis but was not expressed in Sertoli cells of human adult testis. In patients with TGCC, cytokeratin was not expressed in Sertoli cells in tubules with active spermatogenesis but was detected in Sertoli cells in tubules containing GCNIS cells in patients with both pre-invasive and invasive TGCC. In conclusion, desmin was not associated with Sertoli cell maturation or progression to TGCC. However, cytokeratin appeared to be an indicator of impaired Sertoli cell maturation.

Inactivation of vimentin in satellite glial cells affects dorsal root ganglion intermediate filament expression and neuronal axon growth in vitro

Peripheral nerve trauma and regeneration are complex events, and little is known concerning how occurrences in the distal stump affect the cell body's response to injury. Intermediate filament (IF) proteins underpin cellular architecture and take part in nerve cell proliferation, differentiation and axon regeneration, but their role in these processes is not yet fully understood. The present study aimed to investigate the regulation and interrelationship of major neural IFs in adult dorsal root ganglion (DRG) neurons and satellite glial cells (SGCs) following sciatic nerve injury. We demonstrated that the expression of neural IFs in DRG neurons and SGCs after axotomy depends on vimentin activity. In intact DRGs, synemin M and peripherin proteins are detected in small neurons while neurofilament L (NFL) and synemin L characterize large neurons. Both neuronal populations are surrounded by vimentin positive- and glial fibrillary acidic protein (GFAP)-negative SGCs. In response to axotomy, synemin M and peripherin were upregulated in large wild-type DRG neurons and, to a lesser extent, in vim−/− and synm−/− DRG neurons, suggesting the role for these IFs in axon regeneration. However, an increase in the number of NFL-positive small neurons was observed in vim−/− mice, accompanied by a decrease of peripherin-positive small neurons. These findings suggest that vimentin is required for injury-induced neuronal IF remodeling. We further show that vimentin is also indispensable for nerve injury-induced GFAP upregulation in perineuronal SGCs and that inactivation of vimentin and synemin appears to accelerate the rate of DRG neurite regeneration at early stages in vitro.

Altered interaction between enteric glial cells and mast cells in the colon of women with irritable bowel syndrome.

Enteric glial cells (EGC) and mast cells (MC) are intimately associated with gastrointestinal physiological functions. We aimed to investigate EGC-MC interaction in irritable bowel syndrome (IBS), a gut-brain disorder linked to increased intestinal permeability, and MC. Parallel approaches were used to quantify EGC markers in colonic biopsies from healthy controls (HC) and patients with IBS. Data were correlated with MC, vasoactive intestinal polypeptide (VIP) and VIP receptors (VPAC1/VPAC2) expressions, and bacterial translocation through biopsies mounted in Ussing chambers. In addition, we investigated the effects of EGC mediators on colonic permeability and the pharmacological-induced responses of EGC and MC cell lines. Immunofluorescence of IBS colonic mucosa, as well as Western blotting and ELISA of IBS biopsy lysates, revealed increased glial fibrillary intermediate filament (GFAP) expression, indicating EGC activation. Mucosal GFAP correlated with increased MC and VPAC1+ MC numbers and decreased VIP+ MC, which seemed to control bacterial translocation in HC. In the contrary, EGC activation in IBS correlated with less MC and VPAC1+ MC numbers, and more VIP+ MC. In vitro, MC and EGC cell lines showed intracellular calcium responses to each other's mediators. Furthermore, EGC mediators prevented VIP-induced MC degranulation, while MC mediators induced a reactive EGC phenotype. In Ussing chambers, EGC mediators decreased paracellular passage through healthy colonic biopsies. Findings suggest the involvement of EGC and MC in the control of barrier function in the human colon and indicate a potential EGC-MC interaction that seems altered in IBS, with detrimental consequences to colonic permeability. Altogether, results suggest that imbalanced EGC-MC communication contributes to the pathophysiology of IBS.

Influence of cadmium contamination on brain glial cells: consequences and bioindication possibilities

Cadmium (Cd) is a heavy metal that currently presents in almost all components of the environment. Cd is a ubiquitous pollutant that is constantly entering the environment from industry and agriculture, mining, forest fires and many more sources. Some occupational diseases have aftereffects associated with Cd cytotoxicity. Despite long-term studies of the toxic effects of Cd, its cytotoxicity of low doses and the chronic effects on the nerve tissue cells remain undiscovered. The results of determining the Cd neurotoxicity indicate a disturbance of the permeability of the blood-brain barrier, the accumulation of Cd in the brain and the deterioration of the functional activity of the central nervous system. One of the main cellular targets for Cd in the brain are astrocytes. Astrocytes provide nutrition and functional activity of neurons, as well as recovery of physical and metabolic damage. The cytoskeleton of astrocytes is built of glial fibrillary acidic protein (GFAP). GFAP participates in important functions of astrocytes and its condition reflects the astrocytes reactivity. The molecular mechanisms of the neurotoxic effects of Cd on the glial cytoskeleton remain unknown. Glioblastomas are widely used to study the cytotoxic mechanisms of various compounds, including heavy metals, as cellular models of astrocytes. Taking into account the role of oxidative stress in a cell damage, as well as the reactive response of glial cells, we study the influence of low doses of Cd on oxidative stress and expression of GFAP and glucose-6-phosphate dehydrogenase (G6PD) in U373GM cells. Doses of 2-10 μM Cd induced a dose-dependent increase in reactive oxygen species and lipid peroxidation products. The same doses inhibited the expression of the cytoskeletal marker of astrocytes (GFAP) and metabolic marker of glucose utilization (G6PD). The obtained results indicate a pronounced cytotoxic effect of low doses of Cd in the astrocytic cell model U373GM. In addition, the astroglial cytotoxicity of Cd may be mediated by oxidative damage, inhibition of glial intermediate filament expression, and glucose utilization disorders. These parameters can be promising biomarkers of toxic effects both for the assessment of human and animal health and for determining the state of the environment as a whole.

The Effects of Long-Term Immunosuppressive Therapies on the Structure of the Rat Prostate

Background: Little is known about the overall impact of immunosuppressive drugs on the prostate. The study aimed to determine the impact of different protocols of immunosuppressive treatment on the structure of the rat ventral prostate. Methods: For 6 months, 48 male Wistar rats received immunosuppressive drugs: cyclosporin A, tacrolimus, mycophenolate mofetil, rapamycin, and prednisone, according to three-drug protocols. Light and transmission electron microscopic studies, and quantitative evaluation of immunohistochemical expression of selected intermediate filaments, CD117+ mast cells, and CD138+ plasma cells were performed in the rat ventral prostate. Results: In all experimental groups, acini focal hyperplasia, changes to the ultrastructure of the glandular epithelium, changes in the expression of cytokeratins and desmin, and numerous mast and plasma cells in the prostate stroma were found. In cyclosporine-A-based groups, atrophy and numerous intracellular vacuoles were observed. In groups where a three-drug treatment was replaced with rapamycin, morphological alterations were less severe compared to those without conversion. Conclusions: In the rat ventral prostate, (1) immunosuppressive protocols affect the morphology and immunohistochemical expression of intermediate filaments, (2) morphological alterations, expression, and localization of selected proteins are not connected with adenocarcinoma development, and (3) conversion of the treatment to rapamycin may prevent hyperplastic abnormalities.

Coordinated Intervention of Microglial and Müller Cells in Light-Induced Retinal Degeneration.

PurposeTo analyze the role of microglial and Müller cells in the formation of rings of photoreceptor degeneration caused by phototoxicity.MethodsTwo-month-old Sprague-Dawley rats were exposed to light and processed 1, 2, or 3 months later. Retinas were dissected as whole-mounts, immunodetected for microglial cells, Müller cells, and S- and L/M-cones and analyzed using fluorescence, thunder imaging, and confocal microscopy. Cone populations were automatically counted and isodensity maps constructed to document cone topography.ResultsPhototoxicity causes a significant progressive loss of S- and L/M-cones of up to 68% and 44%, respectively, at 3 months after light exposure (ALE). One month ALE, we observed rings of cone degeneration in the photosensitive area of the superior retina. Two and 3 months ALE, these rings had extended to the central and inferior retina. Within the rings of cone degeneration, there were degenerating cones, often activated microglial cells, and numerous radially oriented processes of Müller cells that showed increased expression of intermediate filaments. Between 1 and 3 months ALE, the rings coalesced, and at the same time the microglial cells resumed a mosaic-like distribution, and there was a decrease of Müller cell gliosis at the areas devoid of cones.ConclusionsLight-induced photoreceptor degeneration proceeds with rings of cone degeneration, as observed in inherited retinal degenerations in which cone death is secondary to rod degeneration. The spatiotemporal relationship of cone death microglial cell activation and Müller cell gliosis within the rings of cone degeneration suggests that, although both glial cells are involved in the formation of the rings, they may have coordinated actions and, while microglial cells may be more involved in photoreceptor phagocytosis, Müller cells may be more involved in cone and microglial cell migration, retinal remodeling and glial seal formation.

Ferulic acid attenuates arsenic-induced cardiotoxicity in rats.

Arsenic (As), a potent environmental toxin, causes cardiac functional impairments. Ferulic acid (FA), a ubiquitous dietary hydroxycinnamate, exerts beneficial effects on human health. Hence, the present study investigated the effect of FA on myocardial oxidative stress parameters, ATP level, the status of cardiac cytoskeleton intermediate filaments-desmin and vimentin, and AMPK signaling proteins in As-intoxicated rats. Wistar rats were administered orally with FA-40mg/kg and As-5mg/kg alone and in combination for 30 days. Myocardial As content, serum cardiac marker enzyme activities including creatine kinase-isoenzyme, lactate dehydrogenase, and aspartate aminotransferase were increased in As-exposed rats. An accumulation of myocardial oxidants such as reactive oxygen species, lipid peroxidation, nitric oxide, protein carbonyl content, and histological aberrations was observed. A significant decrease of myocardial antioxidants comprises superoxide dismutase, catalase, glutathione peroxidase, reduced glutathione, and ascorbic acid and declined expression of desmin and vimentin was noted. Impaired energy signaling molecules AMPKα (Thr172), AMPKβ1/2 (Ser108), ACC (Ser79), and intracellular myocardial ATP depletion were observed in As-intoxicated animals. FA attenuates As-induced cardiac dysfunction by restoring the expression of intermediate filaments and AMPK proteins. Based on the above findings, FA treatment could be used as a novel therapeutic against As-induced cardiac dysfunction.

Astrocyte Mechano-Activation by High-Rate Overpressure Involves Alterations in Structural and Junctional Proteins.

Primary blast neurotrauma represents a unique injury paradigm characterized by high-rate overpressure effects on brain tissue. One major hallmark of blast neurotrauma is glial reactivity, notably prolonged astrocyte activation. This cellular response has been mainly defined in primary blast neurotrauma by increased intermediate filament expression. Because the intermediate filament networks physically interface with transmembrane proteins for junctional support, it was hypothesized that cell junction regulation is altered in the reactive phenotype as well. This would have implications for downstream transcriptional regulation via signal transduction pathways like nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Therefore, a custom high-rate overpressure simulator was built for in vitro testing using mechanical conditions based on intracranial pressure measurements in a rat model of blast neurotrauma. Primary rat astrocytes were exposed to isolated high-rate mechanical stimulation to study cell junction dynamics in relation to their mechano-activation. First, a time course for “classical” features of reactivity was devised by evaluation of glial fibrillary acidic protein (GFAP) and proliferating cell nuclear antigen (PCNA) expression. This was followed by gene and protein expression for both gap junction (connexins) and anchoring junction proteins (integrins and cadherins). Signal transduction analysis was carried out by nuclear localization of two molecules, NF-κB p65 and mitogen-activated protein kinase (MAPK) p38. Results indicated significant increases in connexin-43 expression and PCNA first at 24 h post-overpressure (p < 0.05), followed by structural reactivity (via increased GFAP, p < 0.05) corresponding to increased anchoring junction dynamics at 48 h post-overpressure (p < 0.05). Moreover, increased phosphorylation of focal adhesion kinase (FAK) was observed in addition to increased nuclear localization of both p65 and p38 (p < 0.05) during the period of structural reactivity. To evaluate the transcriptional activity of p65 in the nucleus, electrophoretic mobility shift assay was conducted for a binding site on the promoter region for intracellular adhesion molecule-1 (ICAM-1), an antagonist of tight junctions. A significant increase in the interaction of nuclear proteins with the NF-κB site on the ICAM-1 corresponded to increased gene and protein expression of ICAM-1 (p < 0.05). Altogether, these results indicate multiple targets and corresponding signaling pathways which involve cell junction dynamics in the mechano-activation of astrocytes following high-rate overpressure.

Low-level blast exposure disrupts gliovascular and neurovascular connections and induces a chronic vascular pathology in rat brain

Much concern exists over the role of blast-induced traumatic brain injury (TBI) in the chronic cognitive and mental health problems that develop in veterans and active duty military personnel. The brain vasculature is particularly sensitive to blast injury. The aim of this study was to characterize the evolving molecular and histologic alterations in the neurovascular unit induced by three repetitive low-energy blast exposures (3 × 74.5 kPa) in a rat model mimicking human mild TBI or subclinical blast exposure. High-resolution two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry of purified brain vascular fractions from blast-exposed animals 6 weeks post-exposure showed decreased levels of vascular-associated glial fibrillary acidic protein (GFAP) and several neuronal intermediate filament proteins (α-internexin and the low, middle, and high molecular weight neurofilament subunits). Loss of these proteins suggested that blast exposure disrupts gliovascular and neurovascular interactions. Electron microscopy confirmed blast-induced effects on perivascular astrocytes including swelling and degeneration of astrocytic endfeet in the brain cortical vasculature. Because the astrocyte is a major sensor of neuronal activity and regulator of cerebral blood flow, structural disruption of gliovascular integrity within the neurovascular unit should impair cerebral autoregulation. Disrupted neurovascular connections to pial and parenchymal blood vessels might also affect brain circulation. Blast exposures also induced structural and functional alterations in the arterial smooth muscle layer. Interestingly, by 8 months after blast exposure, GFAP and neuronal intermediate filament expression had recovered to control levels in isolated brain vascular fractions. However, despite this recovery, a widespread vascular pathology was still apparent at 10 months after blast exposure histologically and on micro-computed tomography scanning. Thus, low-level blast exposure disrupts gliovascular and neurovascular connections while inducing a chronic vascular pathology.

The intestinal intermediate filament network responds to and protects against microbial insults and toxins.

The enrichment of intermediate filaments in the apical cytoplasm of intestinal cells is evolutionarily conserved, forming a sheath that is anchored to apical junctions and positioned below the microvillar brush border, which suggests a protective intracellular barrier function. To test this, we used Caenorhabditiselegans, the intestinal cells of which are endowed with a particularly dense intermediate filament-rich layer that is referred to as the endotube. We found alterations in endotube structure and intermediate filament expression upon infection with nematicidal B.thuringiensis or treatment with its major pore-forming toxin crystal protein Cry5B. Endotube impairment due to defined genetic mutations of intermediate filaments and their regulators results in increased Cry5B sensitivity as evidenced by elevated larval arrest, prolonged time of larval development and reduced survival. Phenotype severity reflects the extent of endotube alterations and correlates with reduced rescue upon toxin removal. The results provide in vivo evidence for a major protective role of a properly configured intermediate filament network as an intracellular barrier in intestinal cells. This notion is further supported by increased sensitivity of endotube mutants to oxidative and osmotic stress.

ZEB1 and ZEB2 transcription factors are potential therapeutic targets of canine mammary cancer cells.

Mammary tumours are the most frequent in female dogs as in women and half are malignant. Tumorigenicity and invasiveness are important acquired characteristics for the development and progression of cancers and could be regulated by transcription factors associated with epithelial-mesenchymal transition (EMT) as ZEB1, ZEB2, SNAI1, SLUG and STAT3. Thus, here, we evaluate the expression of EMT-associated transcription factors in canine mammary cancer (CMC) cell lines characterized for invasiveness and tumorigenicity to determine if these could be considered good targets for future development of therapies. Five CMC cell lines were characterized regarding their morphology, doubling time and expression of intermediate and actin filaments. In addition, gene expression of SLUG, STAT3, ZEB1, ZEB2 and CDH1, tumorigenicity and invasiveness were assessed. Two of these cells presented an epithelial-like morphology (E20 and E37) and three a mesenchymal-like morphology (M5, M25 and CF41.Mg). M25 and CF41.Mg presented higher invasiveness. Furthermore, only mesenchymal-like cells formed tumorspheres and CF41.Mg made more and larger tumorspheres. The mesenchymal-like cells are more malignant than the epithelial-like cells being the CF41.Mg the most malignant. This cell presented higher ZEB1 and ZEB2 and lower CDH1 gene expression. Finally, our results revealed that there is a positive correlation between ZEBs and the tumorsphere number and size. In conclusion, these findings support ZEB1 and ZEB2 as potential therapeutic targets for CMC cells, demonstrating a great potential of canine models for comparative and translational studies.

A proteomic profile of postpartum cervical repair in mice.

A timely and complete uterine cervical tissue repair postpartum is of necessity to prevent obstetrical complications, such as cervicitis, ectropion, hemorrhage, repeated miscarriages or abortions and possibly preterm labor and malignancies. We recently characterized the morphological alterations, as well as changes in angiogenic expression profile in a mice uterine cervix during the immediate postpartum period. Here, we build on this previous study using a proteomic analysis to profile postpartum tissue changes in mice cervix during the same period, the first 48 h of postpartum. The current proteomics data reveal a variable expression of several intermediate filaments, cytoskeletal modulators and proteins with immune and/or wound-healing properties. We conclude that postpartum cervical repair involves a rapid and tightly regulated balance between a host of biological factors, notably between anti- and pro-inflammatory factors, executed by the M1 and M2 macrophage cells, as revealed by proteomics and verified by confocal immunofluorescence. Future studies will assess the suitability of some of the key proteins identified in this study as potential markers for determining the phase of postpartum cervical repair in obstetrical complications, such as cervical lacerations.

Human Primary Epithelial Cells Acquire an Epithelial-Mesenchymal-Transition Phenotype during Long-Term Infection by the Oral Opportunistic Pathogen, Porphyromonas gingivalis.

Porphyromonas gingivalis is a host-adapted oral pathogen associated with chronic periodontitis that successfully survives and persists in the oral epithelium. Recent studies have positively correlated periodontitis with increased risk and severity of oral squamous cell carcinoma (OSCC). Intriguingly, the presence of P. gingivalis enhances tumorigenic properties independently of periodontitis and has therefore been proposed as a potential etiological agent for OSCC. However, the initial host molecular changes induced by P. gingivalis infection which promote predisposition to cancerous transformation through EMT (epithelial-mesenchymal-transition), has never been studied in human primary cells which more closely mimic the physiological state of cells in vivo. In this study, we examine for the first time in primary oral epithelial cells (OECs) the expression and activation of key EMT mediators during long-term P. gingivalis infection in vitro. We examined the inactive phosphorylated state of glycogen synthase kinase-3 beta (p-GSK3β) over 120 h P. gingivalis infection and found p-GSK3β, an important EMT regulator, significantly increases over the course of infection (p < 0.01). Furthermore, we examined the expression of EMT-associated transcription factors, Slug, Snail, and Zeb1 and found significant increases (p < 0.01) over long-term P. gingivalis infection in protein and mRNA expression. Additionally, the protein expression of mesenchymal intermediate filament, Vimentin, was substantially increased over 120 h of P. gingivalis infection. Analysis of adhesion molecule E-cadherin showed a significant decrease (p < 0.05) in expression and a loss of membrane localization along with β-catenin in OECs. Matrix metalloproteinases (MMPs) 2, 7, and 9 are all markedly increased with long-term P. gingivalis infection. Finally, migration of P. gingivalis infected cells was evaluated using scratch assay in which primary OEC monolayers were wounded and treated with proliferation inhibitor, Mitomycin C. The cellular movement was determined by microscopy. Results displayed P. gingivalis infection promoted cell migration which was slightly enhanced by co-infection with Fusobacterium nucleatum, another oral opportunistic pathogen. Therefore, this study demonstrates human primary OECs acquire initial molecular/cellular changes that are consistent with EMT induction during long-term infection by P. gingivalis and provides a critically novel framework for future mechanistic studies.

Immunohistochemical expression of intermediate filaments in diagnostics of diffuse gliomas.

Immunohistochemical expression of intermediate filaments in diagnostics of diffuse gliomas.

Expression of Epithelial and Mesenchymal Differentiation Markers in the Early Human Gonadal Development.

Expressions of cytokeratin 8 (CK8), vimentin, nestin, and alpha-smooth-muscle-actin (alpha-SMA) were analyzed in the developing gonads of 12, 5-9 week old (W) human conceptuses by immunohistochemistry and immunofluorescence. During the investigated period, the number of CK8 positive cells increased from 56% to 92% in the gonadal surface epithelium, from 50% to 60% in the stroma, and from 23% to 42% in the medulla. In the early fetal period, the cell expression of CK8 increased in all gonadal parts, whereas primordial germ cells (PGC) remained negative. The expression of vimentin increased in the gonad stroma (gs) from 73% to 88%, and in the surface epithelium from 18% to 97% until ninth W. The medulla had the highest expression of vimentin in the seventh to eighth W (93%). Vimentin and CK8 colocalized in the somatic cells, while some PGCs showed vimentin expression only. Initially, nestin was positive in the gonad surface epithelium (8%) and stroma (52%), however during further development it decreased to 1% and 33%, respectively. In the early fetal period, the nestin positive cells decreased from 44% to 31% in the gonad medulla. Alpha-SMA was positive only in the blood vessels and mesonephros. The described pattern of expression of intermediate filaments (IF) in developing human gonads suggests their role in the control of PGC apoptosis, early differentiation of gs cells and cell migration. Both epithelial and mesenchymal origins of follicular cells and possible epithelial-to-mesenchymal transition of somatic cells is proposed. Lastly, IF intensity expression varies depending on the cell type and developmental period analyzed. Anat Rec, 300:1315-1326, 2017. © 2017 Wiley Periodicals, Inc.

Vimentin Intermediate Filaments Template Microtubule Networks to Enhance Persistence in Cell Polarity and Directed Migration

Increased expression of vimentin intermediate filaments (VIFs) enhances directed cell migration, but the mechanism behind VIFs' effect on motility is not understood. VIFs interact with microtubules, whose organization contributes to polarity maintenance in migrating cells. Here, we characterize the dynamic coordination of VIF and microtubule networks in wounded monolayers of retinal pigment epithelial cells. By genome editing, we fluorescently labeled endogenous vimentin and α-tubulin, and we developed computational image analysis to delineate architecture and interactions of the two networks. Our results show that VIFs assemble an ultrastructural copy of the previously polarized microtubule network. Because the VIF network is long-lived compared to the microtubule network, VIFs template future microtubule growth along previous microtubule tracks, thus providing a feedback mechanism that maintains cell polarity. VIF knockdown prevents cells from polarizing and migrating properly during wound healing. We suggest that VIFs' templating function establishes a memory in microtubule organization that enhances persistence in cell polarization in general and migration in particular.