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BACKGROUND: The promoters of mammalian genes contain clusters of CG dinucleotides known as CpG islands. Most mammalian housekeeping genes predominantly contain CpG islands (CGIs), facilitating gene transcription. Numerous studies have explored the physiological implications of the relationship between CGIs and gene expression. However, the evolutionary implications of this relationship remain largely unexplored. Pseudogenes, in contrast, are genomic remnants that have lost their function over evolutionary time. METHODS: In our current research, we employed comparative genomic techniques to demonstrate a correlation between the absence of gene expression due to a lack of CGIs in the gene promoters and pseudogenization. RESULTS: We showed that there is a significant enrichment of tissue-specific genes in the functional orthologs of pseudogenes. We also found a significant correlation between the lack of CGIs and enriched tissue specificity in these functional orthologs of pseudogenes. CONCLUSIONS: We inferred that perhaps tissue-specific genes are more prone to the process of pseudogenization. In this way, because of their impact on gene expression, CGIs may affect the fate of a gene. To our knowledge, this is the first study to propose a connection between CGIs, gene expression, and the pseudogenization process and discuss the evolutionary implications of this potential trilogy.
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Genoma , Genómica , Animales , Islas de CpG/genética , Mamíferos/genética , Expresión GénicaRESUMEN
The stomach's colonization by Helicobacter pylori (H. pylori) results in gastritis, ulcers, and stomach cancer. Frequently, pain is treated with medication, but resistant H. pylori infections are not. Therefore, it is important to find pharmacological targets and improved treatments for resistant H. pylori strains. The aim of the current study was sampling, identification, drug susceptibility testing following genome sequencing and comparative genome-wide analysis of selected H. pylori strains from Pakistan with three representative strains for virulence and drug-resistant characteristics. Based on culture, biochemistry, and molecular biology, 84 strains of H. pylori were identified, which made up 47% of the enrolled cases. Among all H. pylori strains, the highest resistance was reported for metronidazole with 82 H. pylori strains (98%), followed by clarithromycin with 62 resistant strains (74%). Among metronidazole-resistant strains, 38 strains (46%) were also resistant to clarithromycin, contributing 61% of clarithromycin resistant cases. Two strains, HPA1 and HPA2, isolated from 'gastritis' and 'gastric ulcer' patients, respectively, were further processed for WGS. The draft genome sequences of H. pylori strains HPA1 and HPA2 encode 1.66 Mbp and 1.67 Mbp genome size, 24 and 4 contiguous DNA sequences, and 1650 and 1625 coding sequences, respectively. Both the genomes showed greater than 90% similarity with the reference strain H. pylori ATCC 43504/PMSS1. The antibiotic-resistant genes were identified among all the strains with overall similarity above 95%, with minor differences in the sequence similarity. Using the virulent gene data obtained from the Virulence Factor Database, 75 to 85 virulent genes were identified in the five genome assemblies with various key genes such as cytolethal distending toxin (cdt), type IV secretion system, cag PAI, plasticity region, cell-motility- and flagellar-associated genes, neutrophil-activating protein (HP-NAP), T4SS effector cytotoxin-associated gene A (cagA), and urease-associated genes ureA and ureB, etc. Sequence similarity between the virulence factors found in this study and reference genes was at least 90%. In summary, the results of our study showed the relationship between clinical results and specific H. pylori strains' (HPA1 and HPA2) genetics such as antibiotic resistance and specific virulence factors. These findings provide valued understanding of the epidemiology of H. pylori-associated diseases. Moreover, identification and genomics analysis have provided insights into the epidemiology, genetic diversity, pathogenicity, and potential drug resistance genes of H. pylori strains, offering a foundation for developing more targeted and effective medical interventions, including anti-virulent medications.
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CpG islands (CGIs) are aggregation of CpG dinucleotides in the promoters of mammalian genes. These CGIs are present in almost all the housekeeping genes and some tissue-specific genes in the mammalian genome. Extensive research has been done on the prevalence and role of CGIs in protein-coding genes. However, little is known about CGIs in pseudogenes. In the current research project, we focused on CGIs in three main classes of pseudogenes e.g., duplicated pseudogenes (DPGs), processed pseudogenes (PPGs), and unitary pseudogenes (UPGs). We discovered a predominant absence of CGIs in the promoters of all three pseudogenes. We also compared the CGI profile of these pseudogenes with their parent genes and found that unitary pseudogenes (UPGs) differ from the DPGs and PPGs in the sense that in the latter, lack of CGIs is a consequential event while in UPGs, this lack of CGIs in their promoters is not a result of pseudogenization process. We also discussed the implication of the results obtained from this comparison. To our knowledge, this is the first-ever study highlighting this aspect of UPGs throwing new insights into the evolution of genome in general and especially in the context of pseudogenes.
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Genoma , Seudogenes , Animales , Islas de CpG/genética , Metilación de ADN/genética , Humanos , Mamíferos/genética , Regiones Promotoras Genéticas , Seudogenes/genéticaRESUMEN
The lymphatic vasculature is essential for maintaining interstitial fluid homeostasis, and dysfunctional lymphangiogenesis contributes to various pathological processes, including inflammatory disease and tumor metastasis. Mutations in FOXC2 are dominantly associated with late-onset lymphedema; however, the precise role of FOXC2 and a closely related factor, FOXC1, in the lymphatic system remains largely unknown. Here we identified a molecular cascade by which FOXC1 and FOXC2 regulate ERK signaling in lymphatic vessel growth. In mice, lymphatic endothelial cell-specific (LEC-specific) deletion of Foxc1, Foxc2, or both resulted in increased LEC proliferation, enlarged lymphatic vessels, and abnormal lymphatic vessel morphogenesis. Compared with LECs from control animals, LECs from mice lacking both Foxc1 and Foxc2 exhibited aberrant expression of Ras regulators, and embryos with LEC-specific deletion of Foxc1 and Foxc2, alone or in combination, exhibited ERK hyperactivation. Pharmacological ERK inhibition in utero abolished the abnormally enlarged lymphatic vessels in FOXC-deficient embryos. Together, these results identify FOXC1 and FOXC2 as essential regulators of lymphangiogenesis and indicate a new potential mechanistic basis for lymphatic-associated diseases.
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Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Factores de Transcripción Forkhead/metabolismo , Eliminación de Gen , Regulación de la Expresión Génica , Linfangiogénesis , Animales , Proliferación Celular , Femenino , Perfilación de la Expresión Génica , Inflamación , Masculino , Ratones , Ratones Noqueados , Piel/metabolismoRESUMEN
Embryonic Stem Cells (ESCs) hold great potential for regeneration of damaged myocardium, however the molecular circuitry that guides ESC differentiation into cardiomyocytes remains poorly understood. This is exemplified by the elusive role of the transcription factor, Foxc1, during cardiac development. The only known Foxc1 target during heart development is Tbx1. Because Foxc1 null mice contain heart mutations that are far more severe than Tbx1 null mice, it is likely that Foxc1 has additional regulatory roles during heart development. The goal of our study was to test whether Foxc1 is critical for ESC differentiation into functional cardiomyocytes through proper regulation of specific downstream gene networks. Converging evidence from Foxc1 deficient and overexpression ESC models reveals a close relationship between Foxc1 levels and early cardiomyogenic factors Isl1, Mef2c, and Nkx2.5 and also the production of functional cardiomyocytes. We show Foxc1 regulates early cardiomyogenesis during a specific window of differentiation, D4-D6. Through whole transcriptome RNA-sequencing analysis, we report pathways regulated by Foxc1 involved in cardiac function including actin cytoskeleton, cell adhesion, tight and gap junctions, and calcium signaling. Our data indicate a novel Foxc1 direct gene target, Myh7, which encodes the predominant myosin heavy chain isoform, MHCß, expressed during cardiac development. These data lead us to conclude that Foxc1 regulates both early cardiomyogenesis and the functional properties of ESC-derived cardiomyocytes. Our findings shed light on the molecular circuitry governing cardiomyogenesis that may lead to the development of better translational strategies for the use of pluripotent stem cells in regenerative medicine towards repairing damaged myocardium. Stem Cells 2016;34:1487-1500.
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Factores de Transcripción Forkhead/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Organogénesis , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Doxiciclina/farmacología , Endodermo/efectos de los fármacos , Endodermo/metabolismo , Factores de Transcripción Forkhead/deficiencia , Proteína Homeótica Nkx-2.5/metabolismo , Mesodermo/efectos de los fármacos , Mesodermo/metabolismo , Ratones , Células Madre Embrionarias de Ratones/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Cadenas Pesadas de Miosina/genética , Cadenas Pesadas de Miosina/metabolismo , Organogénesis/efectos de los fármacos , Organogénesis/genética , Análisis de Secuencia de ARN , Transcriptoma/genética , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Glaucoma is a leading cause of blindness, afflicting more than 60 million people worldwide. Increased intraocular pressure (IOP) due to impaired aqueous humor drainage is a major risk factor for the development of glaucoma. Here, we demonstrated that genetic disruption of the angiopoietin/TIE2 (ANGPT/TIE2) signaling pathway results in high IOP, buphthalmos, and classic features of glaucoma, including retinal ganglion degeneration and vision loss. Eyes from mice with induced deletion of Angpt1 and Angpt2 (A1A2Flox(WB) mice) lacked drainage pathways in the corneal limbus, including Schlemm's canal and lymphatic capillaries, which share expression of the PROX1, VEGFR3, and FOXC family of transcription factors. VEGFR3 and FOXCs have been linked to lymphatic disorders in patients, and FOXC1 has been linked to glaucoma. In contrast to blood endothelium, in which ANGPT2 is an antagonist of ANGPT1, we have shown that both ligands cooperate to regulate TIE2 in the lymphatic network of the eye. While A1A2Flox(WB) mice developed high IOP and glaucoma, expression of ANGPT1 or ANGPT2 alone was sufficient for ocular drainage. Furthermore, we demonstrated that loss of FOXC2 from lymphatics results in TIE2 downregulation, suggesting a mechanism for ocular defects in patients with FOXC mutations. These data reveal a pathogenetic and molecular basis for glaucoma and demonstrate the importance of angiopoietin ligand cooperation in the lymphatic endothelium.
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Angiopoyetina 1/genética , Glaucoma/patología , Hipertensión Ocular/patología , Receptor TIE-2/genética , Angiopoyetina 2/genética , Animales , Humor Acuoso , Separación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Citometría de Flujo , Factores de Transcripción Forkhead/metabolismo , Proteínas de Homeodominio/metabolismo , Presión Intraocular , Ligandos , Sistema Linfático/patología , Ratones , Ratones Noqueados , Mutación , Malla Trabecular/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The transmembrane receptor Notch1 is a critical regulator of arterial differentiation and blood vessel sprouting. Recent evidence shows that functional blockade of Notch1 and its ligand, Dll4, leads to postnatal lymphatic defects in mice. However, the precise role of the Notch signaling pathway in lymphatic vessel development has yet to be defined. Here we show the developmental role of Notch1 in lymphatic vascular morphogenesis by analyzing lymphatic endothelial cell (LEC)-specific conditional Notch1 knockout mice crossed with an inducible Prox1CreER(T2) driver. RESULTS: LEC-specific Notch1 mutant embryos exhibited enlarged lymphatic vessels. The phenotype of lymphatic overgrowth accords with increased LEC sprouting from the lymph sacs and increased filopodia formation. Furthermore, cell death was significantly reduced in Notch1-mutant LECs, whereas proliferation was increased. RNA-seq analysis revealed that expression of cytokine/chemokine signaling molecules was upregulated in Notch1-mutant LECs isolated from E15.5 dorsal skin, whereas VEGFR3, VEGFR2, VEGFC, and Gja4 (Connexin 37) were downregulated. CONCLUSIONS: The lymphatic phenotype of LEC-specific conditional Notch1 mouse mutants indicates that Notch activity in LECs controls lymphatic sprouting and growth during development. These results provide evidence that similar to postnatal and pathological lymphatic vessel formation, the Notch signaling pathway plays a role in inhibiting developmental lymphangiogenesis.
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Linfangiogénesis/fisiología , Vasos Linfáticos/embriología , Vasos Linfáticos/metabolismo , Morfogénesis/fisiología , Receptor Notch1/metabolismo , Animales , Femenino , Citometría de Flujo , Inmunohistoquímica , Linfangiogénesis/genética , Masculino , Ratones , Morfogénesis/genética , Embarazo , Receptor Notch1/genéticaRESUMEN
BACKGROUND: Cultivated limbal epithelium for reconstruction of corneal surface is a well-established procedure; however, it is not adequate for damage which also extensively involves the conjunctiva. In severe cases of ocular surface damage that warrant additional conjunctival transplantation apart from cultivated limbal stem cell transplantation, we describe the long-term survival of a novel method of cocultivating autologous limbal and conjunctival epithelium on a single substrate. MATERIALS AND METHODS: Forty eyes of 39 patients with severe limbal stem cell deficiency and conjunctival scarring or symblepharon underwent transplantation of autologous cocultivated epithelium on human amniotic membrane. A ring barrier was used to segregate the central limbal and peripheral conjunctival epithelia in vitro. Patients were followed up at regular intervals to assess stability of the ocular surface, defined by absence of conjunctivalization into the central 4 mm of the cornea and absence of diffuse fluorescein staining. Penetrating keratoplasty (PKP) was subsequently performed, where indicated, in patients with surface stability. RESULTS: The cumulative survival probability was 60% at 1 year and 45% at 4 years by Kaplan-Meier analysis (mean follow-up duration: 33 ± 29 months, range: 1-87 months). Best-corrected visual acuity improved to greater than 20/200 in 38% eyes at the last follow-up, compared with 5% eyes before surgery. Immunohistochemistry in five of the corneal buttons excised for PKP showed an epithelial phenotype similar to cornea in all five. CONCLUSIONS: Synchronous use of cultured limbal and conjunctival epithelium offers a feasible alternative and a simpler one-step surgical approach to treat severe ocular surface disorders involving limbus and conjunctiva.
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Conjuntiva/trasplante , Enfermedades de la Córnea/cirugía , Trasplante de Córnea/métodos , Epitelio Corneal/citología , Supervivencia de Injerto , Limbo de la Córnea/citología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Células Cultivadas , Niño , Técnicas de Cocultivo , Conjuntiva/citología , Femenino , Estudios de Seguimiento , Humanos , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Factores de Tiempo , Trasplante Autólogo , Resultado del Tratamiento , Agudeza Visual , Adulto JovenRESUMEN
Heterometrus xanthopus (Scorpion) is one of the most venomous and ancient arthropods. Its venom contains anti-microbial peptides like hadrurin, scorpine, Pandinin 1, and Pandinin 2 that are able to effectively kill multidrug-resistant pathogens. The present study was conducted to evaluate the anti-bacterial activity of H. xanthopus venom. Six Gram-positive and Gram-negative bacterial strains were tested against 1/100, 1/10, and 1/1 fractions of distilled water diluted and crude venom. 1/100 and 1/10 dilutions were not successful in any of the six bacterial strains studied while the 1/1 dilution was effective on Bacillus subtilis ATCC 6633, Salmonella typhimurium ATCC 14028, and Pseudomonas aeruginosa ATCC 27853 with highest zone of inhibition were obtained on B. subtilis. Crude venom was effective against Enterococcus faecalis ATCC 14506, B. subtilis, S. typhimurium, and P. aeruginosa. The most effective results were observed on B. subtilis.
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Antibacterianos/aislamiento & purificación , Antibacterianos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Venenos de Escorpión/aislamiento & purificación , Venenos de Escorpión/farmacología , Animales , Relación Dosis-Respuesta a Droga , Enterococcus faecalis/efectos de los fármacos , Enterococcus faecalis/fisiología , Escherichia coli/efectos de los fármacos , Escherichia coli/fisiología , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Pruebas de Sensibilidad Microbiana/métodos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Escorpiones , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/fisiologíaRESUMEN
PURPOSE: Eph/ephrin signaling proteins are present in the corneal epithelium, where their function remains unknown. The authors examined the role of the EphA2 receptor and ephrin-A1 ligand in human corneal epithelial cell migration. METHODS: Immunohistochemical analysis of EphA2 and ephrin-A1 in healthy and diabetic corneas was performed in concert with linear scratch wound healing studies in primary and telomerase-immortalized human corneal epithelial cells. Corneal epithelial cells were exposed to a soluble ephrin-A1-Fc peptide mimetic that targets EphA2 to trigger receptor phosphorylation and subsequent downregulation. Genetic modulation of EphA2 and ephrin-A1 levels was combined with manipulation of Erk1/2 or Akt signaling during wound healing. RESULTS: EphA2 was immunolocalized to human corneal epithelial cells in vivo and in vitro. Ephrin-A1 ligand targeting of EphA2 restricted the ability of corneal epithelial cells to seal linear scratch wounds in a manner that was associated with a transient reduction in Erk1/2 and Akt activation state. Ephrin-A1-Fc treatment delayed wound healing independently of Mek-Erk1/2 signaling but was no longer capable of restricting migration after pharmacologic blockade of the PI3K-Akt pathway. Interestingly, ephrin-A1 immunoreactivity was increased in the corneal epithelia of diabetic individuals, mice maintained on a high-fat diet, or cultured corneal epithelial cells exposed to high glucose, which exhibit impaired Akt signaling and slower wound healing responses. CONCLUSIONS: EphA2 attenuates corneal epithelial cell migration when stimulated by ephrin-A1 ligand in a manner that involves the suppression of Akt. Elevated levels of ephrin-A1 may contribute to diabetic keratopathies by persistently engaging EphA2 and prohibiting Akt-dependent corneal epithelial repair processes.
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Neovascularización de la Córnea/patología , ADN/genética , Efrina-A1/genética , Epitelio Corneal/metabolismo , Regulación de la Expresión Génica , Receptor EphA2/genética , Animales , Western Blotting , Movimiento Celular , Células Cultivadas , Neovascularización de la Córnea/genética , Neovascularización de la Córnea/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Electroforesis en Gel de Poliacrilamida , Efrina-A1/biosíntesis , Epitelio Corneal/patología , Humanos , Inmunohistoquímica , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptor EphA2/biosíntesis , Transducción de Señal/genéticaRESUMEN
Normal vision requires the precise control of vascular growth to maintain corneal transparency. Here we provide evidence for a unique mechanism by which the Forkhead box transcription factor FoxC1 regulates corneal vascular development. Murine Foxc1 is essential for development of the ocular anterior segment, and in humans, mutations have been identified in Axenfeld-Rieger syndrome, a disorder characterized by anterior segment dysgenesis. We show that FOXC1 mutations also lead to corneal angiogenesis, and that mice homozygous for either a global (Foxc1(-/-)) or neural crest (NC)-specific (NC-Foxc1(-/-)) null mutation display excessive growth of corneal blood and lymphatic vessels. This is associated with disorganization of the extracellular matrix and increased expression of multiple matrix metalloproteinases. Heterozygous mutants (Foxc1(+/-) and NC-Foxc1(+/-)) exhibit milder phenotypes, such as disrupted limbal vasculature. Moreover, environmental exposure to corneal injury significantly increases growth of both blood and lymphatic vessels in both Foxc1(+/-) and NC-Foxc1(+/-) mice compared with controls. Notably, this amplification of the angiogenic response is abolished by inhibition of VEGF receptor 2. Collectively, these findings identify a role for FoxC1 in inhibiting corneal angiogenesis, thereby maintaining corneal transparency by regulating VEGF signaling.
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Vasos Sanguíneos/crecimiento & desarrollo , Córnea/irrigación sanguínea , Factores de Transcripción Forkhead/metabolismo , Álcalis , Animales , Segmento Anterior del Ojo/anomalías , Segmento Anterior del Ojo/metabolismo , Segmento Anterior del Ojo/patología , Vasos Sanguíneos/patología , Quemaduras/patología , Córnea/patología , Sustancia Propia/enzimología , Sustancia Propia/patología , Anomalías del Ojo/complicaciones , Anomalías del Ojo/metabolismo , Anomalías del Ojo/patología , Enfermedades Hereditarias del Ojo , Haploinsuficiencia/genética , Heterocigoto , Linfangiogénesis , Metaloproteinasas de la Matriz/metabolismo , Ratones , Mutación/genética , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología , Cresta Neural/metabolismo , Cresta Neural/patología , Fenotipo , Pupila , Transducción de Señal , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Limbal stem cells (LSCs) have an important role in the maintenance of the corneal surface epithelium, and autologous cultured limbal epithelial cell transplantations have contributed substantially to the treatment of the visually disabling condition known as LSC deficiency. In this protocol, we describe a method of establishing human limbal epithelial cell cultures by a feeder-free explant culture technique using a small limbal biopsy specimen and human amniotic membrane (hAM) as the culture substrate. This protocol is free of animal-derived products and involves the use of human recombinant growth factors. In addition, the recombinant cell dissociation enzyme TrypLE is used to replace trypsin and autologous serum replaces FBS. It takes approximately 2 weeks to establish a confluent monolayer from which approximately 3 x 10(6) cells can be harvested. This procedure can be adopted for both basic research purposes and clinical applications.
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Técnicas de Cultivo de Célula , Epitelio Corneal/citología , Amnios , Medios de Cultivo , HumanosRESUMEN
microRNA-205 (miR-205) and miR-184 coordinately regulate the lipid phosphatase SHIP2 for Akt survival signaling in keratinocytes. As the PI3K-Akt pathway has also been implicated in regulating the actin cytoskeleton and cell motility, we investigated the role that these 2 miRNAs play in keratinocyte migration. We used antagomirs (antago) to reduce the levels of miR-205 and miR-184 in primary human epidermal keratinocytes (HEKs) and corneal epithelial keratinocytes (HCEKs) as well as direct SHIP2 silencing using siRNA oligos. Treatment of HEKs and HCEKs with antago-205 increased SHIP2 levels and impaired the ability of these cells to seal linear scratch wounds compared with untreated or irrelevant-antago treatments. In contrast, AKT signaling was enhanced and wounds sealed faster in HCEKs where miR-184 was suppressed, enabling miR-205 to inhibit SHIP2. Similar increases in migration were observed following direct SHIP2 silencing in HEKs. Furthermore, down-regulation of miR-205 resulted in an increase in Rho-ROCKI activity, phosphorylation of the actin severing protein cofilin, and a corresponding diminution of filamentous actin. The connection among miR-205, RhoA-ROCKI-cofilin inactivation, and the actin cytoskeleton represents a novel post-translational mechanism for the regulation of normal human keratinocyte migration.
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Movimiento Celular/fisiología , Regulación de la Expresión Génica/genética , Cabello/metabolismo , Queratinocitos/citología , MicroARNs/fisiología , Piel/metabolismo , Animales , Secuencia de Bases , Western Blotting , Células Cultivadas , Cartilla de ADN , Citometría de Flujo , Cabello/citología , Hibridación in Situ , Ratones , Piel/citologíaRESUMEN
Corneal opacification due to limbal stem cell deficiency (LSCD) is an important cause for ocular morbidity, resulting from a number of intrinsic and extrinsic factors. While the extrinsic factors include conditions such as chemical or thermal injuries, intrinsic include dysfunction, or reduction in the number of stem cells either due to pathological changes in autoimmune diseases or secondary to certain clinical conditions such as diabetes, dry eye disorders, or multiple previous eye surgeries. LSCD is characterized by a classic triad of signs -- conjunctivalization, neovascularization and decrease in vision. With the increasing knowledge of limbal stem cells, the treatment of this condition has evolved from simple debridement to use of biological materials, direct transplantation of the healthy limbal tissue from the contralateral eye, or allogenic source to the use of cultivated limbal epithelial sheets. This chapter provides an update on the disease pathology, various treatment methodologies, with specific emphasis on the fast developing field of cell therapy and tissue engineering.
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Enfermedades de la Córnea/cirugía , Epitelio Corneal/citología , Limbo de la Córnea/citología , Trasplante de Células Madre , Animales , Trasplante de Células , Células Epiteliales/trasplante , Humanos , Células MadreRESUMEN
Despite their potential to regulate approximately one-third of the whole genome, relatively few microRNA (miRNA) targets have been experimentally validated, particularly in stratified squamous epithelia. Here we demonstrate not only that the lipid phosphatase SHIP2 is a target of miRNA-205 (miR-205) in epithelial cells, but, more importantly, that the corneal epithelial-specific miR-184 can interfere with the ability of miR-205 to suppress SHIP2 levels. This is the first example of a miRNA negatively regulating another to maintain levels of a target protein. Interfering with miR-205 function by using a synthetic antagomir, or by the ectopic expression of miR-184, leads to a coordinated damping of the Akt signaling pathway via SHIP2 induction. This was associated with a marked increase in keratinocyte apoptosis and cell death. Aggressive squamous cell carcinoma (SCC) cells exhibited elevated levels of miR-205. This was associated with a concomitant reduction in SHIP2 levels. Partial knockdown of endogenous miR-205 in SCCs markedly decreased phosphorylated Akt and phosphorylated BAD levels and increased apoptosis. We were able to increase SHIP2 levels in SCC cells after inhibition of miR-205. Therefore, miR-205 might have diagnostic value in determining the aggressivity of SCCs. Blockage of miR-205 activity with an antagomir or via ectopic expression of miR-184 could be novel therapeutic approaches for treating aggressive SCCs.
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Queratinocitos/fisiología , MicroARNs/genética , MicroARNs/metabolismo , Monoéster Fosfórico Hidrolasas/fisiología , Apoptosis/fisiología , Carcinoma de Células Escamosas/fisiopatología , Supervivencia Celular/fisiología , Regulación hacia Abajo/genética , Epitelio Corneal/citología , Regulación Neoplásica de la Expresión Génica , Células HeLa , Humanos , Queratinocitos/citología , Riñón/citología , Fosfatidilinositol-3,4,5-Trifosfato 5-Fosfatasas , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Interferente Pequeño , Factor de Transcripción STAT1/genética , Neoplasias Cutáneas/fisiopatología , TransfecciónRESUMEN
PURPOSE: Mesenchymal stem cells (MSC) are self-renewing, multipotent cells that are present in many adult tissues, including bone marrow, trabecular bone, adipose, and muscle. The presence of such cells of mesenchymal origin and their role during the wound healing of ocular injuries are currently being explored by many studies worldwide. In this study, we aimed to report the presence of mesenchymal cells resembling bone marrow-derived cells (MSC-BM) in the limbus of the human eye. METHODS: Fresh limbal tissues obtained from human subjects undergoing limbal biopsy for ocular surface reconstruction were used to establish limbal mesenchymal cell (MC-L) cultures. The spindle cell outgrowths observed in extended limbal epithelial cultures (LECs) and from deepithelialized limbal tissues were serially passaged using a human corneal epithelial (HCE) medium, which contained epidermal growth factor (EGF) and insulin, and supplemented with fetal bovine serum (FBS). MSC cultures were established from human bone marrow samples using Dulbecco's Modified Eagles Medium (DMEM) supplemented with FBS. The mesenchymal cells from both extended limbal cultures (MC-L) and bone marrow (MSC-BM) were characterized by morphology and immunophenotyping using epithelial, mesenchymal, hematopoietic, and endothelial markers using fluorescent-activated cell sorting (FACS). Selective markers were further confirmed by immunostaining and reverse transcription polymerase chain reaction (RT-PCR). Stromal cells of both origins (limbal and bone marrow-derived) were also evaluated for colony forming ability and population doubling. Attempts were made to differentiate these into adipocytes and osteocytes using conditioned medium. RESULTS: Spindle cells from extended limbal epithelial cultures as well as de-epithelialized human limbal tissues appeared elongated and fibroblast-like with oval vesicular nuclei. Both MC-L and MSC-BM showed colony forming ability in 14 days of plating. MC-L showed a population doubling of 22.95 while in MSC-BM, it was 30.98. Immunophenotyping of these cells by FACS and immunocytochemistry showed that the MC-L were positive for mesenchymal markers and negative for epithelial and hematopoietic markers similar to MSC-BM. The MC-L phenotype has thus been defined as MC-L(CD105, CD106, CD54, CD166, CD90, CD29, CD71, pax -6 +/ p75, SSEA1, Tra-1-61, Tra-1-81, CD31, CD34, CD45, CD11a, CD11c, CD14, CD138, Flk1, Flt1, VE-Cadherin -). The profile was further confirmed by RT-PCR. These cells also showed differentiation into adipocytes and osteocytes. CONCLUSIONS: We demonstrated the presence of mesenchymal cells in the human limbus, similar to the bone marrow-derived MSC-BM. This presence suggests that these cells are unique to the adult stem cell niche.
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Limbo de la Córnea/citología , Células Madre Mesenquimatosas/citología , Células del Estroma/citología , Adipocitos/citología , Biomarcadores/metabolismo , Células de la Médula Ósea/citología , Diferenciación Celular , Proliferación Celular , Células Cultivadas , Ensayo de Unidades Formadoras de Colonias , Células Epiteliales/citología , Citometría de Flujo , Humanos , Microscopía Confocal , Osteocitos/citología , Fenotipo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
A 6-year-old Bangladeshi girl presented with total limbal stem cell deficiency in the left eye, secondary to a 6-month-old chemical injury. The patient had also previously undergone two limbal transplantation surgeries. At the authors' centre the child underwent autologous cultured limbal epithelium transplantation, on human amniotic membrane, without the use of air-lift technique. Symptomatic relief, re-epithelialization of the ocular surface, regression of corneal pannus and slight improvement in vision were all noted. The corneal button obtained at the time of keratoplasty (performed 4 months later) revealed stratified epithelium with basement membrane. Thirty-seven months post keratoplasty, the best-corrected visual acuity was 6/15 with clear graft and stable ocular surface. Herein, a case of limbal stem cell deficiency successfully managed by monolayer of cultured limbal epithelium is presented.
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Quemaduras Químicas/cirugía , Enfermedades de la Córnea/cirugía , Epitelio Corneal/trasplante , Quemaduras Oculares/inducido químicamente , Limbo de la Córnea/citología , Trasplante de Células Madre , Quemaduras Químicas/patología , Compuestos de Calcio , Supervivencia Celular , Células Cultivadas , Niño , Enfermedades de la Córnea/patología , Células Epiteliales/trasplante , Quemaduras Oculares/cirugía , Femenino , Humanos , Queratoplastia Penetrante , Óxidos , Cicatrización de HeridasRESUMEN
A 12-year-old girl with total limbal stem cell deficiency in the right eye following chemical burns underwent autologous cultivated limbal epithelium transplantation from the healthy left eye. Postoperatively at 6 weeks a mass at the limbus was noted, which increased in size and involved infero-nasal limbus extending over 5 mm on bulbar conjunctiva. It was a gelatinous, placoid freely movable mass with irregular surface, multiple intralesional cysts without feeder vessels or intrinsic vascularization and stained brilliantly with rose bengal. Histopathology following excision biopsy showed hyperplastic epithelium with stratified columnar cells and goblet cells. At the last follow-up, 6 months following cultivated limbal epithelium transplantation the ocular surface was stable without any recurrence of the lesion. We herein report a rare complication of epithelial hyperplasia presenting as leukoplakia following cultivated limbal epithelium transplantation mimicking ocular surface squamous neoplasia.
Asunto(s)
Carcinoma de Células Escamosas/diagnóstico , Trasplante de Córnea/efectos adversos , Epitelio Corneal/patología , Neoplasias del Ojo/diagnóstico , Limbo de la Córnea/cirugía , Complicaciones Posoperatorias/patología , Niño , Diagnóstico Diferencial , Femenino , Humanos , Hiperplasia/etiología , Hiperplasia/patología , Complicaciones Posoperatorias/etiologíaRESUMEN
PURPOSE: To report the clinical outcome of autologous cultivated limbal epithelial transplantation. METHODS: Eighty-six patients' records and their clinical photographs were reviewed for demographics, primary etiology, type of limbal transplantation, ocular surface stability, visual acuity, final outcome and possible factors affecting outcome and complications. RESULTS: Eighty-eight eyes of 86 patients with limbal stem cell deficiency (LSCD) underwent autologous cultivated limbal epithelium transplantation between March 2001 and May 2003, with a mean follow-up of 18.3 months. The etiology of LSCD was alkali burns in 64% patients. Sixty-one eyes had total LSCD. Thirty-two of the 88 eyes had undergone amniotic membrane transplantation and 10 eyes had previously undergone limbal transplantation with unfavorable outcome. Nineteen eyes underwent penetrating keratoplasty, of which 11 grafts survived at the final follow-up. Finally, 57 eyes (73.1%, 95% CI: 63.3-82.9) had a successful outcome with a stable ocular surface without conjunctivalization, 21 eyes (26.9%, 95%CI: 17.1-36.7) were considered failures and 10 patients were lost to follow-up. CONCLUSION: LSCD can be successfully treated by autologous cultivated limbal epithelium transplantation in majority of the cases.