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1.
Nat Commun ; 14(1): 8452, 2023 Dec 19.
Artículo en Inglés | MEDLINE | ID: mdl-38114516

RESUMEN

Lung epithelial regeneration after acute injury requires coordination cellular coordination to pattern the morphologically complex alveolar gas exchange surface. During adult lung regeneration, Wnt-responsive alveolar epithelial progenitor (AEP) cells, a subset of alveolar type 2 (AT2) cells, proliferate and transition to alveolar type 1 (AT1) cells. Here, we report a refined primary murine alveolar organoid, which recapitulates critical aspects of in vivo regeneration. Paired scRNAseq and scATACseq followed by transcriptional regulatory network (TRN) analysis identified two AT1 transition states driven by distinct regulatory networks controlled in part by differential activity of Nkx2-1. Genetic ablation of Nkx2-1 in AEP-derived organoids was sufficient to cause transition to a proliferative stressed Krt8+ state, and AEP-specific deletion of Nkx2-1 in adult mice led to rapid loss of progenitor state and uncontrolled growth of Krt8+ cells. Together, these data implicate dynamic epigenetic maintenance via Nkx2-1 as central to the control of facultative progenitor activity in AEPs.


Asunto(s)
Epigenómica , Pulmón , Animales , Ratones , Diferenciación Celular , Células Epiteliales , Homeostasis , Células Madre
2.
Nat Commun ; 14(1): 1975, 2023 04 08.
Artículo en Inglés | MEDLINE | ID: mdl-37031202

RESUMEN

Persistent HPV16 infection is a major cause of the global cancer burden. The viral life cycle is dependent on the differentiation program of stratified squamous epithelium, but the landscape of keratinocyte subpopulations which support distinct phases of the viral life cycle has yet to be elucidated. Here, single cell RNA sequencing of HPV16 infected compared to uninfected organoids identifies twelve distinct keratinocyte populations, with a subset mapped to reconstruct their respective 3D geography in stratified squamous epithelium. Instead of conventional terminally differentiated cells, an HPV-reprogrammed keratinocyte subpopulation (HIDDEN cells) forms the surface compartment and requires overexpression of the ELF3/ESE-1 transcription factor. HIDDEN cells are detected throughout stages of human carcinogenesis including primary human cervical intraepithelial neoplasias and HPV positive head and neck cancers, and a possible role in promoting viral carcinogenesis is supported by TCGA analyses. Single cell transcriptome information on HPV-infected versus uninfected epithelium will enable broader studies of the role of individual keratinocyte subpopulations in tumor virus infection and cancer evolution.


Asunto(s)
Carcinoma de Células Escamosas , Proteínas Oncogénicas Virales , Infecciones por Papillomavirus , Femenino , Humanos , Papillomavirus Humano 16/genética , Papillomavirus Humano 16/metabolismo , Transcriptoma , Epitelio/metabolismo , Queratinocitos/metabolismo , Carcinogénesis/genética , Carcinoma de Células Escamosas/genética , Proteínas Oncogénicas Virales/genética
3.
Front Immunol ; 13: 1061544, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-36505495

RESUMEN

Introduction: Neutrophils are critical for host immune defense; yet, aberrant neutrophil tissue infiltration triggers tissue damage. Neutrophils are heterogeneous functionally, and adopt 'normal' or 'pathogenic' effector function responses. Understanding neutrophil heterogeneity could provide specificity in targeting inflammation. We previously identified a signaling pathway that suppresses neutrophilmediated inflammation via integrin-mediated Rap1b signaling pathway. Methods: Here, we used Rap1-deficient neutrophils and proteomics to identify pathways that specifically control pathogenic neutrophil effector function. Results: We show neutrophil acidity is normally prevented by Rap1b during normal immune response with loss of Rap1b resulting in increased neutrophil acidity via enhanced Ldha activity and abnormal neutrophil behavior. Acidity drives the formation of abnormal invasive-like protrusions in neutrophils, causing a shift to transcellular migration through endothelial cells. Acidity increases neutrophil extracellular matrix degradation activity and increases vascular leakage in vivo. Pathogenic inflammatory condition of ischemia/reperfusion injury is associated with increased neutrophil transcellular migration and vascular leakage. Reducing acidity with lactate dehydrogenase inhibition in vivo limits tissue infiltration of pathogenic neutrophils but less so of normal neutrophils, and reduces vascular leakage. Discussion: Acidic milieu renders neutrophils more dependent on Ldha activity such that their effector functions are more readily inhibited by small molecule inhibitor of Ldha activity, which offers a therapeutic window for antilactate dehydrogenase treatment in specific targeting of pathogenic neutrophils in vivo.


Asunto(s)
Células Endoteliales , Neutrófilos , Humanos , Movimiento Celular , Infiltración Neutrófila , Inflamación , L-Lactato Deshidrogenasa , Proteínas de Unión al GTP rap
4.
Viruses ; 13(1)2021 Jan 06.
Artículo en Inglés | MEDLINE | ID: mdl-33418959

RESUMEN

Juvenile-onset recurrent respiratory papillomatosis (JoRRP) is driven by human papillomavirus (HPV) low-risk strains and is associated with significant morbidity. While previous studies of 2D cultures have shed light on disease pathogenesis and demonstrated the utility of personalized medicine approaches, monolayer cultures lack the 3D tissue architecture and physiology of stratified, sequentially differentiated mucosal epithelium important in RRP disease pathogenesis. Herein we describe the establishment of JoRRP-derived primary cell populations that retain HPV genomes and viral gene expression in culture. These were directly compared to cells from matched adjacent non-diseased tissue, given the known RRP patient-to-patient variability. JoRRP papilloma versus control cells displayed decreased growth at subconfluency, with a switch to increased growth after reaching confluency, suggesting relative resistance to cell-cell contact and/or differentiation. The same papilloma cells grown as 3D organotypic rafts harbored hyperproliferation as compared to controls, with increased numbers of proliferating basal cells and inappropriately replicating suprabasal cells, mimicking phenotypes in the patient biopsies from which they were derived. These complementary model systems provide novel opportunities to elucidate disease mechanisms at distinct stages in JoRRP progression and to identify diagnostic, prognostic and therapeutic factors to personalize patient management and treatment.


Asunto(s)
Alphapapillomavirus/genética , Alphapapillomavirus/aislamiento & purificación , Células Epiteliales/virología , Infecciones por Papillomavirus/virología , Infecciones del Sistema Respiratorio/patología , Humanos , Técnicas de Cultivo de Órganos , Infecciones por Papillomavirus/patología , Fenotipo , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones del Sistema Respiratorio/virología , Factores de Riesgo
5.
Cell Stem Cell ; 26(3): 420-430.e6, 2020 03 05.
Artículo en Inglés | MEDLINE | ID: mdl-32059807

RESUMEN

The metabolic requirements of hematopoietic stem cells (HSCs) change with their cell cycle activity. However, the underlying role of mitochondria remains ill-defined. Here we found that, after mitochondrial activation with replication, HSCs irreversibly remodel the mitochondrial network and that this network is not repaired after HSC re-entry into quiescence, contrary to hematopoietic progenitors. HSCs keep and accumulate dysfunctional mitochondria through asymmetric segregation during active division. Mechanistically, mitochondria aggregate and depolarize after stress because of loss of activity of the mitochondrial fission regulator Drp1 onto mitochondria. Genetic and pharmacological studies indicate that inactivation of Drp1 causes loss of HSC regenerative potential while maintaining HSC quiescence. Molecularly, HSCs carrying dysfunctional mitochondria can re-enter quiescence but fail to synchronize the transcriptional control of core cell cycle and metabolic components in subsequent division. Thus, loss of fidelity of mitochondrial morphology and segregation is one type of HSC divisional memory and drives HSC attrition.


Asunto(s)
Células Madre Hematopoyéticas , Mitocondrias , Ciclo Celular , División Celular , Autorrenovación de las Células , Células Madre Hematopoyéticas/metabolismo
6.
Hepatology ; 72(5): 1800-1818, 2020 11.
Artículo en Inglés | MEDLINE | ID: mdl-32064648

RESUMEN

BACKGROUND AND AIMS: During liver injury, quiescent hepatic stellate cells (qHSCs) transdifferentiate into proliferative and fibrogenic activated myofibroblastic phenotype (activated hepatic stellate cell; aHSCs) expressing smooth muscle α-actin (αSMA) and platelet-derived growth factor beta receptor (PDGFßR). Their interactions with gut-derived bacterial lipopolysaccharide (LPS) are implicated in hepatic fibrogenesis. However, LPS can also attenuate fibrogenic characteristics of aHSCs. APPROACH AND RESULTS: We examined molecular mechanisms of antifibrogenic effects of LPS on aHSCs in vitro and in vivo. Culture-activated rat HSCs were exposed to 0-100 ng/mL of LPS or its active component, diphosphoryl-lipid A (DPLA), and parameters of fibrosis and inflammatory cytokines/chemokines were determined by qRT-PCR, western, and immunohistochemical analyses. In vivo, HSCs were activated by repeated CCl4 administration to rats every 3 days for 3 or 8 weeks, then challenged with LPS (5 mg/kg; IP). HSCs were isolated 24 hours later, and fibrogenic/inflammatory parameters were analyzed. LPS induced phenotypic changes in aHSCs (rounding, size reduction) and loss of proliferation. LPS down-regulated expression of αSMA, PDGFßR, transforming growth factor beta receptor 1 (TGFßR1), collagen 1α1 (Col1α1), and fibronectin while up-regulating tumor necrosis factor alpha, interleukin-6, and C-X-C motif chemokine ligand 1 expression. LPS did not increase peroxisome proliferation-activated receptor gamma expression or lipid accumulation typical of qHSCs. DPLA elicited the same effects as LPS on aHSCs, indicating specificity, and monophosphoryl lipid A down-regulated fibrogenic markers, but elicited very weak inflammatory response. LPS down-regulated the expression of cMyb, a transcription factor for αSMA, and up-regulated small mother against decapentaplegic (SMAD)7 and CCAAT/enhancer-binding protein (C/EBP)δ, the transcriptional inhibitors of Col1α1 expression. In vivo LPS treatment of aHSCs inhibited their proliferation, down-regulated PDGFßR, αSMA, TGFßR1, Col1α1, and cMyb expression, and increased expression of SMAD7, C/EBPα, and C/EBPδ. CONCLUSIONS: In conclusion, LPS induces a unique phenotype in aHSCs associated with down-regulation of key fibrogenic mechanisms and thus may have an important role in limiting fibrosis.


Asunto(s)
Regulación de la Expresión Génica/inmunología , Células Estrelladas Hepáticas/inmunología , Lípido A/análogos & derivados , Cirrosis Hepática Experimental/inmunología , Hígado/patología , Animales , Proteína delta de Unión al Potenciador CCAAT/metabolismo , Tetracloruro de Carbono/administración & dosificación , Tetracloruro de Carbono/toxicidad , Transdiferenciación Celular/inmunología , Células Cultivadas , Citocinas/genética , Citocinas/inmunología , Regulación hacia Abajo , Silenciador del Gen , Células Estrelladas Hepáticas/patología , Humanos , Lípido A/inmunología , Lípido A/metabolismo , Hígado/citología , Hígado/inmunología , Cirrosis Hepática Experimental/inducido químicamente , Cirrosis Hepática Experimental/patología , Ratones , Ratones Noqueados , Miofibroblastos/inmunología , Miofibroblastos/patología , Oxidorreductasas actuantes sobre Donantes de Grupos Sulfuro/genética , Cultivo Primario de Células , Proteínas Proto-Oncogénicas c-myb/metabolismo , Ratas , Transducción de Señal/genética , Transducción de Señal/inmunología , Proteína smad7/genética , Proteína smad7/metabolismo , Regulación hacia Arriba/inmunología
7.
Dev Cell ; 51(6): 665-674.e6, 2019 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-31813796

RESUMEN

The trachea and esophagus arise from the separation of a common foregut tube during early fetal development. Mutations in key signaling pathways such as Hedgehog (HH)/Gli can disrupt tracheoesophageal (TE) morphogenesis and cause life-threatening birth defects (TEDs); however, the underlying cellular mechanisms are unknown. Here, we use mouse and Xenopus to define the HH/Gli-dependent processes orchestrating TE morphogenesis. We show that downstream of Gli the Foxf1+ splanchnic mesenchyme promotes medial constriction of the foregut at the boundary between the presumptive Sox2+ esophageal and Nkx2-1+ tracheal epithelium. We identify a unique boundary epithelium co-expressing Sox2 and Nkx2-1 that fuses to form a transient septum. Septum formation and resolution into distinct trachea and esophagus requires endosome-mediated epithelial remodeling involving the small GTPase Rab11 and localized extracellular matrix degradation. These are disrupted in Gli-deficient embryos. This work provides a new mechanistic framework for TE morphogenesis and informs the cellular basis of human TEDs.


Asunto(s)
Endosomas/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Proteínas Hedgehog/metabolismo , Morfogénesis/fisiología , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Sistema Digestivo/metabolismo , Endodermo/metabolismo , Endosomas/genética , Esófago/embriología , Factores de Transcripción Forkhead/metabolismo , Humanos , Mesodermo/metabolismo , Mutación/genética , Xenopus
8.
Lab Chip ; 18(20): 3079-3085, 2018 10 09.
Artículo en Inglés | MEDLINE | ID: mdl-30238091

RESUMEN

Current in vitro approaches and animal models have critical limitations for modeling human gastrointestinal diseases because they may not properly represent multicellular human primary tissues. Therefore, there is a need for model platforms that recapitulate human in vivo development, physiology, and disease processes to validate new therapeutics. One of the major steps toward this goal was the generation of three-dimensional (3D) human gastric organoids (hGOs) via the directed differentiation of human pluripotent stem cells (hPSCs). The normal functions and diseases of the stomach occur in the luminal epithelium, however accessing the epithelium on the inside of organoids is challenging. We sought to develop a bioengineered platform to introduce luminal flow through hGOs to better model in vivo gastric functions. Here, we report an innovative microfluidic imaging platform housing hGOs with peristaltic luminal flow in vitro. This human stomach-on-a-chip allows robust, long-term, 3D growth of hGOs with the capacity for luminal delivery via a peristaltic pump. Organoids were cannulated and medium containing fluorescent dextran was delivered through the lumen using a peristaltic pump. This system also allowed us to rhythmically introduce stretch and contraction to the organoid, reminiscent of gastric motility. Our platform has the potential for long-term delivery of nutrients or pharmacological agents into the gastric lumen in vitro for the study of human gastric physiology, disease modeling, and drug screening, among other possibilities.


Asunto(s)
Motilidad Gastrointestinal , Estómago/citología , Estómago/fisiología , Análisis de Matrices Tisulares/métodos , Humanos , Organoides/citología , Análisis de Matrices Tisulares/instrumentación
9.
Nat Cell Biol ; 20(11): 1328, 2018 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-30190576

RESUMEN

In the version of this Article originally published, in ref. 34 the first author's name was spelled incorrectly. The correct reference is: Rodón, L. et al. Active CREB1 promotes a malignant TGFß2 autocrine loop in glioblastoma. Cancer Discov. 10, 1230-1241 (2014). This has now been amended in all online versions of the Article.

10.
Nat Cell Biol ; 20(10): 1228, 2018 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-30089841

RESUMEN

In the version of this Article originally published, the competing interests statement was missing. The authors declare no competing interests; this statement has now been added in all online versions of the Article.

11.
Curr Protoc Cytom ; 86(1): e38, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-30005145

RESUMEN

Biologic tissues are generally opaque due to optical properties that result in scattering and absorption of light. Preparation of tissues for optical microscopy often involves sectioning to a thickness of 50-100 µm, the practical limits of light penetration and recovery. A researcher who wishes to image a whole tissue must acquire potentially hundreds of individual sections before rendering them into a three-dimensional volume. Clearing removes strongly light-scattering and light-absorbing components of a tissue and equalizes the refractive index of the imaging medium to that of the tissue. After clearing, the maximum depth of imaging is often defined by the microscope optics rather than the tissue. Such visibility enables the interrogation of whole tissues and even animals without the need to section. Researchers can study a biological process in the context of its three-dimensional environment, identify rare events in large volumes of tissues, and trace cells and cell-cell interactions over large distances. This article describes four popular clearing protocols that are relevant to a wide variety of scenarios across biologic disciplines: CUBIC, CLARITY, 3DISCO, and SeeDB. © 2018 by John Wiley & Sons, Inc.


Asunto(s)
Imagenología Tridimensional/métodos , Animales , Árboles de Decisión , Fluorescencia , Ratones , Solventes , Coloración y Etiquetado
12.
Nat Cell Biol ; 20(7): 823-835, 2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29915361

RESUMEN

Stress is integral to tumour evolution, and cancer cell survival depends on stress management. We found that cancer-associated stress chronically activates the bioenergetic sensor AMP kinase (AMPK) and, to survive, tumour cells hijack an AMPK-regulated stress response pathway conserved in normal cells. Analysis of The Cancer Genome Atlas data revealed that AMPK isoforms are highly expressed in the lethal human cancer glioblastoma (GBM). We show that AMPK inhibition reduces viability of patient-derived GBM stem cells (GSCs) and tumours. In stressed (exercised) skeletal muscle, AMPK is activated to cooperate with CREB1 (cAMP response element binding protein-1) and promote glucose metabolism. We demonstrate that oncogenic stress chronically activates AMPK in GSCs that coopt the AMPK-CREB1 pathway to coordinate tumour bioenergetics through the transcription factors HIF1α and GABPA. Finally, we show that adult mice tolerate systemic deletion of AMPK, supporting the use of AMPK pharmacological inhibitors in the treatment of GBM.


Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Neoplasias Encefálicas/enzimología , Proliferación Celular , Metabolismo Energético , Glioblastoma/enzimología , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/genética , Animales , Antineoplásicos/farmacología , Apoptosis , Autofagia , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Metabolismo Energético/efectos de los fármacos , Femenino , Factor de Transcripción de la Proteína de Unión a GA/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Regulación Neoplásica de la Expresión Génica , Glioblastoma/tratamiento farmacológico , Glioblastoma/genética , Glioblastoma/patología , Células HEK293 , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Masculino , Ratones Endogámicos NOD , Ratones SCID , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/enzimología , Células Madre Neoplásicas/patología , Inhibidores de Proteínas Quinasas/farmacología , Transducción de Señal , Factores de Tiempo , Transcripción Genética , Carga Tumoral , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de Xenoinjerto
13.
Hepatology ; 65(1): 174-188, 2017 01.
Artículo en Inglés | MEDLINE | ID: mdl-27641439

RESUMEN

Biliary atresia (BA) is a fibroinflammatory obstruction of the extrahepatic biliary tree in neonates. While intrahepatic bile duct proliferation is universal at diagnosis, bile duct paucity develops later. We hypothesized that polarized T helper lymphocyte responses orchestrate progression of intrahepatic biliary injury in this disease. Interleukin 17A (IL-17A)-green fluorescent protein, cluster of differentiation 11c (CD11c)/diphtheria toxin receptor, and IL-17 receptor A-/- mice were used to examine T-lymphocyte polarization, inflammatory leukocyte recruitment, and biliary injury in rhesus rotavirus-induced BA. Multiparameter flow cytometry and automated image analysis of immunostaining were applied to liver tissue samples from infants with BA. In the mouse model, activated CD4+ lymphocytes started to emerge in the liver on day 8 after viral challenge, while innate immune responses were waning. Plasma IL-17A levels rose concomitantly with hepatic accumulation of T helper 17 lymphocytes and myeloid dendritic cells. Targeted depletion of CD11c+ dendritic cells diminished hepatic IL-17A production and ameliorated intrahepatic bile duct injury. Recombinant IL-17A induced expression of chemokine (C-C motif) ligand 2 in neonatal cholangiocytes in vitro, and blockade of the corresponding chemokine (C-C motif) receptor 2 reduced recruitment of inflammatory macrophages to the liver in vivo. Genetic disruption of IL-17A signaling was associated with down-regulation of hepatic Ccl2/Ccr2 messenger RNA expression, reduced infiltration of the liver with inflammatory Ly6Chi macrophages, and improved survival. In the liver of infants with BA, cholangiocytes were found to express IL-17 receptor A, and the prevalence of IL-17A+ cells was positively correlated with the degree of CD68+ macrophage infiltration at diagnosis. Hepatic CD4+ lymphocytes were chief producers of IL-17A in patients with progressive disease undergoing liver transplantation. CONCLUSION: These findings identify the dendritic cell-T helper 17-macrophage axis as a target for the development of strategies to block progression of intrahepatic bile duct injury in patients with BA. (Hepatology 2017;65:174-188).


Asunto(s)
Atresia Biliar/inmunología , Células Dendríticas/fisiología , Macrófagos/fisiología , Células Th17/fisiología , Animales , Conductos Biliares Intrahepáticos/citología , Progresión de la Enfermedad , Células Epiteliales/patología , Humanos , Ratones , Ratones Endogámicos BALB C
14.
Genes Brain Behav ; 16(2): 250-259, 2017 02.
Artículo en Inglés | MEDLINE | ID: mdl-27594048

RESUMEN

Development of the mammalian forebrain requires a significant contribution from tubulin proteins to physically facilitate both the large number of mitoses in the neurogenic brain (in the form of mitotic spindles) as well as support cellular scaffolds to guide radial migration (radial glial neuroblasts). Recent studies have identified a number of mutations in human tubulin genes affecting the forebrain, including TUBB2B . We previously identified a mouse mutation in Tubb2b and we show here that mice heterozygous for this missense mutation in Tubb2b have significant cognitive defects in spatial learning and memory. We further showed reduced hippocampal long-term potentiation consistent with these defects. In addition to the behavioural and physiological deficits, we show here abnormal hippocampal morphology. Taken together, these phenotypes suggest that heterozygous mutations in tubulin genes result in cognitive deficits not previously appreciated. This has implications for design and interpretation of genetic testing for humans with intellectual disability disorders.


Asunto(s)
Trastornos del Conocimiento/genética , Hipocampo/patología , Tubulina (Proteína)/genética , Animales , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Trastornos del Conocimiento/metabolismo , Heterocigoto , Hipocampo/metabolismo , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Memoria/fisiología , Ratones , Mutación , Mutación Missense , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Neurogénesis/genética , Neuronas/metabolismo , Neuronas/patología , Fenotipo , Aprendizaje Espacial/fisiología , Tubulina (Proteína)/metabolismo
15.
J Immunol ; 197(11): 4228-4239, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27794000

RESUMEN

KIF3A, the gene encoding kinesin family member 3A, is a susceptibility gene locus associated with asthma; however, mechanisms by which KIF3A might influence the pathogenesis of the disorder are unknown. In this study, we deleted the mouse Kif3a gene in airway epithelial cells. Both homozygous and heterozygous Kif3a gene-deleted mice were highly susceptible to aeroallergens from Aspergillus fumigatus and the house dust mite, resulting in an asthma-like pathology characterized by increased goblet cell metaplasia, airway hyperresponsiveness, and Th2-mediated inflammation. Deletion of the Kif3a gene increased the severity of pulmonary eosinophilic inflammation and expression of cytokines (Il-4, Il-13, and Il-17a) and chemokine (Ccl11) RNAs following pulmonary exposure to Aspergillus extract. Inhibition of Kif3a disrupted the structure of motile cilia and impaired mucociliary clearance, barrier function, and epithelial repair, demonstrating additional mechanisms by which deficiency of KIF3A in respiratory epithelial cells contributes to pulmonary pathology. Airway epithelial KIF3A suppresses Th2 pulmonary inflammation and airway hyperresponsiveness following aeroallergen exposure, implicating epithelial microtubular functions in the pathogenesis of Th2-mediated lung pathology.


Asunto(s)
Alérgenos/inmunología , Aspergillus fumigatus/inmunología , Asma/inmunología , Células Epiteliales/inmunología , Cinesinas/inmunología , Mucosa Respiratoria/inmunología , Células Th2/inmunología , Animales , Asma/inducido químicamente , Asma/genética , Asma/patología , Citocinas/genética , Citocinas/inmunología , Células Epiteliales/patología , Cinesinas/genética , Pulmón/inmunología , Pulmón/patología , Ratones , Ratones Transgénicos , Mucosa Respiratoria/patología , Células Th2/patología
16.
Open Biol ; 6(8)2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27488374

RESUMEN

Nodal class TGF-ß signalling molecules play essential roles in establishing the vertebrate body plan. In all vertebrates, nodal family members have specific waves of expression required for tissue specification and axis formation. In Xenopus laevis, six nodal genes are expressed before gastrulation, raising the question of whether they have specific roles or act redundantly with each other. Here, we examine the role of Xnr5. We find it acts at the late blastula stage as a mesoderm inducer and repressor of ectodermal gene expression, a role it shares with Vg1. However, unlike Vg1, Xnr5 depletion reduces the expression of the nodal family member xnr1 at the gastrula stage. It is also required for left/right laterality by controlling the expression of the laterality genes xnr1, antivin (lefty) and pitx2 at the tailbud stage. In Xnr5-depleted embryos, the heart field is established normally, but symmetrical reduction in Xnr5 levels causes a severely stunted midline heart, first evidenced by a reduction in cardiac troponin mRNA levels, while left-sided reduction leads to randomization of the left/right axis. This work identifies Xnr5 as the earliest step in the signalling pathway establishing normal heart laterality in Xenopus.


Asunto(s)
Blástula/metabolismo , Tipificación del Cuerpo , Corazón/crecimiento & desarrollo , Ligandos de Señalización Nodal/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Animales , Regulación del Desarrollo de la Expresión Génica , Factores de Determinación Derecha-Izquierda/metabolismo , Ligandos de Señalización Nodal/genética , Transducción de Señal , Factores de Transcripción/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis/genética , Xenopus laevis/metabolismo
17.
Biofabrication ; 7(3): 035007, 2015 Sep 03.
Artículo en Inglés | MEDLINE | ID: mdl-26335389

RESUMEN

Multiphoton fabrication is a powerful technique for three-dimensional (3D) printing of structures at the microscale. Many polymers and proteins have been successfully structured and patterned using this method. Type I collagen comprises a large part of the extracellular matrix for most tissue types and is a widely used cellular scaffold material for tissue engineering. Current methods for creating collagen tissue scaffolds do not allow control of local geometry on a cellular scale. This means the environment experienced by cells may be made up of the native material but unrelated to native cellular-scale structure. In this study, we present a novel method to allow multiphoton crosslinking of type I collagen with flavin mononucleotide photosensitizer. The method detailed allows full 3D printing of crosslinked structures made from unmodified type I collagen and uses only demonstrated biocompatible materials. Resolution of 1 µm for both standing lines and high-aspect ratio gaps between structures is demonstrated and complex 3D structures are fabricated. This study demonstrates a means for 3D printing with one of the most widely used tissue scaffold materials. High-resolution, 3D control of the fabrication of collagen scaffolds will facilitate higher fidelity recreation of the native extracellular environment for engineered tissues.


Asunto(s)
Materiales Biocompatibles/química , Bioimpresión/métodos , Colágeno Tipo I/química , Impresión Tridimensional , Ingeniería de Tejidos/métodos , Andamios del Tejido/química , Reactivos de Enlaces Cruzados , Mononucleótido de Flavina , Fármacos Fotosensibilizantes
18.
J Appl Physiol (1985) ; 118(7): 921-31, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25614601

RESUMEN

Synthesis and remodeling of the lung matrix is necessary for primary and compensatory lung growth. Because cyclic negative force is applied to developing lung tissue during the respiratory cycle, we hypothesized that stretch is a critical regulator of lung matrix remodeling. By using quantitative image analysis of whole-lung and whole-lobe elastin in situ zymography images, we demonstrated that elastase activity increased twofold during the alveolar stage of postnatal lung morphogenesis in the mouse. Remodeling was restricted to alveolar walls and ducts and was nearly absent in dense elastin band structures. In the mouse pneumonectomy model of compensatory lung growth, elastase activity increased threefold, peaking at 14 days postpneumonectomy and was higher in the accessory lobe compared with other lobes. Remodeling during normal development and during compensatory lung growth was different with increased major airway and pulmonary arterial remodeling during development but not regeneration, and with homogenous remodeling throughout the parenchyma during development, but increased remodeling only in subpleural regions during compensatory lung growth. Left lung wax plombage prevented increased lung elastin during compensatory lung growth. To test whether the adult lung retains an innate capacity to remodel elastin, we developed a confocal microscope-compatible stretching device. In ex vivo adult mouse lung sections, lung elastase activity increased exponentially with strain and in peripheral regions of lung more than in central regions. Our study demonstrates that lung elastase activity is stretch-dependent and supports a model in which externally applied forces influence the composition, structure, and function of the matrix during periods of alveolar septation.


Asunto(s)
Pulmón/enzimología , Pulmón/crecimiento & desarrollo , Mecanotransducción Celular/fisiología , Morfogénesis/fisiología , Elastasa Pancreática/fisiología , Animales , Módulo de Elasticidad/fisiología , Activación Enzimática , Técnicas In Vitro , Ratones , Ratones Endogámicos C57BL , Estrés Mecánico , Resistencia a la Tracción/fisiología , Distribución Tisular
19.
Biotechniques ; 57(5): 254-6, 2014 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-25391914

RESUMEN

Two-color fluorescent in situ hybridization (FISH) is a widely used technique for comparing relative gene expression patterns. Current two-color FISH protocols are not ideal for detecting weakly expressed transcripts or monitoring signal strength and background levels during the course of the reaction. Here we describe an improved FISH protocol using the conventional highly sensitive chromogenic substrates nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Vector Red in zebrafish embryos. This protocol substantially improves on existing FISH techniques by combining the advantages of long reactivity of alkaline phosphatase, chromogenic monitoring of both developing reactions, and the ability to perform subsequent high-resolution fluorescent imaging. Although tested in zebrafish, a similar approach is expected to be applicable to ISH in any model organism.


Asunto(s)
Compuestos Cromogénicos/análisis , Embrión no Mamífero/química , Hibridación Fluorescente in Situ/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Animales , Embrión no Mamífero/citología , Embrión no Mamífero/embriología , Pez Cebra
20.
PLoS One ; 9(8): e104675, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-25144761

RESUMEN

SOX17 is a key transcriptional regulator that can act by regulating other transcription factors including HNF1ß and FOXA2, which are known to regulate postnatal ß cell function. Given this, we investigated the role of SOX17 in the developing and postnatal pancreas and found a novel role for SOX17 in regulating insulin secretion. Deletion of the Sox17 gene in the pancreas (Sox17-paLOF) had no observable impact on pancreas development. However, Sox17-paLOF mice had higher islet proinsulin protein content, abnormal trafficking of proinsulin, and dilated secretory organelles suggesting that Sox17-paLOF adult mice are prediabetic. Consistant with this, Sox17-paLOF mice were more susceptible to aged-related and high fat diet-induced hyperglycemia and diabetes. Overexpression of Sox17 in mature ß cells using Ins2-rtTA driver mice resulted in precocious secretion of proinsulin. Transcriptionally, SOX17 appears to broadly regulate secretory networks since a 24-hour pulse of SOX17 expression resulted in global transcriptional changes in factors that regulate hormone transport and secretion. Lastly, transient SOX17 overexpression was able to reverse the insulin secretory defects observed in MODY4 animals and restored euglycemia. Together, these data demonstrate a critical new role for SOX17 in regulating insulin trafficking and secretion and that modulation of Sox17-regulated pathways might be used therapeutically to improve cell function in the context of diabetes.


Asunto(s)
Proteínas HMGB/metabolismo , Células Secretoras de Insulina/metabolismo , Insulina/metabolismo , Factores de Transcripción SOXF/metabolismo , Animales , Femenino , Técnica del Anticuerpo Fluorescente , Proteínas HMGB/genética , Secreción de Insulina , Masculino , Ratones , Microscopía Confocal , Microscopía Electrónica , Páncreas/citología , Páncreas/metabolismo , Páncreas/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción SOXF/genética
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