RESUMO
Age-related hearing loss (ARHL) is a threat to future human wellbeing. Multiple factors contributing to the terminal auditory decline have been identified; but a unified understanding of ARHL - or the homeostatic maintenance of hearing before its breakdown - is missing. We here present an in-depth analysis of homeostasis and ageing in the antennal ears of the fruit fly Drosophila melanogaster. We show that Drosophila, just like humans, display ARHL. By focusing on the phase of dynamic stability prior to the eventual hearing loss we discovered a set of evolutionarily conserved homeostasis genes. The transcription factors Onecut (closest human orthologues: ONECUT2, ONECUT3), Optix (SIX3, SIX6), Worniu (SNAI2) and Amos (ATOH1, ATOH7, ATOH8, NEUROD1) emerged as key regulators, acting upstream of core components of the fly's molecular machinery for auditory transduction and amplification. Adult-specific manipulation of homeostatic regulators in the fly's auditory neurons accelerated - or protected against - ARHL.
Assuntos
Envelhecimento , Antenas de Artrópodes/fisiologia , Drosophila melanogaster/fisiologia , Perda Auditiva/genética , Audição/genética , Homeostase , Neurônios/fisiologia , Animais , Proteínas de Drosophila/genética , Feminino , Genótipo , Proteínas de Homeodomínio/genética , Humanos , Masculino , Camundongos , Fatores de Crescimento Neural/genética , Proteínas do Tecido Nervoso/genética , Interferência de RNA , Análise de Sequência de RNA , Som , Fatores de Tempo , Transativadores/genética , Fatores de Transcrição/genética , TranscriptomaRESUMO
3-dimensional (3D) laboratory tissue cultures have emerged as an alternative to traditional 2-dimensional (2D) culture systems that do not recapitulate native cell behavior. The discrepancy between in vivo and in vitro tissue-cell-molecular responses impedes understanding of human physiology in general and creates roadblocks for the discovery of therapeutic solutions. Two parallel approaches have emerged for the design of 3D culture systems. The first is biomedical engineering methodology, including bioengineered materials, bioprinting, microfluidics and bioreactors, used alone or in combination, to mimic the microenvironments of native tissues. The second approach is organoid technology, in which stem cells are exposed to chemical and/or biological cues to activate differentiation programs that are reminiscent of human (prenatal) development. This review article describes recent technological advances in engineering 3D cultures that more closely resemble the human brain. The contributions of in vitro 3D tissue culture systems to new insights in neurophysiology, neurological diseases and regenerative medicine are highlighted. Perspectives on designing improved tissue models of the human brain are offered, focusing on an integrative approach merging biomedical engineering tools with organoid biology.
RESUMO
Hair cells in the auditory organ of the vertebrate inner ear are the sensory receptors that convert acoustic stimuli into electrical signals that are conveyed along the auditory nerve to the brainstem. Hair cells are highly susceptible to ototoxic drugs, infection, and acoustic trauma, which can cause cellular degeneration. In mammals, hair cells that are lost after damage are not replaced, leading to permanent hearing impairments. By contrast, supporting cells in birds and other non-mammalian vertebrates regenerate hair cells after damage, which restores hearing function. The cellular mechanisms that regulate hair cell regeneration are not well understood. We investigated the role of vascular endothelial growth factor (VEGF) during regeneration of auditory hair cells in chickens after ototoxic injury. Using RNA-Seq, immunolabeling, and in situ hybridization, we found that VEGFA, VEGFC, VEGFR1, VEGFR2, and VEGFR3 were expressed in the auditory epithelium, with VEGFA expressed in hair cells and VEGFR1 and VEGFR2 expressed in supporting cells. Using organotypic cultures of the chicken cochlear duct, we found that blocking VEGF receptor activity during hair cell injury reduced supporting cell proliferation as well as the numbers of regenerated hair cells. By contrast, addition of recombinant human VEGFA to organ cultures caused an increase in both supporting cell division and hair cell regeneration. VEGF's effects on supporting cells were preserved in isolated supporting cell cultures, indicating that VEGF can act directly upon supporting cells. These observations demonstrate a heretofore uncharacterized function for VEGF signaling as a critical positive regulator of hair cell regeneration in the avian inner ear.
Assuntos
Proteínas Aviárias/metabolismo , Proliferação de Células , Células Ciliadas Auditivas Internas/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Regeneração , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Apoptose , Proteínas Aviárias/genética , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Galinhas , Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/efeitos dos fármacos , Células Labirínticas de Suporte/efeitos dos fármacos , Células Labirínticas de Suporte/metabolismo , Células Labirínticas de Suporte/patologia , Mecanotransdução Celular , Regeneração/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , Fator A de Crescimento do Endotélio Vascular/genética , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
Human vestibular sensory epithelia in explant culture were incubated in gentamicin to ablate hair cells. Subsequent transduction of supporting cells with ATOH1 using an Ad-2 viral vector resulted in generation of highly significant numbers of cells expressing the hair cell marker protein myosin VIIa. Cells expressing myosin VIIa were also generated after blocking the Notch signalling pathway with TAPI-1 but less efficiently. Transcriptomic analysis following ATOH1 transduction confirmed up-regulation of 335 putative hair cell marker genes, including several downstream targets of ATOH1. Morphological analysis revealed numerous cells bearing dense clusters of microvilli at the apical surfaces which showed some hair cell-like characteristics confirming a degree of conversion of supporting cells. However, no cells bore organised hair bundles and several expected hair cell markers genes were not expressed suggesting incomplete differentiation. Nevertheless, the results show a potential to induce conversion of supporting cells in the vestibular sensory tissues of humans.
Assuntos
Epitélio/fisiologia , Células Ciliadas Vestibulares/fisiologia , Regeneração/fisiologia , Adenoviridae/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Epitélio/ultraestrutura , Regulação da Expressão Gênica , Gentamicinas/efeitos adversos , Proteínas de Fluorescência Verde/metabolismo , Células Ciliadas Vestibulares/patologia , Células Ciliadas Vestibulares/ultraestrutura , Humanos , Miosina VIIa , Miosinas/metabolismo , Receptores Notch/metabolismo , Sáculo e Utrículo/fisiologia , Sáculo e Utrículo/ultraestrutura , Transdução de Sinais , Transdução GenéticaRESUMO
The loss of sensory hair cells from the inner ear is a leading cause of hearing and balance disorders. The mammalian ear has a very limited ability to replace lost hair cells, but the inner ears of non-mammalian vertebrates can spontaneously regenerate hair cells after injury. Prior studies have shown that replacement hair cells are derived from epithelial supporting cells and that the differentiation of new hair cells is regulated by the Notch signaling pathway. The present study examined molecular influences on regeneration in the avian utricle, which has a particularly robust regenerative ability. Chicken utricles were placed in organotypic culture and hair cells were lesioned by application of the ototoxic antibiotic streptomycin. Cultures were then allowed to regenerate in vitro for seven days. Some specimens were treated with small molecule inhibitors of γ-secretase or ADAM10, proteases which are essential for transmission of Notch signaling. As expected, treatment with both inhibitors led to increased numbers of replacement hair cells. However, we also found that inhibition of both proteases resulted in increased regenerative proliferation. Subsequent experiments showed that inhibition of γ-secretase or ADAM10 could also trigger proliferation in undamaged utricles. To better understand these phenomena, we used RNA-Seq profiling to characterize changes in gene expression following γ-secretase inhibition. We observed expression patterns that were consistent with Notch pathway inhibition, but we also found that the utricular sensory epithelium contains numerous γ-secretase substrates that might regulate cell cycle entry and possibly supporting cell-to-hair cell conversion. Together, our data suggest multiple roles for γ-secretase and ADAM10 in vestibular hair cell regeneration.
Assuntos
Proteína ADAM10/antagonistas & inibidores , Secretases da Proteína Precursora do Amiloide/antagonistas & inibidores , Células Ciliadas Vestibulares/citologia , Receptores Notch/metabolismo , Regeneração/fisiologia , Sáculo e Utrículo/crescimento & desenvolvimento , Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células , Embrião de Galinha , Galinhas , Células Epiteliais/fisiologia , Técnicas de Cultura de Órgãos , Sáculo e Utrículo/citologiaRESUMO
Behavioural anomalies suggesting an inner ear disorder were observed in a colony of transgenic mice. Affected animals were profoundly deaf. Severe hair bundle defects were identified in all outer and inner hair cells (OHC, IHC) in the cochlea and in hair cells of vestibular macular organs, but hair cells in cristae were essentially unaffected. Evidence suggested the disorder was likely due to gene disruption by a randomly inserted transgene construct. Whole-genome sequencing identified interruption of the SorCS2 (Sortilin-related VPS-10 domain containing protein) locus. Real-time-qPCR demonstrated disrupted expression of SorCS2 RNA in cochlear tissue from affected mice and this was confirmed by SorCS2 immuno-labelling. In all affected hair cells, stereocilia were shorter than normal, but abnormalities of bundle morphology and organisation differed between hair cell types. Bundles on OHC were grossly misshapen with significantly fewer stereocilia than normal. However, stereocilia were organised in rows of increasing height. Bundles on IHC contained significantly more stereocilia than normal with some longer stereocilia towards the centre, or with minimal height differentials. In early postnatal mice, kinocilia (primary cilia) of IHC and of OHC were initially located towards the lateral edge of the hair cell surface but often became surrounded by stereocilia as bundle shape and apical surface contour changed. In macular organs the kinocilium was positioned in the centre of the cell surface throughout maturation. There was disruption of the signalling pathway controlling intrinsic hair cell apical asymmetry. LGN and Gαi3 were largely absent, and atypical Protein Kinase C (aPKC) lost its asymmetric distribution. The results suggest that SorCS2 plays a role upstream of the intrinsic polarity pathway and that there are differences between hair cell types in the deployment of the machinery that generates a precisely organised hair bundle.
Assuntos
Regulação da Expressão Gênica , Células Ciliadas Auditivas Internas/metabolismo , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Estereocílios/genética , Fatores Etários , Animais , Células Ciliadas Auditivas Internas/patologia , Perda Auditiva/genética , Perda Auditiva/metabolismo , Perda Auditiva/fisiopatologia , Imuno-Histoquímica , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Microscopia Confocal , Microscopia Eletrônica de Varredura , Proteínas do Tecido Nervoso/metabolismo , Órgão Espiral/metabolismo , Órgão Espiral/fisiopatologia , Órgão Espiral/ultraestrutura , Receptores de Superfície Celular/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estereocílios/metabolismo , Estereocílios/patologiaRESUMO
Silk hydrogels were formulated with anti-vascular endothelial growth factor (anti-VEGF) therapeutics for sustained ocular drug delivery. Using silk fibroin as a vehicle for delivery, bevacizumab-loaded hydrogel formulations demonstrated sustained release of 3 months or greater in experiments in vitro as well as in vivo using an intravitreal injection model in Dutch-belted rabbits. Using both standard dose (1.25mg bevacizumab/50 µL injection) and high dose (5.0mg bevacizumab/50 µL injection) hydrogel formulations, release concentrations were achieved at day 90 that were equivalent or greater than those achieved at day 30 with the positive standard dose control (single injection (50 µL) of 1.25mg bevacizumab solution), which is estimated to be the therapeutic threshold based on the current dosage administration schedule of 1 injection/month. These gels also demonstrated signs of biodegradation after 3 months, suggesting that repeated injections may be possible (e.g., one injection every 3-6 months or longer). Due to its pharmacokinetic and biodegradation profiles, this delivery system may be used to reduce the frequency of dosing for patients currently enduring treatment using bevacizumab or other anti-VEGF therapeutics.
Assuntos
Inibidores da Angiogênese/administração & dosagem , Bevacizumab/administração & dosagem , Sistemas de Liberação de Medicamentos , Fibroínas/química , Administração Oftálmica , Inibidores da Angiogênese/farmacocinética , Inibidores da Angiogênese/farmacologia , Animais , Bevacizumab/farmacocinética , Bevacizumab/farmacologia , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Liberação Controlada de Fármacos , Hidrogéis , Injeções Intravítreas , Coelhos , Seda/química , Fatores de Tempo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidoresAssuntos
Técnicas de Planejamento , Administração da Prática Médica/organização & administração , Melhoria de Qualidade , Agendamento de Consultas , Humanos , Liderança , Objetivos Organizacionais , Avaliação de Processos e Resultados em Cuidados de Saúde , Administração da Prática Médica/economia , Estresse Psicológico/prevenção & controleRESUMO
A silk-protein based reservoir rod was developed for zero-order and long-term sustained drug delivery applications. Silk reservoir rod formulations were processed in three steps. First, a regenerated silk fibroin solution, rich in random-coil content was transformed into a tubular silk film with controllable dimensions, uniform film morphology and a structure rich in silk II, ß-sheet content via "film-spinning." Second, the drug powder was loaded into swollen silk tubes followed by tube end clamping. Last, clamped silk tube ends were sealed completely via dip coating. Anastrozole, an FDA approved active ingredient for the treatment of breast cancer, was used as a model drug to investigate viability of the silk reservoir rod technology for sustained drug delivery. The in vitro and in vivo pharmacokinetic data (in a female Sprague-Dawley rat model) analyzed via liquid chromatography-tandem mass spectroscopy indicated zero-order release for 91 days. Both in vitro and in vivo anastrozole release rates could be controlled simply by varying silk rod dimensions. The swelling behavior of silk films and zero-order anastrozole release kinetics indicated practically immediate film hydration and formation of a linear anastrozole concentration gradient along the silk film thickness. The dependence of anastrozole release rate on the overall silk rod dimensions was in good agreement with an essentially diffusion-controlled sustained release from a reservoir cylindrical geometry. In vivo results highlighted a strong in vitro-in vivo pharmacokinetic correlation and a desirable biocompatibility profile of silk reservoir rods. During a 6-month implantation in rats, the apparent silk molecular weight values decreased gradually, while rod dry mass and ß-sheet crystal content values remained essentially constant, providing a suitable timeframe for controlled, long-term sustained delivery applications. Overall, the silk reservoir rod may be a viable candidate for sustained delivery of breast cancer therapeutics.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Fibroínas/química , Anastrozol , Animais , Antineoplásicos/farmacologia , Biodegradação Ambiental , Neoplasias da Mama/patologia , Feminino , Cinética , Microscopia Eletrônica de Varredura , Nitrilas/farmacocinética , Nitrilas/farmacologia , Ratos Sprague-Dawley , Espectroscopia de Infravermelho com Transformada de Fourier , Fatores de Tempo , Triazóis/farmacocinética , Triazóis/farmacologiaRESUMO
Silk presents a rare combination of desirable properties for sustained drug delivery, including aqueous-based purification and processing options without chemical cross-linkers, compatibility with common sterilization methods, controllable and surface-mediated biodegradation into non-inflammatory by-products, biocompatibility, utility in drug stabilization, and robust mechanical properties. A versatile silk-based toolkit is currently available for sustained drug delivery formulations of small molecule through macromolecular drugs, with a promise to mitigate several drawbacks associated with other degradable sustained delivery technologies in the market. Silk-based formulations utilize silk's well-defined nano- through microscale structural hierarchy, stimuli-responsive self-assembly pathways and crystal polymorphism, as well as sequence and genetic modification options towards targeted pharmaceutical outcomes. Furthermore, by manipulating the interactions between silk and drug molecules, near-zero order sustained release may be achieved through diffusion- and degradation-based release mechanisms. Because of these desirable properties, there has been increasing industrial interest in silk-based drug delivery systems currently at various stages of the developmental pipeline from pre-clinical to FDA-approved products. Here, we discuss the unique aspects of silk technology as a sustained drug delivery platform and highlight the current state of the art in silk-based drug delivery. We also offer a potential early development pathway for silk-based sustained delivery products.
Assuntos
Materiais Biocompatíveis/química , Sistemas de Liberação de Medicamentos , Seda/química , Preparações de Ação Retardada , Estabilidade de Medicamentos , Humanos , Hidrogéis/química , Seda/farmacocinéticaRESUMO
The auditory systems of animals that perceive sounds in air are organized to separate sound stimuli into their component frequencies. Individual tones then stimulate mechanosensory hair cells located at different positions on an elongated frequency (tonotopic) axis. During development, immature hair cells located along the axis must determine their tonotopic position in order to generate frequency-specific characteristics. Expression profiling along the developing tonotopic axis of the chick basilar papilla (BP) identified a gradient of Bmp7. Disruption of that gradient in vitro or in ovo induces changes in hair cell morphologies consistent with a loss of tonotopic organization and the formation of an organ with uniform frequency characteristics. Further, the effects of Bmp7 in determination of positional identity are shown to be mediated through activation of the Mapk, Tak1. These results indicate that graded, Bmp7-dependent, activation of Tak1 signalling controls the determination of frequency-specific hair cell characteristics along the tonotopic axis.
Assuntos
Proteína Morfogenética Óssea 7/genética , Regulação da Expressão Gênica no Desenvolvimento , MAP Quinase Quinase Quinases/genética , Órgão Espiral/metabolismo , RNA Mensageiro/metabolismo , Animais , Proteína Morfogenética Óssea 7/metabolismo , Embrião de Galinha , Orelha Interna/embriologia , Orelha Interna/metabolismo , Células Ciliadas Auditivas/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Órgão Espiral/embriologia , Organogênese/genética , Transdução de SinaisRESUMO
Precise frequency discrimination is a hallmark of auditory function in birds and mammals and is required for distinguishing similar sounding words, like 'bat,' 'cat' and 'hat.' In the cochlea, tuning and spectral separation result from longitudinal differences in basilar membrane stiffness and numerous individual gradations in sensory hair cell phenotypes, but it is unknown what patterns the phenotypes. Here we used RNA-seq to compare transcriptomes from proximal, middle and distal regions of the embryonic chicken cochlea, and found opposing longitudinal gradients of expression for retinoic acid (RA)-synthesizing and degrading enzymes. In vitro experiments showed that RA is necessary and sufficient to induce the development of distal-like hair cell phenotypes and promotes expression of the actin-crosslinking proteins, Espin and Fscn2. These and other findings highlight a role for RA signalling in patterning the development of a longitudinal gradient of frequency-tuned hair cell phenotypes in the cochlea.
Assuntos
Membrana Basilar/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células Ciliadas Auditivas/metabolismo , RNA Mensageiro/metabolismo , Tretinoína/metabolismo , Aldeído Oxirredutases/genética , Animais , Proteínas de Transporte/genética , Embrião de Galinha , Sistema Enzimático do Citocromo P-450/genética , Proteínas dos Microfilamentos/genética , Receptores do Ácido Retinoico/genética , Transdução de SinaisRESUMO
Despite declining sequencing costs, few methods are available for cost-effective single-nucleotide polymorphism (SNP), insertion/deletion (INDEL) and copy number variation (CNV) discovery in a single assay. Commercially available methods require a high investment to a specific region and are only cost-effective for large samples. Here, we introduce a novel, flexible approach for multiplexed targeted sequencing and CNV analysis of large genomic regions called multiplexed direct genomic selection (MDiGS). MDiGS combines biotinylated bacterial artificial chromosome (BAC) capture and multiplexed pooled capture for SNP/INDEL and CNV detection of 96 multiplexed samples on a single MiSeq run. MDiGS is advantageous over other methods for CNV detection because pooled sample capture and hybridization to large contiguous BAC baits reduces sample and probe hybridization variability inherent in other methods. We performed MDiGS capture for three chromosomal regions consisting of â¼ 550 kb of coding and non-coding sequence with DNA from 253 patients with congenital lower limb disorders. PITX1 nonsense and HOXC11 S191F missense mutations were identified that segregate in clubfoot families. Using a novel pooled-capture reference strategy, we identified recurrent chromosome chr17q23.1q23.2 duplications and small HOXC 5' cluster deletions (51 kb and 12 kb). Given the current interest in coding and non-coding variants in human disease, MDiGS fulfills a niche for comprehensive and low-cost evaluation of CNVs, coding, and non-coding variants across candidate regions of interest.
Assuntos
Variações do Número de Cópias de DNA , Genômica/métodos , Mutação INDEL , Polimorfismo de Nucleotídeo Único , Cromossomos Artificiais Bacterianos , Exoma , Humanos , Deformidades Congênitas das Extremidades Inferiores/genética , Análise de Sequência de DNARESUMO
Sensory hair cell loss is the major cause of hearing and balance disorders. Mammals are incapable of sustained hair cell regeneration, but lower vertebrates can regenerate these mechano-electrical transducers. We present the first comprehensive transcriptome (by mRNA-Seq) of hair cell regeneration in the chick utricle. We provide pathway and pattern annotations and correlate these with the phenotypic events that occur during regeneration. These patterns are surprisingly synchronous and highly punctuated. We show how these patterns are a new resource for identifying components of the hair cell transcriptome and identify 494 new putative hair-cell-specific genes and validate three of these (of three tested) by immunohistochemical staining. We describe many surprising new components and dynamic expression patterns, particularly within NOTCH signaling. For example, we show that HES7 is specifically expressed during utricle hair cell regeneration and closely parallels the expression of HES5. Likewise, the expression of ATOH1 is closely correlated with HEYL and the HLH inhibitory transcription factors ID1, ID2, and ID4. We investigate the correlation between fibroblast growth factor signaling and supporting cell proliferation and show that FGF20 inhibits supporting cell proliferation. We also present an analysis of 212 differentially expressed transcription factor genes in the regenerative time course that fall into nine distinct gene expression patterns, many of which correlate with phenotypic events during regeneration and represent attractive candidates for future analysis and manipulation of the regenerative program in sensory epithelia and other vertebrate neuroepithelia.
Assuntos
Células Ciliadas Auditivas Internas/fisiologia , Regeneração/fisiologia , Sáculo e Utrículo/fisiologia , Transcriptoma/fisiologia , Animais , Aves , Galinhas , Orelha Interna/fisiologia , Feminino , Masculino , Técnicas de Cultura de Órgãos , Transdução de Sinais/fisiologiaRESUMO
We all start out as a single totipotent cell that is programmed to produce a multicellular organism. How do individual cells make those complex developmental switches? How do single cells within a tissue or organ differ, how do they coordinate their actions or go astray in a disease process? These are long-standing and fundamental questions in biology that are now becoming tractable because of advances in microfluidics, DNA amplification and DNA sequencing. Methods for studying single-cell transcriptomes (or at least the polyadenylated mRNA fraction of it) are by far the furthest ahead and reveal remarkable heterogeneity between morphologically identical cells. The analysis of genomic DNA variation is not far behind. The other 'omics' of single cells pose greater technological obstacles, but they are progressing and promise to yield highly integrated large data sets in the near future.
Assuntos
Perfilação da Expressão Gênica/métodos , Genômica/métodos , Metabolômica/métodos , Análise de Célula Única , Transcriptoma , Fenômenos Fisiológicos Celulares , Variação Genética , Proteômica/métodos , Biologia de Sistemas/métodosRESUMO
The perturbation of myocardial transcriptome homeostasis is the hallmark of pathological hypertrophy, underlying the maladaptive myocardial remodeling secondary to pathological stresses. Classic and novel therapeutics that provide beneficial effects against pathological remodeling likely impact myocardial transcriptome architecture, including miRNA and mRNA expression profiles. Microarray and PCR-based technologies, although employed extensively, cannot provide adequate sequence coverage or quantitative accuracy to test this hypothesis directly. The goal of this study was to develop and exploit next-generation sequencing approaches for comprehensive and quantitative analyses of myocardial miRNAs and mRNAs to test the hypothesis that augmented phosphoinositide-3-kinase-p110α (PI3Kα) signaling in the setting of pathological hypertrophy provides beneficial effects through remodeling of the myocardial transcriptome signature. In these studies, a molecular and bioinformatic pipeline permitting comprehensive analysis and quantification of myocardial miRNA and mRNA expression with next-generation sequencing was developed and the impact of enhanced PI3Kα signaling on the myocardial transcriptome signature of pressure overload-induced pathological hypertrophy was explored. These analyses identified multiple miRNAs and mRNAs that were abnormally expressed in pathological hypertrophy and partially or completely normalized with increased PI3Kα signaling. Additionally, several novel miRNAs potentially linked to remodeling in cardiac hypertrophy were identified. Additional experiments revealed that increased PI3Kα signaling reduces cardiac fibrosis in pathological hypertrophy through modulating TGF-ß signaling and miR-21 expression. In conclusion, using the approach of combined miRNA and mRNA sequencing, we identify the protective transcriptome signature of enhanced PI3Kα signaling in the context of pathological hypertrophy, and demonstrate the regulation of TGF-ß/miR-21 by which enhanced PI3Kα signaling protects against cardiac fibrosis.
Assuntos
Cardiomegalia/genética , Classe Ia de Fosfatidilinositol 3-Quinase/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , MicroRNAs/química , RNA Mensageiro/química , Análise de Sequência de RNA , Animais , Sequência de Bases , Cardiomegalia/enzimologia , Cardiomegalia/metabolismo , Análise por Conglomerados , Fibrose Endomiocárdica/genética , Fibrose Endomiocárdica/metabolismo , Perfilação da Expressão Gênica , Ventrículos do Coração/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcriptoma , Remodelação Ventricular/genéticaRESUMO
We present a case of leptospirosis in a previously healthy girl following a trip to Costa Rica. While she was clinically asymptomatic, she had spirochetes cultured from her urine six weeks following her trip. Prolonged urinary shedding following infection with Leptospira is possible in humans and often has subtle manifestations in children.
RESUMO
Higher vertebrates use similar genetic tools to derive very different facial features. This diversity is believed to occur through temporal, spatial and species-specific changes in gene expression within cranial neural crest (NC) cells. These contribute to the facial skeleton and contain species-specific information that drives morphological variation. A few signaling molecules and transcription factors are known to play important roles in these processes, but little is known regarding the role of micro-RNAs (miRNAs). We have identified and compared all miRNAs expressed in cranial NC cells from three avian species (chicken, duck, and quail) before and after species-specific facial distinctions occur. We identified 170 differentially expressed miRNAs. These include thirty-five novel chicken orthologs of previously described miRNAs, and six avian-specific miRNAs. Five of these avian-specific miRNAs are conserved over 120 million years of avian evolution, from ratites to galliforms, and their predicted target mRNAs include many components of Wnt signaling. Previous work indicates that mRNA gene expression in NC cells is relatively static during stages when the beak acquires species-specific morphologies. However, miRNA expression is remarkably dynamic within this timeframe, suggesting that the timing of specific developmental transitions is altered in birds with different beak shapes. We evaluated one miRNA:mRNA target pair and found that the cell cycle regulator p27(KIP1) is a likely target of miR-222 in frontonasal NC cells, and that the timing of this interaction correlates with the onset of phenotypic variation. Our comparative genomic approach is the first comprehensive analysis of miRNAs in the developing facial primordial, and in species-specific facial development.
Assuntos
Aves/genética , Animais , Evolução Biológica , Embrião de Galinha , Galinhas/genética , Patos/genética , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Crista Neural/embriologia , Osteogênese/genética , Codorniz/genética , Análise de Sequência de RNA , Via de Sinalização Wnt/genéticaRESUMO
Silk fibroin, derived from Bombyx mori cocoons, is a widely used and studied protein polymer for biomaterial applications. Silk fibroin has remarkable mechanical properties when formed into different materials, demonstrates biocompatibility, has controllable degradation rates from hours to years and can be chemically modified to alter surface properties or to immobilize growth factors. A variety of aqueous or organic solvent-processing methods can be used to generate silk biomaterials for a range of applications. In this protocol, we include methods to extract silk from B. mori cocoons to fabricate hydrogels, tubes, sponges, composites, fibers, microspheres and thin films. These materials can be used directly as biomaterials for implants, as scaffolding in tissue engineering and in vitro disease models, as well as for drug delivery.