RESUMO
Loss of functional hairless (HR) transcriptional repressor leads to utricle formation and congenital hair loss both in mice and men. Studies in mice have shown that this is preceded by overexpression of caspase-14 at the infundibulum in the hair follicle before conversion to utricle occurs. In this report, we show that HR regulates caspase-14 expression dependent on its interaction with histone deacetylases, implicating chromatin remodelling in the transcriptional regulation of caspase-14. However, crossing hairless mutant mice with caspase-14-deficient mice revealed that caspase-14 overexpression is not the cause of utricle formation.
Assuntos
Caspases/metabolismo , Regulação Enzimológica da Expressão Gênica , Folículo Piloso/fisiologia , Histona Desacetilases/metabolismo , Sáculo e Utrículo/fisiologia , Animais , Linhagem Celular , Cruzamentos Genéticos , Humanos , Camundongos , Camundongos Pelados , Camundongos Transgênicos , Microscopia de Fluorescência , Mutação , FenótipoRESUMO
Avian pathogenic Escherichia coli (APEC) can cause colibacillosis in poultry, characterised by localised or systemic infections. Colibacillosis is considered one of the leading causes of economic losses in the poultry industry due to reduced performance, increased mortality, treatment costs and carcass condemnations. A live attenuated Escherichia coli O78 aroA gene mutant is widely used to prevent disease. However, no effective strategies to differentiate the vaccine strain from field strains are available, hampering follow-up of vaccination campaigns. In the current study, we report a PCR-based method to simultaneously detect the vaccine strain by targeting the vaccine-specific mutation in the aroA gene, as well as the wild type E. coli strains by targeting the xanQ gene. The specificity of this PCR was evaluated using 123 E. coli isolates, form which 5 WT aroA auxotrophic strains (WT strains with a natural aroA deficiency), as well as 7 non-Escherichia isolates. The PCR showed 100% sensitivity of the xanQ primers for E. coli detection and 100% sensitivity of the ΔaroA primers for the vaccine strain. In order to allow quantification of the vaccine strain in complex samples containing many different E. coli strains and other related organisms, such as chicken faeces, a probe-based duplex qPCR was developed. The limit of detection (LOD) of this duplex qPCR method was 8.4*103 copies/g faeces. The specificity of the duplex qPCR was confirmed by determining both the vaccine strain levels, and the total E. coli load in intestinal digesta from both vaccinated and non-vaccinated birds. E. coli could be detected in both vaccinated and non-vaccinated birds. The duplex qPCR was specific for the vaccine strain as this strain was detected in all vaccinated birds, whereas no signal was detected in non-vaccinated birds. The duplex qPCR is helpful in monitoring colonization and shedding of the vaccine strain.
Assuntos
Infecções por Escherichia coli , Vacinas contra Escherichia coli , Doenças das Aves Domésticas , Animais , Escherichia coli/genética , Galinhas , Vacinas Atenuadas , Infecções por Escherichia coli/veterinária , Doenças das Aves Domésticas/prevenção & controleRESUMO
In humans, receptor-interacting protein kinase 4 (RIPK4) mutations can lead to the autosomal recessive Bartsocas-Papas and popliteal pterygium syndromes, which are characterized by severe skin defects, pterygia, as well as clefting. We show here that the epithelial fusions observed in RIPK4 full knockout (KO) mice are E-cadherin dependent, as keratinocyte-specific deletion of E-cadherin in RIPK4 full KO mice rescued the tail-to-body fusion and fusion of oral epithelia. To elucidate RIPK4 function in epidermal differentiation and development, we generated epidermis-specific RIPK4 KO mice (RIPK4EKO). In contrast to RIPK4 full KO epidermis, RIPK4EKO epidermis was normally stratified and the outside-in skin barrier in RIPK4EKO mice was largely intact at the trunk, in contrast to the skin covering the head and the outer end of the extremities. However, RIPK4EKO mice die shortly after birth due to excessive water loss because of loss of tight junction protein claudin-1 localization at the cell membrane, which results in tight junction leakiness. In contrast, mice with keratinocyte-specific RIPK4 deletion during adult life remain viable. Furthermore, our data indicate that epidermis-specific deletion of RIPK4 results in delayed keratinization and stratum corneum maturation and altered lipid organization and is thus indispensable during embryonic development for the formation of a functional inside-out epidermal barrier.
Assuntos
Membrana Celular/patologia , Epiderme/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/metabolismo , Perda Insensível de Água/fisiologia , Animais , Caderinas/metabolismo , Diferenciação Celular , Linhagem Celular , Embrião de Mamíferos , Epiderme/patologia , Humanos , Queratinócitos/citologia , Queratinócitos/patologia , Queratinócitos/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Serina-Treonina Quinases/genética , RNA Interferente Pequeno/metabolismo , Junções Íntimas/patologia , Junções Íntimas/ultraestruturaRESUMO
Unlike its family member p53, TP63 is rarely mutated in human cancer. However, ΔNp63α protein levels are often elevated in tumors of epithelial origin, such as squamous cell carcinoma and cholangiocarcinoma. To study the oncogenic properties of ΔNp63α in vivo, we generated transgenic mice overexpressing ΔNp63α from the Rosa26 locus promoter controlled by keratin 5-Cre. We found that these mice spontaneously develop epidermal cysts and ectopic ΔNp63α expression in the bile duct epithelium that leads to dilatation of the intrahepatic biliary ducts, to hepatic cyst formation and bile duct adenoma. Moreover, when subjected to models of 7,12-dimethylbenz[a]anthracene-based carcinogenesis, tumor initiation was increased in ΔNp63α transgenic mice in a gene dosage-dependent manner although ΔNp63α overexpression did not alter the sensitivity to 7,12-dimethylbenz[a]anthracene-induced cytotoxicity in vivo. However, keratinocytes isolated from ΔNp63α transgenic mice displayed increased survival and delayed cellular senescence compared with wild-type keratinocytes, marked by decreased p16Ink4a and p19Arf expression. Taken together, we show that increased ΔNp63α protein levels facilitate oncogenic transformation in the epidermis as well as in the bile duct.
Assuntos
Neoplasias dos Ductos Biliares/etiologia , Transformação Celular Neoplásica , Fosfoproteínas/fisiologia , Neoplasias Cutâneas/etiologia , Transativadores/fisiologia , 9,10-Dimetil-1,2-benzantraceno , Animais , Células Cultivadas , Senescência Celular , Hiperplasia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fosfoproteínas/análise , Pele/patologia , Acetato de Tetradecanoilforbol , Transativadores/análiseRESUMO
In contrast to most common gene delivery techniques, lentiviral vectors allow targeting of almost any mammalian cell type, even non-dividing cells, and they stably integrate in the genome. Therefore, these vectors are a very powerful tool for biomedical research. Here we report the generation of a versatile new set of 22 lentiviral vectors with broad applicability in multiple research areas. In contrast to previous systems, our platform provides a choice between constitutive and/or conditional expression and six different C-terminal fusions. Furthermore, two compatible selection markers enable the easy derivation of stable cell lines co-expressing differently tagged transgenes in a constitutive or inducible manner. We show that all of the vector features are functional and that they contribute to transgene overexpression in proof-of-principle experiments.