RESUMEN

Alcohol use is an independent risk factor for the development of bacterial pneumonia due, in part, to impaired mucus-facilitated clearance, macrophage phagocytosis, and recruitment of neutrophils. Alcohol consumption is also known to reduce peripheral natural killer (NK) cell numbers and compromise NK cell cytolytic activity, especially NK cells with a mature phenotype. However, the role of innate lymphocytes, such as NK cells during host defense against alcohol-associated bacterial pneumonia is essentially unknown. We have previously shown that indole supplementation mitigates increases in pulmonary bacterial burden and improves pulmonary NK cell recruitment in alcohol-fed mice, which were dependent on aryl hydrocarbon receptor (AhR) signaling. Employing a binge-on-chronic alcohol-feeding model we sought to define the role and interaction of indole and NK cells during pulmonary host defense against alcohol-associated pneumonia. We demonstrate that alcohol dysregulates NK cell effector function and pulmonary recruitment via alterations in two key signaling pathways. We found that alcohol increases transforming growth factor beta (TGF-ß) signaling while suppressing AhR signaling. We further demonstrated that NK cells isolated from alcohol-fed mice have a reduced ability to kill Klebsiella pneumoniae. NK cell migratory capacity to chemokines was also significantly altered by alcohol, as NK cells isolated from alcohol-fed mice exhibited preferential migration in response to CXCR3 chemokines but exhibited reduced migration in response to CCR2, CXCR4, and CX3CR1 chemokines. Together this data suggests that alcohol disrupts NK cell-specific TGF-ß and AhR signaling pathways leading to decreased pulmonary recruitment and cytolytic activity thereby increasing susceptibility to alcohol-associated bacterial pneumonia.

Asunto(s)

Células Asesinas Naturales , Ratones Endogámicos C57BL , Neumonía Bacteriana , Receptores de Hidrocarburo de Aril , Transducción de Señal , Animales , Células Asesinas Naturales/inmunología , Neumonía Bacteriana/inmunología , Neumonía Bacteriana/microbiología , Ratones , Receptores de Hidrocarburo de Aril/metabolismo , Pulmón/inmunología , Pulmón/microbiología , Factor de Crecimiento Transformador beta/metabolismo , Etanol , Receptores CCR2/metabolismo , Receptores CCR2/genética , Modelos Animales de Enfermedad , Indoles/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Masculino , Klebsiella pneumoniae , Receptores CXCR3/metabolismo

RESUMEN

The study of cooperating genes in cancer can lead to mechanistic understanding and identifying potential therapeutic targets. To facilitate these types of studies, we developed a new dual-inducible system utilizing the tetracycline- and cumate-inducible systems driving HES3 and the PAX3::FOXO1 fusion-oncogene, respectively, as cooperating genes from fusion-positive rhabdomyosarcoma. With this model, we can independently induce expression of either HES3 or PAX3::FOXO1, as well as simultaneously induce expression of both genes. This new model will allow us to further investigate the cooperation between HES3 and PAX3::FOXO1 including the temporal requirements for genetic cooperation. Functionally, we show that dual-induction of PAX3::FOXO1 and HES3 modifies sphere formation in a HEK293T-based system. More broadly, this lentiviral dual-inducible system can be adapted for any cooperating genes (overexpression or knockdown), allowing for independent, simultaneous, or temporally controlled gene expression.

Asunto(s)

Regulación Neoplásica de la Expresión Génica , Humanos , Células HEK293 , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Rabdomiosarcoma/genética , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Factor de Transcripción PAX3/genética , Factor de Transcripción PAX3/metabolismo

RESUMEN

Lineage plasticity in small cell lung carcinoma (SCLC) causes therapeutic difficulties. This study aimed to investigate the pathological findings of plasticity in SCLC, focusing on combined SCLC, and elucidate the involvement of YAP1 and other transcription factors. We analysed 100 surgically resected SCLCs through detailed morphological observations and immunohistochemistry for YAP1 and other transcription factors. Component-by-component next-generation sequencing (n = 15 pairs) and immunohistochemistry (n = 35 pairs) were performed on the combined SCLCs. Compared with pure SCLCs (n = 65), combined SCLCs (n = 35) showed a significantly larger size, higher expression of NEUROD1, and higher frequency of double-positive transcription factors (p = 0.0009, 0.04, and 0.019, respectively). Notably, 34% of the combined SCLCs showed morphological mosaic patterns with unclear boundaries between the SCLC and its partner. Combined SCLCs not only had unique histotypes as partners but also represented different lineage plasticity within the partner. NEUROD1-dominant combined SCLCs had a significantly higher proportion of adenocarcinomas as partners, whereas POU2F3-dominant combined SCLCs had a significantly higher proportion of squamous cell carcinomas as partners (p = 0.006 and p = 0.0006, respectively). YAP1 expression in SCLC components was found in 80% of combined SCLCs and 62% of pure SCLCs, often showing mosaic-like expression. Among the combined SCLCs with component-specific analysis, the identical TP53 mutation was found in 10 pairs, and the identical Rb1 abnormality was found in 2 pairs. On immunohistochemistry, the same abnormal p53 pattern was found in 34 pairs, and Rb1 loss was found in 24 pairs. In conclusion, combined SCLC shows a variety of pathological plasticity. Although combined SCLC is more plastic than pure SCLC, pure SCLC is also a phenotypically plastic tumour. The morphological mosaic pattern and YAP1 mosaic-like expression may represent ongoing lineage plasticity. This study also identified the relationship between transcription factors and partners in combined SCLC. Transcription factors may be involved in differentiating specific cell lineages beyond just 'neuroendocrine'.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Neoplasias Pulmonares , Carcinoma Pulmonar de Células Pequeñas , Factores de Transcripción , Proteínas Señalizadoras YAP , Humanos , Proteínas Señalizadoras YAP/metabolismo , Carcinoma Pulmonar de Células Pequeñas/patología , Carcinoma Pulmonar de Células Pequeñas/metabolismo , Carcinoma Pulmonar de Células Pequeñas/genética , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Masculino , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Femenino , Persona de Mediana Edad , Anciano , Inmunohistoquímica , Linaje de la Célula , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/metabolismo , Mutación , Plasticidad de la Célula , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética

RESUMEN

The number of neural stem cells reflects the total number of neurons in the mature brain. As neural stem cells arise from neuroepithelial cells, the neuroepithelial cell population must be expanded to secure a sufficient number of neural stem cells. However, molecular mechanisms that regulate timely differentiation from neuroepithelial to neural stem cells are largely unclear. Here, we show that TCF4/Daughterless is a key factor that determines the timing of the differentiation in Drosophila. The neuroepithelial cells initiated but never completed the differentiation in the absence of TCF4/Daughterless. We also found that TCF4/Daughterless binds to the Notch locus, suggesting that Notch is one of its downstream candidate genes. Consistently, Notch expression was ectopically induced in the absence of TCF4/Daughterless. Furthermore, ectopic activation of Notch signaling phenocopied loss of TCF4/Daughterless. Our findings demonstrate that TCF4/Daughterless directly inactivates Notch signaling pathway, resulting in completion of the differentiation from neuroepithelial cells into neural stem cells with optimal timing. Thus, the present results suggest that TCF4/Daughterless is essential for determining whether to move to the next state or stay in the current state in differentiating neuroepithelial cells.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Proteínas de Drosophila , Células-Madre Neurales , Células Neuroepiteliales , Receptores Notch , Transducción de Señal , Animales , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Receptores Notch/metabolismo , Receptores Notch/genética , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Células Neuroepiteliales/metabolismo , Células Neuroepiteliales/citología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Diferenciación Celular/genética , Regulación del Desarrollo de la Expresión Génica , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/citología , Factores de Tiempo , Drosophila/metabolismo

RESUMEN

Acinetobacter baumannii is a pathogenic and multidrug-resistant Gram-negative bacterium that causes severe nosocomial infections. To better understand the mechanism of pathogenesis, we compare the proteomes of uninfected and infected human cells, revealing that transcription factor FOS is the host protein most strongly induced by A. baumannii infection. Pharmacological inhibition of FOS reduces the cytotoxicity of A. baumannii in cell-based models, and similar results are also observed in a mouse infection model. A. baumannii outer membrane vesicles (OMVs) are shown to activate the aryl hydrocarbon receptor (AHR) of host cells by inducing the host enzyme tryptophan-2,3-dioxygenase (TDO), producing the ligand kynurenine, which binds AHR. Following ligand binding, AHR is a direct transcriptional activator of the FOS gene. We propose that A. baumannii infection impacts the host tryptophan metabolism and promotes AHR- and FOS-mediated cytotoxicity of infected cells.

Asunto(s)

Infecciones por Acinetobacter , Acinetobacter baumannii , Quinurenina , Receptores de Hidrocarburo de Aril , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Acinetobacter baumannii/metabolismo , Acinetobacter baumannii/genética , Acinetobacter baumannii/efectos de los fármacos , Humanos , Animales , Ratones , Infecciones por Acinetobacter/microbiología , Infecciones por Acinetobacter/metabolismo , Quinurenina/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Proteínas Proto-Oncogénicas c-fos/genética , Triptófano/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Interacciones Huésped-Patógeno

RESUMEN

Neurodevelopmental disorders are currently one of the major complications faced by patients with congenital heart disease (CHD). Chronic hypoxia in the prenatal and postnatal preoperative brain may be associated with neurological damage and impaired long-term cognitive function, but the exact mechanisms are unknown. In this study, we find that delayed neuronal migration and impaired synaptic development are attributed to altered Atoh1 under chronic hypoxia. This is due to the fact that excessive Atoh1 facilitates expression of Kif21b, which causes excess in free-state α-tubulin, leading to disrupted microtubule dynamic stability. Furthermore, the delay in neonatal brain maturation induces cognitive disabilities in adult mice. Then, by down-regulating Atoh1 we alleviate the impairment of cell migration and synaptic development, improving the cognitive behavior of mice to some extent. Taken together, our work unveil that Atoh1 may be one of the targets to ameliorate hypoxia-induced neurodevelopmental disabilities and cognitive impairment in CHD.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Disfunción Cognitiva , Neuronas , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones , Disfunción Cognitiva/etiología , Disfunción Cognitiva/metabolismo , Neuronas/metabolismo , Hipoxia/metabolismo , Femenino , Neurogénesis , Animales Recién Nacidos , Ratones Endogámicos C57BL , Masculino , Movimiento Celular

RESUMEN

Motor neuron loss is well recognized in amyotrophic lateral sclerosis (ALS), but research on retinal ganglion cells (RGCs) is limited. Ocular symptoms are generally not considered classic ALS symptoms, although RGCs and spinal motor neurons share certain cell pathologies, including hallmark signs of glutamate neurotoxicity, which may be triggered by activation of extrasynaptic NMDA receptors (NMDARs). To explore potential novel strategies to prevent ALS-associated death of RGCs, we utilized inhibition of the TwinF interface, a new pharmacological principle that detoxifies extrasynaptic NMDARs by disrupting the NMDAR/TRPM4 death signaling complex. Using the ALS mouse model SOD1G93A, we found that the small molecule TwinF interface inhibitor FP802 prevents the loss of RGCs, improves pattern electroretinogram (pERG) performance, increases the retinal expression of Bdnf, and restores the retinal expression of the immediate early genes, Inhibin beta A and Npas4. Thus, FP802 not only prevents, as recently described, death of spinal motor neurons in SOD1G93A mice, but it also mitigates ALS-associated retinal damage. TwinF interface inhibitors have great potential for alleviating neuro-ophthalmologic symptoms in ALS patients and offer a promising new avenue for therapeutic intervention.

Asunto(s)

Esclerosis Amiotrófica Lateral , Modelos Animales de Enfermedad , Ratones Transgénicos , Células Ganglionares de la Retina , Animales , Esclerosis Amiotrófica Lateral/patología , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Células Ganglionares de la Retina/efectos de los fármacos , Células Ganglionares de la Retina/patología , Células Ganglionares de la Retina/metabolismo , Ratones , Electrorretinografía , Ratones Endogámicos C57BL , Fármacos Neuroprotectores/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Humanos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo

RESUMEN

Introduction: Colitis is an inflammatory bowel disease (IBD) characterized by immune cell dysregulation and alterations in the gut microbiome. In our previous report, we showed a natural product in cruciferous vegetables and ligand of the aryl hydrocarbon receptor (AhR), indole-3-carbinol (I3C), was able to reduce colitis-induced disease severity and microbial dysbiosis in an interleukin-22 (IL-22) dependent manner. Methods: In the current study, we performed single-cell RNA sequencing (scRNAseq) from colonocytes during colitis induction and supplementation with I3C and show how this treatment alters expression of genes involved in IL-22 signaling. To further define the role of IL-22 signaling in I3C-mediated protection during colitis and disease-associated microbial dysbiosis, we generated mice with AhR deficiency in RAR-related orphan receptor c (Rorc)-expressing cells (AhR ΔRorc ) which depletes this receptor in immune cells involved in production of IL-22. Colitis was induced in wildtype (WT), AhR ΔRorc , and littermate (LM) mice with or without I3C treatment. Results: Results showed AhR ΔRorc mice lost the efficacy effects of I3C treatment which correlated with a loss of ability to increase IL-22 by innate lymphoid type 3 (ILC3s), not T helper 22 (Th22) cells. 16S rRNA microbiome profiling studies showed AhR ΔRorc mice were unable to regulate disease-associated increases in Bacteroides, which differed between males and females. Lastly, inoculation with a specific disease-associated Bacteroides species, Bacteroides acidifaciens (B. acidifaciens), was shown to exacerbate colitis in females, but not males. Discussion: Collectively, this report highlights the cell and sex-specific role of AhR in regulating microbes that can impact colitis disease.

Asunto(s)

Bacteroides , Colitis , Interleucina-22 , Interleucinas , Receptores de Hidrocarburo de Aril , Receptores de Hidrocarburo de Aril/metabolismo , Receptores de Hidrocarburo de Aril/genética , Animales , Interleucinas/metabolismo , Colitis/inmunología , Colitis/microbiología , Femenino , Ratones , Masculino , Bacteroides/inmunología , Microbioma Gastrointestinal/inmunología , Disbiosis/inmunología , Ratones Endogámicos C57BL , Indoles/farmacología , Modelos Animales de Enfermedad , Factores Sexuales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones Noqueados

RESUMEN

Shade avoidance helps plants maximize their access to light for growth under crowding. It is unknown, however, whether a priming shade avoidance mechanism exists that allows plants to respond more effectively to successive shade conditions. Here, we show that the shade-intolerant plant Arabidopsis can remember a first experienced shade event and respond more efficiently to the next event on hypocotyl elongation. The transcriptional regulator PHYTOCHROME-INTERACTING FACTOR 7 (PIF7) and the histone H3K27-demethylase RELATIVE OF EARLY FLOWERING 6 (REF6) are identified as being required for this shade avoidance memory. RNA-sequencing analysis reveals that shade induction of shade-memory-related genes is impaired in the pif7 and ref6 mutants. Based on the analyses of enrichments of H3K27me3, REF6 and PIF7, we find that priming shade treatment induces PIF7 accumulation, which further recruits REF6 to demethylate H3K27me3 on the chromatin of certain shade-memory-related genes, leading to a state poised for their transcription. Upon a second shade treatment, enhanced shade-mediated inductions of these genes result in stronger hypocotyl growth responses. We conclude that the transcriptional memory mediated by epigenetic modification plays a key role in the ability of primed plants to remember previously experienced shade and acquire enhanced responses to recurring shade conditions.

Asunto(s)

Proteínas de Arabidopsis , Arabidopsis , Regulación de la Expresión Génica de las Plantas , Histonas , Hipocótilo , Arabidopsis/genética , Arabidopsis/fisiología , Arabidopsis/metabolismo , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de la radiación , Hipocótilo/crecimiento & desarrollo , Hipocótilo/genética , Hipocótilo/metabolismo , Histonas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Luz , Cromatina/metabolismo , Mutación , Epigénesis Genética , Proteínas de Unión al ADN , Factores de Transcripción

RESUMEN

Metabolic imbalance leading to inflammatory hypoxia and stabilization of hypoxia-inducible transcription factors (HIFs) is a hallmark of inflammatory bowel diseases. We hypothesize that HIF could be stabilized in CD4+ T cells during intestinal inflammation and alter the functional responses of T cells via regulation of microRNAs. Our assays reveal markedly increased T cell-intrinsic hypoxia and stabilization of HIF protein during experimental colitis. microRNA screen in primary CD4+ T cells points us towards miR-29a and our subsequent studies identify a selective role for HIF-2α in CD4-cell-intrinsic induction of miR-29a during hypoxia. Mice with T cell-intrinsic HIF-2α deletion display elevated T-bet (target of miR-29a) levels and exacerbated intestinal inflammation. Mice with miR-29a deficiency in T cells show enhanced intestinal inflammation. T cell-intrinsic overexpression of HIF-2α or delivery of miR-29a mimetic dampen TH1-driven colitis. In this work, we show a previously unrecognized function for hypoxia-dependent induction of miR-29a in attenuating TH1-mediated inflammation.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Colitis , MicroARNs , Células TH1 , Animales , MicroARNs/genética , MicroARNs/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Colitis/genética , Colitis/metabolismo , Colitis/inmunología , Células TH1/inmunología , Células TH1/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas de Dominio T Box/metabolismo , Proteínas de Dominio T Box/genética , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/inmunología , Ratones Noqueados , Humanos , Femenino , Modelos Animales de Enfermedad , Masculino

RESUMEN

Direct neuronal reprogramming is a promising approach to replace neurons lost due to disease via the conversion of endogenous glia reacting to brain injury into neurons. However, it is essential to demonstrate that the newly generated neurons originate from glial cells and/or show that they are not pre-existing endogenous neurons. Here, we use controls for both requirements while comparing two viral vector systems (Mo-MLVs and AAVs) for the expression of the same neurogenic factor, the phosphorylation-resistant form of Neurogenin2. Our results show that Mo-MLVs targeting proliferating glial cells after traumatic brain injury reliably convert astrocytes into neurons, as assessed by genetic fate mapping of astrocytes. Conversely, expressing the same neurogenic factor in a flexed AAV system results in artefactual labelling of endogenous neurons fatemapped by birthdating in development that are negative for the genetic fate mapping marker induced in astrocytes. These results are further corroborated by chronic live in vivo imaging. Taken together, the phosphorylation-resistant form of Neurogenin2 is more efficient in reprogramming reactive glia into neurons than its wildtype counterpart in vivo using retroviral vectors (Mo-MLVs) targeting proliferating glia. Conversely, AAV-mediated expression generates artefacts and is not sufficient to achieve fate conversion.

Asunto(s)

Astrocitos , Reprogramación Celular , Corteza Cerebral , Dependovirus , Vectores Genéticos , Neuronas , Animales , Astrocitos/metabolismo , Neuronas/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/metabolismo , Ratones , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Dependovirus/genética , Reprogramación Celular/genética , Proteínas del Tejido Nervioso/metabolismo , Proteínas del Tejido Nervioso/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Ratones Endogámicos C57BL , Masculino , Retroviridae/genética

RESUMEN

Articular cartilage receives nutrients and oxygen from the synovial fluid to maintain homeostasis. However, compared to tissues with abundant blood flow, articular cartilage is exposed to a hypoxic environment (i.e., physioxia) and has an enhanced hypoxic stress response. Hypoxia-inducible factors (HIFs) play a pivotal role in this physioxic environment. In normoxic conditions, HIFs are downregulated, whereas in physioxic conditions, they are upregulated. The HIF-α family comprises three members: HIF-1α, HIF-2α, and HIF-3α. Each member has a distinct function in articular cartilage. In osteoarthritis, which is primarily caused by degeneration of articular cartilage, HIF-1α is upregulated in chondrocytes and is believed to protect articular cartilage by acting anabolically on it. Conversely, in contrast to HIF-1α, HIF-2α exerts a catabolic influence on articular cartilage. It may therefore be possible to develop a new treatment for OA by controlling the expression of HIF-1α and HIF-2α with drugs or by altering the oxygen environment in the joints.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Cartílago Articular , Condrocitos , Homeostasis , Subunidad alfa del Factor 1 Inducible por Hipoxia , Osteoartritis , Humanos , Cartílago Articular/metabolismo , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Osteoartritis/metabolismo , Condrocitos/metabolismo , Oxígeno/metabolismo , Hipoxia/metabolismo , Hipoxia/fisiopatología

RESUMEN

The bHLH transcription factors are important plant regulators against abiotic stress and involved in plant growth and development. In this study, SlALC, a gene coding for a prototypical DNA-binding protein in the bHLH family, was isolated, and SlALC-overexpression tomato (SlALC-OE) plants were generated by Agrobacterium-mediated genetic transformation. SlALC transgenic lines manifested higher osmotic stress tolerance than the wild-type plants, estimated by higher relative water content and lower water loss rate, higher chlorophyll, reducing sugar, starch, proline, soluble protein contents, antioxidant enzyme activities, and lower MDA and reactive oxygen species contents in the leaves. In SlALC-OE lines, there were more significant alterations in the expression of genes associated with stress. Furthermore, SlALC-OE fruits were more vulnerable to dehiscence, with higher water content, reduced lignin content, SOD/POD/PAL enzyme activity, and lower phenolic compound concentrations, all of which corresponded to decreased expression of lignin biosynthetic genes. Moreover, the dual luciferase reporter test revealed that SlTAGL1 inhibits SlALC expression. This study revealed that SlALC may play a role in controlling plant tolerance to drought and salt stress, as well as fruit lignification, which influences fruit dehiscence. The findings of this study have established a foundation for tomato tolerance breeding and fruit quality improvement.

Asunto(s)

Sequías , Frutas , Regulación de la Expresión Génica de las Plantas , Proteínas de Plantas , Plantas Modificadas Genéticamente , Tolerancia a la Sal , Solanum lycopersicum , Solanum lycopersicum/genética , Solanum lycopersicum/metabolismo , Tolerancia a la Sal/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/genética , Frutas/genética , Frutas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Estrés Fisiológico

RESUMEN

The bHLH (basic helix-loop-helix) transcription factor AtCFLAP2 regulates epidermal wax accumulation, but the underlying molecular mechanism remains unknown. We obtained BnUC1mut (BnaA05g18250D homologous to AtCFLAP2) from a Brassica napus mutant with up-curling leaves (Bnuc1) and epidermal wax deficiency via map-based cloning. BnUC1mut contains a point mutation (N200S) in the conserved dimerization domain. Overexpressing BnUC1mut in ZS11 (Zhongshuang11) significantly decreased the leaf epidermal wax content, resulting in up-curled and glossy leaves. In contrast, knocking out BnUC1mut in ZS11-NIL (Zhongshuang11-near-isogenic line) restored the normal leaf phenotype (i.e., flat) and significantly increased the leaf epidermal wax content. The point mutation weakens the ability of BnUC1mut to bind to the promoters of VLCFA (very-long-chain fatty acids) synthesis-related genes, including KCS (ß-ketoacyl coenzyme synthase) and LACS (long-chain acyl CoA synthetase), as well as lipid transport-related genes, including LTP (non-specific lipid transfer protein). The resulting sharp decrease in the transcription of genes affecting VLCFA biosynthesis and lipid transport disrupts the normal accumulation of leaf epidermal wax. Thus, BnUC1 influences epidermal wax formation by regulating the expression of LTP and genes associated with VLCFA biosynthesis. Our findings provide a foundation for future investigations on the mechanism mediating plant epidermal wax accumulation.

Asunto(s)

Brassica napus , Regulación de la Expresión Génica de las Plantas , Epidermis de la Planta , Proteínas de Plantas , Ceras , Ceras/metabolismo , Brassica napus/metabolismo , Brassica napus/genética , Proteínas de Plantas/metabolismo , Proteínas de Plantas/genética , Epidermis de la Planta/metabolismo , Epidermis de la Planta/genética , Hojas de la Planta/metabolismo , Hojas de la Planta/genética , Metabolismo de los Lípidos/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Transporte Biológico

RESUMEN

A limited understanding of tendon cell biology in healthy and pathological conditions has impeded the development of effective treatments, necessitating in vitro biomimetic models for studying tendon events. We established a dynamic culture using fibrin scaffolds, bioengineered with tendon stem/progenitor cells (hTSPCs) from healthy or diseased human biopsies and perfused with 20 ng/mL of human transforming growth factor-ß1 for 21 days. Both cell types showed long-term viability and upregulated Scleraxis (SCX-A) and Tenomodulin (TNMD) gene expressions, indicating tenogenic activity. However, diseased hTSPCs underexpressed collagen type I and III (COL1A1 and COL3A1) genes and exhibited lower SCX-A and TNMD protein levels, but increased type I collagen production, with a type I/type III collagen ratio > 1.5 by day 14, matching healthy cells. Diseased hTSPCs also showed constant high levels of pro-inflammatory cytokines, such as IL-8 and IL-6. This biomimetic environment is a valuable tool for studying tenogenic and inflammatory events in healthy and diseased tendon cells and identifying new therapeutic targets.

Asunto(s)

Colágeno Tipo I , Fibrina , Células Madre , Tendones , Andamios del Tejido , Factor de Crecimiento Transformador beta1 , Humanos , Tendones/citología , Tendones/metabolismo , Andamios del Tejido/química , Células Madre/metabolismo , Células Madre/citología , Fibrina/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Colágeno Tipo I/metabolismo , Colágeno Tipo I/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Tendinopatía/metabolismo , Tendinopatía/patología , Células Cultivadas , Colágeno Tipo III/metabolismo , Colágeno Tipo III/genética , Cadena alfa 1 del Colágeno Tipo I/metabolismo , Persona de Mediana Edad , Masculino , Supervivencia Celular/efectos de los fármacos , Ingeniería de Tejidos/métodos , Proteínas de la Membrana

RESUMEN

Diabetes mellitus, a chronic and non-transmissible disease, triggers a wide range of micro- and macrovascular complications. The differentiation of pancreatic ß-like cells (PßLCs) from induced pluripotent stem cells (iPSCs) offers a promising avenue for regenerative medicine aimed at treating diabetes. Current differentiation protocols strive to emulate pancreatic embryonic development by utilizing cytokines and small molecules at specific doses to activate and inhibit distinct molecular signaling pathways, directing the differentiation of iPSCs into pancreatic ß cells. Despite significant progress and improved protocols, the full spectrum of molecular signaling pathways governing pancreatic development and the physiological characteristics of the differentiated cells are not yet fully understood. Here, we report a specific combination of cofactors and small molecules that successfully differentiate iPSCs into PßLCs. Our protocol has shown to be effective, with the resulting cells exhibiting key functional properties of pancreatic ß cells, including the expression of crucial molecular markers (pdx1, nkx6.1, ngn3) and the capability to secrete insulin in response to glucose. Furthermore, the addition of vitamin C and retinoic acid in the final stages of differentiation led to the overexpression of specific ß cell genes.

Asunto(s)

Ácido Ascórbico , Diferenciación Celular , Diabetes Mellitus , Células Madre Pluripotentes Inducidas , Células Secretoras de Insulina , Tretinoina , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/efectos de los fármacos , Células Secretoras de Insulina/citología , Ácido Ascórbico/farmacología , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Tretinoina/farmacología , Diferenciación Celular/efectos de los fármacos , Humanos , Diabetes Mellitus/metabolismo , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Transactivadores/metabolismo , Transactivadores/genética , Insulina/metabolismo , Proteínas del Tejido Nervioso

RESUMEN

Worldwide incidence of kidney diseases has been rising. Thus, recent research has focused on zebrafish, whose fast development and innate regeneration capacity allow identifying factors influencing renal processes. Among these poorly studied factors are extracellular matrix (ECM) proteins like Fibronectin (Fn) essential in various tissues but not yet evaluated in a renal context. We utilized early nat and han zebrafish mutant embryos and carrier adults to investigate Fn's role during kidney development and regeneration. The locus natter (nat) encodes Fn and the locus han encodes Hand2, which results in increased Fn deposition. Our results show that Fn impacts identity maintenance and morphogenesis during development and influences conditions for neonephrogenic cluster formation during regeneration. Histological analysis revealed disrupted pronephric structures and increased blood cell accumulation in Fn mutants. Despite normal expression of specification markers (pax2, ATPα1a.1), structural abnormalities were evident. Differences between wild-type and mutation-carriers suggest a haploinsufficiency scenario. These findings reveal a novel function for ECM in renal development and regeneration, with potential implications for understanding and treating kidney diseases.

Asunto(s)

Fibronectinas , Pronefro , Regeneración , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Pronefro/metabolismo , Pronefro/embriología , Fibronectinas/metabolismo , Fibronectinas/genética , Mesonefro/metabolismo , Mutación , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Túbulos Renales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Organogénesis/genética

RESUMEN

Proinflammatory hepatic macrophage activation plays a key role in the development of nonalcoholic steatohepatitis (NASH). This involves increased embryonic hepatic Kupffer cell (KC) death, facilitating the replacement of KCs with bone marrow-derived recruited hepatic macrophages (RHMs) that highly express proinflammatory genes. Moreover, phago/efferocytic activity of KCs is diminished in NASH, enhancing liver inflammation. However, the molecular mechanisms underlying these changes in KCs are not known. Here, we show that hypoxia-inducible factor 2α (HIF-2α) mediates NASH-associated decreased KC growth and efferocytosis by enhancing lysosomal stress. At the molecular level, HIF-2α stimulated mammalian target of rapamycin (mTOR)- and extracellular signal-regulated kinase-dependent inhibitory transcription factor EB (TFEB) phosphorylation, leading to decreased lysosomal and phagocytic gene expression. With increased metabolic stress and phago/efferocytic burden in NASH, these changes were sufficient to increase lysosomal stress, causing decreased efferocytosis and lysosomal cell death. Of interest, HIF-2α-dependent TFEB regulation only occurred in KCs but not RHMs. Instead, in RHMs, HIF-2α promoted mitochondrial reactive oxygen species production and proinflammatory activation by increasing ANT2 expression and mitochondrial permeability transition. Consequently, myeloid lineage-specific or KC-specific HIF-2α depletion or the inhibition of mTOR-dependent TFEB inhibition using antisense oligonucleotide treatment protected against the development of NASH in mice. Moreover, treatment with an HIF-2α-specific inhibitor reduced inflammatory and fibrogenic gene expression in human liver spheroids cultured under a NASH-like condition. Together, our results suggest that macrophage subtype-specific effects of HIF-2α collectively contribute to the proinflammatory activation of liver macrophages, leading to the development of NASH.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Macrófagos del Hígado , Hígado , Activación de Macrófagos , Enfermedad del Hígado Graso no Alcohólico , Macrófagos del Hígado/metabolismo , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hígado/metabolismo , Hígado/patología , Ratones , Muerte Celular , Lisosomas/metabolismo , Fagocitosis , Humanos , Especies Reactivas de Oxígeno/metabolismo , Inflamación/patología , Inflamación/metabolismo , Ratones Endogámicos C57BL , Serina-Treonina Quinasas TOR/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Macrófagos/metabolismo , Mitocondrias/metabolismo

RESUMEN

Notch signaling is a conserved pathway crucial for nervous system development. Disruptions in this pathway are linked to neurodevelopmental disorders, neurodegenerative diseases, and brain tumors. Hairy/E(spl) (HES) genes, major downstream targets of Notch, are commonly used as markers for Notch activation. However, these genes can be activated, inhibited, or function independently of Notch signaling, and their response to Notch disruption varies across tissues and developmental stages. MIB1/Mib1 is an E3 ubiquitin ligase that enables Notch receptor activation by processing ligands like Delta and Serrate. We investigated Notch signaling disruption using the zebrafish Mib1 mutant line, mib1ta52b, focusing on changes in the expression of Hairy/E(spl) (her) genes. Our findings reveal significant variability in her gene expression across different neural cell types, regions, and developmental stages following Notch disruption. This variability questions the reliability of Hairy/E(spl) genes as universal markers for Notch activation, as their response is highly context-dependent. This study highlights the complex and context-specific nature of Notch signaling regulation. It underscores the need for a nuanced approach when using Hairy/E(spl) genes as markers for Notch activity. Additionally, it provides new insights into Mib1's role in Notch signaling, contributing to a better understanding of its involvement in Notch signaling-related disorders.

Asunto(s)

Regulación del Desarrollo de la Expresión Génica , Receptores Notch , Transducción de Señal , Proteínas de Pez Cebra , Pez Cebra , Animales , Pez Cebra/genética , Pez Cebra/metabolismo , Receptores Notch/metabolismo , Receptores Notch/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neurogénesis/genética

RESUMEN

Autoimmune diseases represent a complex array of conditions where the body's immune system mistakenly attacks its own tissues. These disorders, affecting millions worldwide, encompass a broad spectrum of conditions ranging from rheumatoid arthritis and multiple sclerosis to lupus and type 1 diabetes. The Aryl hydrocarbon receptor (AhR) translocator, expressed across immune and other cell types, plays crucial roles in immune disorders and inflammatory diseases. With a realm towards natural remedies in modern medicine for disease prevention, this study investigates the electronic properties and behaviors of bioactive compounds from dietary sources, including Apium graveolens L. (Celery), Coriandrum sativum seeds (Coriander), and Mentha longifolia, as AhR modulators. Through comprehensive analysis (HOMO-LUMO, ESP, LOL, and ELF), electron-rich and -poor regions, electron localization, and delocalization are identified, contrasting these compounds with the toxic AhR ligand, TCDD. Evaluation of Absorption, Distribution, Metabolism, Excretion, and Toxicity (ADMET) properties reveals favorable pharmacokinetics without blood-brain barrier penetration, indicating drug-like characteristics. Molecular docking demonstrates stronger interactions of dietary flavonoid ligands with AhR transcription compared to TCDD. Molecular dynamics simulations confirm the stability of complexes and the sustainability of interactions formed. This research underscores the potential of natural compounds as effective AhR modulators for therapeutic interventions in immune-related disorders.

Asunto(s)

Apium , Coriandrum , Receptores de Hidrocarburo de Aril , Coriandrum/química , Receptores de Hidrocarburo de Aril/metabolismo , Humanos , Apium/química , Inmunoterapia , Extractos Vegetales/química , Extractos Vegetales/farmacología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Plantas Comestibles/química , Simulación del Acoplamiento Molecular