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1.
Development ; 147(21)2020 11 05.
Artículo en Inglés | MEDLINE | ID: mdl-33033118

RESUMEN

Mitchell-Riley syndrome (MRS) is caused by recessive mutations in the regulatory factor X6 gene (RFX6) and is characterised by pancreatic hypoplasia and neonatal diabetes. To determine why individuals with MRS specifically lack pancreatic endocrine cells, we micro-CT imaged a 12-week-old foetus homozygous for the nonsense mutation RFX6 c.1129C>T, which revealed loss of the pancreas body and tail. From this foetus, we derived iPSCs and show that differentiation of these cells in vitro proceeds normally until generation of pancreatic endoderm, which is significantly reduced. We additionally generated an RFX6HA reporter allele by gene targeting in wild-type H9 cells to precisely define RFX6 expression and in parallel performed in situ hybridisation for RFX6 in the dorsal pancreatic bud of a Carnegie stage 14 human embryo. Both in vitro and in vivo, we find that RFX6 specifically labels a subset of PDX1-expressing pancreatic endoderm. In summary, RFX6 is essential for efficient differentiation of pancreatic endoderm, and its absence in individuals with MRS specifically impairs formation of endocrine cells of the pancreas head and tail.


Asunto(s)
Diferenciación Celular , Diabetes Mellitus/genética , Diabetes Mellitus/patología , Endodermo/embriología , Enfermedades de la Vesícula Biliar/genética , Enfermedades de la Vesícula Biliar/patología , Células Madre Pluripotentes Inducidas/patología , Atresia Intestinal/genética , Atresia Intestinal/patología , Mutación/genética , Páncreas/embriología , Factores de Transcripción del Factor Regulador X/genética , Alelos , Secuencia de Bases , Diferenciación Celular/genética , Cromatina/metabolismo , Consanguinidad , Diabetes Mellitus/diagnóstico por imagen , Embrión de Mamíferos/metabolismo , Desarrollo Embrionario , Familia , Femenino , Enfermedades de la Vesícula Biliar/diagnóstico por imagen , Genoma Humano , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Atresia Intestinal/diagnóstico por imagen , Masculino , Linaje , Transcripción Genética , Transcriptoma/genética , Microtomografía por Rayos X
2.
Development ; 142(18): 3126-37, 2015 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-26395141

RESUMEN

A wealth of data and comprehensive reviews exist on pancreas development in mammals, primarily mice, and other vertebrates. By contrast, human pancreatic development has been less comprehensively reviewed. Here, we draw together those studies conducted directly in human embryonic and fetal tissue to provide an overview of what is known about human pancreatic development. We discuss the relevance of this work to manufacturing insulin-secreting ß-cells from pluripotent stem cells and to different aspects of diabetes, especially permanent neonatal diabetes, and its underlying causes.


Asunto(s)
Diferenciación Celular/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Células Secretoras de Insulina/citología , Morfogénesis/fisiología , Páncreas/embriología , Páncreas/crecimiento & desarrollo , Células Madre Pluripotentes/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Especificidad de la Especie
3.
Biochim Biophys Acta ; 1861(9 Pt B): 1365-1378, 2016 09.
Artículo en Inglés | MEDLINE | ID: mdl-26825689

RESUMEN

Plants possess effective mechanisms to quickly respond to biotic and abiotic stresses. The rapid activation of phosphatidylinositol-specific phospholipase C (PLC) enzymes occurs early after the stimulation of plant immune-receptors. Genomes of different plant species encode multiple PLC homologs belonging to one class, PLCζ. Here we determined whether all tomato homologs encode active enzymes and whether they can generate signals that are distinct from one another. We searched the recently completed tomato (Solanum lycopersicum) genome sequence and identified a total of seven PLCs. Recombinant proteins were produced for all tomato PLCs, except for SlPLC7. The purified proteins showed typical PLC activity, as different PLC substrates were hydrolysed to produce diacylglycerol. We studied SlPLC2, SlPLC4 and SlPLC5 enzymes in more detail and observed distinct requirements for Ca(2+) ions and pH, for both their optimum activity and substrate preference. This indicates that each enzyme could be differentially and specifically regulated in vivo, leading to the generation of PLC homolog-specific signals in response to different stimuli. PLC overexpression and specific inhibition of PLC activity revealed that PLC is required for both specific effector- and more general "pattern"-triggered immunity. For the latter, we found that both the flagellin-triggered response and the internalization of the corresponding receptor, Flagellin Sensing 2 (FLS2) of Arabidopsis thaliana, are suppressed by inhibition of PLC activity. Altogether, our data support an important role for PLC enzymes in plant defence signalling downstream of immune receptors. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner.


Asunto(s)
Fosfoinositido Fosfolipasa C/genética , Inmunidad de la Planta/genética , Solanum lycopersicum/genética , Arabidopsis/enzimología , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Solanum lycopersicum/enzimología , Familia de Multigenes , Fosfoinositido Fosfolipasa C/biosíntesis , Fosfoinositido Fosfolipasa C/aislamiento & purificación , Proteínas Quinasas/genética
4.
J Exp Bot ; 65(22): 6487-98, 2014 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-25205577

RESUMEN

Pathogens can colonize all plant organs and tissues. To prevent this, each cell must be capable of autonomously triggering defence. Therefore, it is generally assumed that primary sensors of the immune system are constitutively present. One major primary sensor against bacterial infection is the flagellin sensing 2 (FLS2) pattern recognition receptor (PRR). To gain insights into its expression pattern, the FLS2 promoter activity in ß-glucuronidase (GUS) reporter lines was monitored. The data show that pFLS2::GUS activity is highest in cells and tissues vulnerable to bacterial entry and colonization, such as stomata, hydathodes, and lateral roots. GUS activity is also high in the vasculature and, by monitoring Ca(2+) responses in the vasculature, it was found that this tissue contributes to flg22-induced Ca(2+) burst. The FLS2 promoter is also regulated in a tissue- and cell type-specific manner and is responsive to hormones, damage, and biotic stresses. This results in stimulus-dependent expansion of the FLS2 expression domain. In summary, a tissue- and cell type-specific map of FLS2 expression has been created correlating with prominent entry sites and target tissues of plant bacterial pathogens.


Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Bacterias/metabolismo , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética , Raíces de Plantas/microbiología , Brotes de la Planta/genética , Brotes de la Planta/microbiología , Proteínas Quinasas/metabolismo , Arabidopsis/microbiología , Proteínas de Arabidopsis/genética , Flagelina/metabolismo , Ácidos Indolacéticos/metabolismo , Modelos Biológicos , Especificidad de Órganos/genética , Raíces de Plantas/crecimiento & desarrollo , Estomas de Plantas/fisiología , Proteínas Quinasas/genética , Estrés Fisiológico/genética
5.
Microbiol Spectr ; : e0135023, 2023 Aug 30.
Artículo en Inglés | MEDLINE | ID: mdl-37646508

RESUMEN

Assuring that cell therapy products are safe before releasing them for use in patients is critical. Currently, compendial sterility testing for bacteria and fungi can take 7-14 days. The goal of this work was to develop a rapid untargeted approach for the sensitive detection of microbial contaminants at low abundance from low volume samples during the manufacturing process of cell therapies. We developed a long-read sequencing methodology using Oxford Nanopore Technologies MinION platform with 16S and 18S amplicon sequencing to detect USP <71> organisms and other microbial species. Reads are classified metagenomically to predict the microbial species. We used an extreme gradient boosting machine learning algorithm (XGBoost) to first assess if a sample is contaminated, and second, determine whether the predicted contaminant is correctly classified or misclassified. The model was used to make a final decision on the sterility status of the input sample. An optimized experimental and bioinformatics pipeline starting from spiked species through to sequenced reads allowed for the detection of microbial samples at 10 colony-forming units (CFU)/mL using metagenomic classification. Machine learning can be coupled with long-read sequencing to detect and identify sample sterility status and microbial species present in T-cell cultures, including the USP <71> organisms to 10 CFU/mL. IMPORTANCE This research presents a novel method for rapidly and accurately detecting microbial contaminants in cell therapy products, which is essential for ensuring patient safety. Traditional testing methods are time-consuming, taking 7-14 days, while our approach can significantly reduce this time. By combining advanced long-read nanopore sequencing techniques and machine learning, we can effectively identify the presence and types of microbial contaminants at low abundance levels. This breakthrough has the potential to improve the safety and efficiency of cell therapy manufacturing, leading to better patient outcomes and a more streamlined production process.

6.
Stem Cell Reports ; 9(5): 1387-1394, 2017 11 14.
Artículo en Inglés | MEDLINE | ID: mdl-29056335

RESUMEN

To interrogate the alternative fates of pancreas and liver in the earliest stages of human organogenesis, we developed laser capture, RNA amplification, and computational analysis of deep sequencing. Pancreas-enriched gene expression was less conserved between human and mouse than for liver. The dorsal pancreatic bud was enriched for components of Notch, Wnt, BMP, and FGF signaling, almost all genes known to cause pancreatic agenesis or hypoplasia, and over 30 unexplored transcription factors. SOX9 and RORA were imputed as key regulators in pancreas compared with EP300, HNF4A, and FOXA family members in liver. Analyses implied that current in vitro human stem cell differentiation follows a dorsal rather than a ventral pancreatic program and pointed to additional factors for hepatic differentiation. In summary, we provide the transcriptional codes regulating the start of human liver and pancreas development to facilitate stem cell research and clinical interpretation without inter-species extrapolation.


Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Hígado/embriología , Páncreas/embriología , Activación Transcripcional , Transcriptoma , Diferenciación Celular , Perfilación de la Expresión Génica , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Hígado/metabolismo , Páncreas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
7.
PLoS One ; 10(2): e0117202, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25699671

RESUMEN

Biopolymers, such as poly-3-hydroxybutyrate (P(3HB)) are produced as a carbon store in an array of organisms and exhibit characteristics which are similar to oil-derived plastics, yet have the added advantages of biodegradability and biocompatibility. Despite these advantages, P(3HB) production is currently more expensive than the production of oil-derived plastics, and therefore, more efficient P(3HB) production processes would be desirable. In this study, we describe the model-guided design and experimental validation of several engineered P(3HB) producing operons. In particular, we describe the characterization of a hybrid phaCAB operon that consists of a dual promoter (native and J23104) and RBS (native and B0034) design. P(3HB) production at 24 h was around six-fold higher in hybrid phaCAB engineered Escherichia coli in comparison to E. coli engineered with the native phaCAB operon from Ralstonia eutropha H16. Additionally, we describe the utilization of non-recyclable waste as a low-cost carbon source for the production of P(3HB).


Asunto(s)
Escherichia coli/genética , Hidroxibutiratos/metabolismo , Poliésteres/metabolismo , Reactores Biológicos , Medios de Cultivo , Escherichia coli/metabolismo , Expresión Génica , Genes Bacterianos , Ingeniería Genética , Operón , Regiones Promotoras Genéticas
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