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The development of wearable devices for sweat analysis has experienced significant growth in the last two decades, being the main focus the monitoring of athletes health during workouts. One of the main challenges of these approaches has been to attain the continuous monitoring of sweat for time periods over 1 h. This is the main challenge addressed in this work by designing an analytical platform that combines the high performance of potentiometric sensors and a fluidic structure made of a plastic fabric into a multiplexed wearable device. The platform comprises Ion-Sensitive Field-Effect Transistors (ISFETs) manufactured on silicon, a tailor-made solid-state reference electrode, and a temperature sensor integrated into a patch-like polymeric substrate, together with the component that easily collects and drives samples under continuous capillary flow to the sensor areas. ISFET sensors for measuring pH, sodium, and potassium ions were fully characterized in artificial sweat solutions, providing reproducible and stable responses. Then, the real-time and continuous monitoring of the biomarkers in sweat with the wearable platform was assessed by comparing the ISFETs responses recorded during an 85-min continuous exercise session with the concentration values measured using commercial Ion-Selective Electrodes (ISEs) in samples collected at certain times during the session. The developed sensing platform enables the continuous monitoring of biomarkers and facilitates the study of the effects of various real working conditions, such as cycling power and skin temperature, on the target biomarker concentration levels.
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Biomarcadores , Técnicas Biosensibles , Silicio , Sudor , Transistores Electrónicos , Dispositivos Electrónicos Vestibles , Sudor/química , Técnicas Biosensibles/instrumentación , Humanos , Silicio/química , Biomarcadores/análisis , Diseño de Equipo , Sodio/análisis , Potasio/análisis , Concentración de Iones de Hidrógeno , Monitoreo Fisiológico/instrumentaciónRESUMEN
Insulinomas are rare neuroendocrine tumors arising from pancreatic ß cells, characterized by aberrant proliferation and altered insulin secretion, leading to glucose homeostasis failure. With the aim of uncovering the role of noncoding regulatory regions and their aberrations in the development of these tumors, we coupled epigenetic and transcriptome profiling with whole-genome sequencing. As a result, we unraveled somatic mutations associated with changes in regulatory functions. Critically, these regions impact insulin secretion, tumor development, and epigenetic modifying genes, including polycomb complex components. Chromatin remodeling is apparent in insulinoma-selective domains shared across patients, containing a specific set of regulatory sequences dominated by the SOX17 binding motif. Moreover, many of these regions are H3K27me3 repressed in ß cells, suggesting that tumoral transition involves derepression of polycomb-targeted domains. Our work provides a compendium of aberrant cis-regulatory elements affecting the function and fate of ß cells in their progression to insulinomas and a framework to identify coding and noncoding driver mutations.
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Insulinoma , Humanos , Insulinoma/genética , Insulinoma/patología , Insulinoma/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Mutación , Regulación Neoplásica de la Expresión Génica , Epigénesis Genética , Ensamble y Desensamble de Cromatina/genéticaRESUMEN
BACKGROUND & AIMS: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. METHODS: We used single cell RNA sequencing to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models, and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. RESULTS: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro, including a Wnt-responsive population, a ciliated population, and Flrt3+ cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples. The expression of Wnt-responsive, interferon-responsive, and epithelial-to-mesenchymal transition population markers increases in chronic pancreatitis and tumor samples. CONCLUSIONS: In light of our discovery of previously unidentified ductal populations, we unmask potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis. Thus, novel lineage-tracing models are needed to investigate ductal-specific populations in vivo.
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Background and aims: Pancreatic ducts form an intricate network of tubules that secrete bicarbonate and drive acinar secretions into the duodenum. This network is formed by centroacinar cells, terminal, intercalated, intracalated ducts, and the main pancreatic duct. Ductal heterogeneity at the single-cell level has been poorly characterized; therefore, our understanding of the role of ductal cells in pancreas regeneration and exocrine pathogenesis has been hampered by the limited knowledge and unexplained diversity within the ductal network. Methods: We used scRNA-seq to comprehensively characterize mouse ductal heterogeneity at single-cell resolution of the entire ductal epithelium from centroacinar cells to the main duct. Moreover, we used organoid cultures, injury models and pancreatic tumor samples to interrogate the role of novel ductal populations in pancreas regeneration and exocrine pathogenesis. Results: We have identified the coexistence of 15 ductal populations within the healthy pancreas and characterized their organoid formation capacity and endocrine differentiation potential. Cluster isolation and subsequent culturing let us identify ductal cell populations with high organoid formation capacity and endocrine and exocrine differentiation potential in vitro , including Wnt-responsive-population, ciliated-population and FLRT3 + cells. Moreover, we have characterized the location of these novel ductal populations in healthy pancreas, chronic pancreatitis, and tumor samples, highlighting a putative role of WNT-responsive, IFN-responsive and EMT-populations in pancreatic exocrine pathogenesis as their expression increases in chronic pancreatitis and PanIN lesions. Conclusions: In light of our discovery of previously unidentified ductal populations, we unmask the potential roles of specific ductal populations in pancreas regeneration and exocrine pathogenesis.
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Transient reprogramming by the expression of OCT4, SOX2, KLF4 and MYC (OSKM) is a therapeutic strategy for tissue regeneration and rejuvenation, but little is known about its metabolic requirements. Here we show that OSKM reprogramming in mice causes a global depletion of vitamin B12 and molecular hallmarks of methionine starvation. Supplementation with vitamin B12 increases the efficiency of reprogramming both in mice and in cultured cells, the latter indicating a cell-intrinsic effect. We show that the epigenetic mark H3K36me3, which prevents illegitimate initiation of transcription outside promoters (cryptic transcription), is sensitive to vitamin B12 levels, providing evidence for a link between B12 levels, H3K36 methylation, transcriptional fidelity and efficient reprogramming. Vitamin B12 supplementation also accelerates tissue repair in a model of ulcerative colitis. We conclude that vitamin B12, through its key role in one-carbon metabolism and epigenetic dynamics, improves the efficiency of in vivo reprogramming and tissue repair.
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Plasticidad de la Célula , Reprogramación Celular , Animales , Ratones , Vitamina B 12 , Cicatrización de Heridas , VitaminasRESUMEN
Sweat secreted by the human eccrine sweat glands can provide valuable biomarker information during exercise. Real-time non-invasive biomarker recordings are therefore useful for evaluating the physiological conditions of an athlete such as their hydration status during endurance exercise. This work describes a wearable sweat biomonitoring patch incorporating printed electrochemical sensors into a plastic microfluidic sweat collector and data analysis that shows the real-time recorded sweat biomarkers can be used to predict a physiological biomarker. The system was placed on subjects carrying out an hour-long exercise session and results were compared to a wearable system using potentiometric robust silicon-based sensors and to commercially available HORIBA-LAQUAtwin devices. Both prototypes were applied to the real-time monitoring of sweat during cycling sessions and showed stable readings for around an hour. Analysis of the sweat biomarkers collected from the printed patch prototype shows that their real-time measurements correlate well (correlation coefficient ≥ 0.65) with other physiological biomarkers such as heart rate and regional sweat rate collected in the same session. We show for the first time, that the real-time sweat sodium and potassium concentration biomarker measurements from the printed sensors can be used to predict the core body temperature with root mean square error (RMSE) of 0.02 °C which is 71% lower compared to the use of only the physiological biomarkers. These results show that these wearable patch technologies are promising for real-time portable sweat monitoring analytical platforms, especially for athletes performing endurance exercise.
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Técnicas Biosensibles , Dispositivos Electrónicos Vestibles , Humanos , Sudor/química , Temperatura Corporal , Electrólitos , Biomarcadores/análisisRESUMEN
Maintenance of stemness is tightly linked to cell cycle regulation through protein phosphorylation by cyclin-dependent kinases (CDKs). However, how this process is reversed during differentiation is unknown. We report here that exit from stemness and differentiation of pluripotent cells along the neural lineage are controlled by CDC14, a CDK-counteracting phosphatase whose function in mammals remains obscure. Lack of the two CDC14 family members, CDC14A and CDC14B, results in deficient development of the neural system in the mouse and impairs neural differentiation from embryonic stem cells (ESCs). Mechanistically, CDC14 directly dephosphorylates specific proline-directed Ser/Thr residues of undifferentiated embryonic transcription Factor 1 (UTF1) during the exit from stemness, triggering its proteasome-dependent degradation. Multiomic single-cell analysis of transcription and chromatin accessibility in differentiating ESCs suggests that increased UTF1 levels in the absence of CDC14 prevent the proper firing of bivalent promoters required for differentiation. CDC14 phosphatases are dispensable for mitotic exit, suggesting that CDC14 phosphatases have evolved to control stemness rather than cell cycle exit and establish the CDK-CDC14 axis as a critical molecular switch for linking cell cycle regulation and self-renewal.
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Proteínas de Ciclo Celular , Proteínas de Saccharomyces cerevisiae , Animales , Ratones , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Tirosina Fosfatasas/genética , Proteínas Tirosina Fosfatasas/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclo Celular , Fosforilación/fisiología , Mitosis , Proteínas de Saccharomyces cerevisiae/metabolismo , MamíferosRESUMEN
Regenerating pancreatic ß-cells is a potential curative approach for diabetes. We previously identified the small molecule CID661578 as a potent inducer of ß-cell regeneration, but its target and mechanism of action have remained unknown. We now screened 257 million yeast clones and determined that CID661578 targets MAP kinase-interacting serine/threonine kinase 2 (MNK2), an interaction we genetically validated in vivo. CID661578 increased ß-cell neogenesis from ductal cells in zebrafish, neonatal pig islet aggregates and human pancreatic ductal organoids. Mechanistically, we found that CID661578 boosts protein synthesis and regeneration by blocking MNK2 from binding eIF4G in the translation initiation complex at the mRNA cap. Unexpectedly, this blocking activity augmented eIF4E phosphorylation depending on MNK1 and bolstered the interaction between eIF4E and eIF4G, which is necessary for both hypertranslation and ß-cell regeneration. Taken together, our findings demonstrate a targetable role of MNK2-controlled translation in ß-cell regeneration, a role that warrants further investigation in diabetes.
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Factor 4E Eucariótico de Iniciación , Factor 4G Eucariótico de Iniciación , Animales , Línea Celular , Factor 4E Eucariótico de Iniciación/química , Factor 4E Eucariótico de Iniciación/genética , Factor 4E Eucariótico de Iniciación/metabolismo , Humanos , Recién Nacido , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Pez Cebra/metabolismoRESUMEN
In recent years, the development of ex vivo organoid cultures has gained substantial attention as a model to study regenerative medicine and diseases in several tissues. Diabetes and pancreatic ductal adenocarcinoma (PDAC) are the two major devastating diseases affecting the pancreas. Suitable models for regenerative medicine in diabetes and to accurately study PDAC biology and treatment response are essential in the pancreatic field. Pancreatic organoids can be generated from healthy pancreas or pancreatic tumors and constitute an important translational bridge between in vitro and in vivo models. Here, we review the rapidly emerging field of pancreatic organoids and summarize the current applications of the technology to tissue regeneration, disease modelling, and drug screening.
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Liquid analysis is key to track conformity with the strict process quality standards of sectors like food, beverage, and chemical manufacturing. In order to analyse product qualities online and at the very point of interest, automated monitoring systems must satisfy strong requirements in terms of miniaturization, energy autonomy, and real time operation. Toward this goal, we present the first implementation of artificial taste running on neuromorphic hardware for continuous edge monitoring applications. We used a solid-state electrochemical microsensor array to acquire multivariate, time-varying chemical measurements, employed temporal filtering to enhance sensor readout dynamics, and deployed a rate-based, deep convolutional spiking neural network to efficiently fuse the electrochemical sensor data. To evaluate performance we created MicroBeTa (Microsensor Beverage Tasting), a new dataset for beverage classification incorporating 7 h of temporal recordings performed over 3 days, including sensor drifts and sensor replacements. Our implementation of artificial taste is 15× more energy efficient on inference tasks than similar convolutional architectures running on other commercial, low power edge-AI inference devices, achieving over 178× lower latencies than the sampling period of the sensor readout, and high accuracy (97%) on a single Intel Loihi neuromorphic research processor included in a USB stick form factor.
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The development of diagnostic tools for measuring a wide spectrum of target analytes, from biomarkers to other biochemical parameters in biological fluids, has experienced a significant growth in the last decades, with a good number of such tools entering the market. Recently, a clear focus has been put on miniaturized wearable devices, which offer powerful capabilities for real-time and continuous analysis of biofluids, mainly sweat, and can be used in athletics, consumer wellness, military, and healthcare applications. Sweat is an attractive biofluid in which different biomarkers could be noninvasively measured to provide rapid information about the physical state of an individual. Wearable devices reported so far often provide discrete (single) measurements of the target analytes, most of them in the form of a yes/no qualitative response. However, quantitative biomarker analysis over certain periods of time is highly demanded for many applications such as the practice of sports or the precise control of the patient status in hospital settings. For this, a feasible combination of fluidic elements and sensor architectures has been sought. In this regard, this paper shows a concise overview of analytical tools based on the use of capillary-driven fluidics taking place on paper or fabric devices integrated with solid-state sensors fabricated by thick film technologies. The main advantages and limitations of the current technologies are pointed out together with the progress towards the development of functional devices. Those approaches reported in the last decade are examined in detail.
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Técnicas Biosensibles , Dispositivos Electrónicos Vestibles , Biomarcadores , Humanos , Sudor , TextilesRESUMEN
Multiple transcription factors have been shown to promote pancreatic ß-cell differentiation, yet much less is known about negative regulators. Earlier epigenomic studies suggested that the transcriptional repressor REST could be a suppressor of endocrinogenesis in the embryonic pancreas. However, pancreatic Rest knockout mice failed to show abnormal numbers of endocrine cells, suggesting that REST is not a major regulator of endocrine differentiation. Using a different conditional allele that enables profound REST inactivation, we observed a marked increase in pancreatic endocrine cell formation. REST inhibition also promoted endocrinogenesis in zebrafish and mouse early postnatal ducts and induced ß-cell-specific genes in human adult duct-derived organoids. We also defined genomic sites that are bound and repressed by REST in the embryonic pancreas. Our findings show that REST-dependent inhibition ensures a balanced production of endocrine cells from embryonic pancreatic progenitors.
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Regulación del Desarrollo de la Expresión Génica , Pez Cebra , Animales , Diferenciación Celular/genética , Ratones , Organogénesis/genética , Páncreas , Pez Cebra/genéticaRESUMEN
OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.
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The Hnf1b-CreERT2 BAC transgenic (Tg(Hnf1b-cre/ERT2)1Jfer) has been used extensively to trace the progeny of pancreatic ducts in developmental, regeneration, or cancer models. Hnf1b-CreERT2 transgenics have been used to show that the cells that form the embryonic pancreas duct-like plexus are bipotent duct-endocrine progenitors, whereas adult mouse duct cells are not a common source of ß cells in various regenerative settings. The interpretation of such genetic lineage tracing studies is critically dependent on a correct understanding of the cell type specificity of recombinase activity with each reporter system. We have reexamined the performance of Hnf1b-CreERT2 with a Rosa26-RFP reporter transgene. This showed inducible recombination of up to 96% adult duct cells, a much higher efficiency than previously used reporter transgenes. Despite this high duct-cell excision, recombination in α and ß cells remained very low, similar to previously used reporters. However, nearly half of somatostatin-expressing δ cells showed reporter activation, which was due to Cre expression in δ cells rather than to duct to δ cell conversions. The high recombination efficiency in duct cells indicates that the Hnf1b-CreERT2 model can be useful for both ductal fate mapping and genetic inactivation studies. The recombination in δ cells does not modify the interpretation of studies that failed to show duct conversions to other cell types, but needs to be considered if this model is used in studies that aim to modify the plasticity of pancreatic duct cells.
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Islotes Pancreáticos , Células Secretoras de Somatostatina , Animales , Factor Nuclear 1-beta del Hepatocito/genética , Integrasas/genética , Ratones , Ratones Transgénicos , Recombinación Genética , TransgenesRESUMEN
In vertebrates, GATA2 is a master regulator of hematopoiesis and is expressed throughout embryo development and in adult life. Although the essential role of GATA2 in mouse hematopoiesis is well established, its involvement during early human hematopoietic development is not clear. By combining time-controlled overexpression of GATA2 with genetic knockout experiments, we found that GATA2, at the mesoderm specification stage, promotes the generation of hemogenic endothelial progenitors and their further differentiation to hematopoietic progenitor cells, and negatively regulates cardiac differentiation. Surprisingly, genome-wide transcriptional and chromatin immunoprecipitation analysis showed that GATA2 bound to regulatory regions, and repressed the expression of cardiac development-related genes. Moreover, genes important for hematopoietic differentiation were upregulated by GATA2 in a mostly indirect manner. Collectively, our data reveal a hitherto unrecognized role of GATA2 as a repressor of cardiac fates, and highlight the importance of coordinating the specification and repression of alternative cell fates.
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Factor de Transcripción GATA2/metabolismo , Hematopoyesis , Mesodermo/metabolismo , Diferenciación Celular , Factor de Transcripción GATA2/genética , Regulación de la Expresión Génica , Hemangioblastos/citología , Hemangioblastos/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Mesodermo/citología , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Unión Proteica , Análisis de la Célula IndividualRESUMEN
Recent advances in ß-cell regeneration in vivo are providing insights into the mechanisms involved in the conversion of distinct pancreatic cell lineages into ß cells. These mechanisms mostly involve reactivation of the gene encoding the pancreatic endocrine cell-specifying transcription factor neurogenin-3.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Células Secretoras de Insulina/fisiología , Proteínas del Tejido Nervioso , Regeneración , Transducción de Señal , Humanos , Células Secretoras de Insulina/metabolismoRESUMEN
The adult pancreas is only capable of limited regeneration. Unlike highly regenerative tissues such as the skin, intestinal crypts and hematopoietic system, no dedicated adult stem cells or stem cell niche have so far been identified within the adult pancreas. New ß cells have been shown to form in the adult pancreas, in response to high physiological demand or experimental ß-cell ablation, mostly by replication of existing ß cells. The possibility that new ß cells are formed from other sources is currently a point of major controversy. Under particular injury conditions, fully differentiated pancreatic duct and acinar cells have been shown to dedifferentiate into a progenitor-like state, however the extent, to which ductal, acinar or other endocrine cells contribute to restoring pancreatic ß-cell mass remains to be resolved. In this review we focus on regenerative events in the pancreas with emphasis on the restoration of ß-cell mass. We present an overview of regenerative responses noted within the different pancreatic lineages, following injury. We also highlight the intrinsic plasticity of the adult pancreas that allows for inter-conversion of fully differentiated pancreatic lineages through manipulation of few genes or growth factors. Taken together, evidence from a number of studies suggest that differentiated pancreatic lineages could act as facultative progenitor cells, but the extent to which these contribute to ß-cell regeneration in vivo is still a matter of contention.
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Células Madre Adultas/citología , Células Secretoras de Insulina/fisiología , Regeneración , Animales , Desdiferenciación Celular , Plasticidad de la Célula , Humanos , Células Secretoras de Insulina/citologíaRESUMEN
Mueller matrix polarimetry distinguishes the different origins of the reversible and irreversible chiroptical effects emerging in stirred solutions of J-aggregate nanoparticles: the reversible effect is due to an anisotropic ordering in the solution and the irreversible one is due to a bias from the racemic composition of intrinsically chiral structures.
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The process of regeneration serves to heal injury by replacing missing cells. Understanding regeneration can help us replace cell populations lost during disease, such as the insulin-producing ß cells lost in diabetic patients. Centroacinar cells (CACs) are a specialized ductal pancreatic cell type that act as progenitors to replace ß cells in the zebrafish. However, whether CACs contribute to ß-cell regeneration in adult mammals remains controversial. Here we review the current understanding of the role of CACs as endocrine progenitors during regeneration in zebrafish and mammals.