ABSTRACT
Neurogenin-3 (NEUROG3) is a helix-loop-helix (HLH) transcription factor involved in the production of endocrine cells in the intestine and pancreas of humans and mice. However, the human NEUROG3 loss-of-function phenotype differs subtly from that in mice, but the reason for this difference remains poorly understood. Because NEUROG3 expression precedes exit of the cell cycle and the expression of endocrine cell markers during differentiation, we investigated the effect of lentivirus-mediated overexpression of the human NEUROG3 gene on the cell cycle of BON4 cells and various human nonendocrine cell lines. NEUROG3 overexpression induced a reversible cell cycle exit, whereas expression of a neuronal lineage homolog, NEUROG1, had no such effect. In endocrine lineage cells, the cellular quiescence induced by short-term NEUROG3 expression required cyclin-dependent kinase inhibitor 1A (CDKN1A)/p21CIP1 expression. Expression of endocrine differentiation markers required sustained NEUROG3 expression in the quiescent, but not in the senescent, state. Inhibition of the phosphatase and tensin homolog (PTEN) pathway reversed quiescence by inducing cyclin-dependent kinase 2 (CDK2) and reducing p21CIP1 and NEUROG3 protein levels in BON4 cells and human enteroids. We discovered that NEUROG3 expression stimulates expression of CDKN2a/p16INK4a and BMI1 proto-oncogene polycomb ring finger (BMI1), with the latter limiting expression of the former, delaying the onset of CDKN2a/p16INK4a -driven cellular senescence. Furthermore, NEUROG3 bound to the promoters of both CDKN1a/p21CIP1 and BMI1 genes, and BMI1 attenuated NEUROG3 binding to the CDKN1a/p21CIP1 promoter. Our findings reveal how human NEUROG3 integrates inputs from multiple signaling pathways and thereby mediates cell cycle exit at the onset of differentiation.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Cycle Checkpoints , Mitogen-Activated Protein Kinase 7/metabolism , Nerve Tissue Proteins/metabolism , PTEN Phosphohydrolase/metabolism , Cell Line , Cellular Senescence , Gene Expression Regulation , Genes, p16 , Humans , Proto-Oncogene MasABSTRACT
Inflammatory bowel disease (IBD) is a complex disorder that is associated with significant morbidity. While many recent advances have been made with new diagnostic and therapeutic tools, a deeper understanding of its basic pathophysiology is needed to continue this trend toward improving treatments. By utilizing an unbiased, high-throughput transcriptomic analysis of two well-established mouse models of colitis, we set out to uncover novel coding and noncoding RNAs that are differentially expressed in the setting of colonic inflammation. RNA-seq analysis was performed using colonic tissue from two mouse models of colitis, a dextran sodium sulfate-induced model and a genetic-induced model in mice lacking IL-10. We identified 81 coding RNAs that were commonly altered in both experimental models. Of these coding RNAs, 12 of the human orthologs were differentially expressed in a transcriptomic analysis of IBD patients. Interestingly, 5 of the 12 of human differentially expressed genes have not been previously identified as IBD-associated genes, including ubiquitin D. Our analysis also identified 15 noncoding RNAs that were differentially expressed in either mouse model. Surprisingly, only three noncoding RNAs were commonly dysregulated in both of these models. The discovery of these new coding and noncoding RNAs expands our transcriptional knowledge of mouse models of IBD and offers additional targets to deepen our understanding of the pathophysiology of IBD. NEW & NOTEWORTHY Much of the genome is transcribed as non-protein-coding RNAs; however, their role in inflammatory bowel disease is largely unknown. This study represents the first of its kind to analyze the expression of long noncoding RNAs in two mouse models of inflammatory bowel disease and correlate them to human clinical samples. Using high-throughput RNA-seq analysis, we identified new coding and noncoding RNAs that were differentially expressed such as ubiquitin D and 5730437C11Rik.
Subject(s)
Colitis/genetics , Inflammatory Bowel Diseases/genetics , MicroRNAs/genetics , RNA, Long Noncoding/genetics , Animals , Caco-2 Cells , Cells, Cultured , Colitis/metabolism , Colon/metabolism , Colon/pathology , Humans , Inflammatory Bowel Diseases/metabolism , Male , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , RNA, Long Noncoding/metabolism , TranscriptomeABSTRACT
Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.
Subject(s)
Aging/physiology , Intestines/cytology , Tissue Culture Techniques/methods , Animals , Cryopreservation , Culture Media, Conditioned/pharmacology , Immunohistochemistry , Intestinal Mucosa/metabolism , Mice , Myofibroblasts/cytology , Myofibroblasts/drug effects , Sus scrofa , Temperature , Transduction, GeneticABSTRACT
BACKGROUND & AIMS: Proprotein convertase 1/3 (PC1/3) deficiency, an autosomal-recessive disorder caused by rare mutations in the proprotein convertase subtilisin/kexin type 1 (PCSK1) gene, has been associated with obesity, severe malabsorptive diarrhea, and certain endocrine abnormalities. Common variants in PCSK1 also have been associated with obesity in heterozygotes in several population-based studies. PC1/3 is an endoprotease that processes many prohormones expressed in endocrine and neuronal cells. We investigated clinical and molecular features of PC1/3 deficiency. METHODS: We studied the clinical features of 13 children with PC1/3 deficiency and performed sequence analysis of PCSK1. We measured enzymatic activity of recombinant PC1/3 proteins. RESULTS: We identified a pattern of endocrinopathies that develop in an age-dependent manner. Eight of the mutations had severe biochemical consequences in vitro. Neonates had severe malabsorptive diarrhea and failure to thrive, required prolonged parenteral nutrition support, and had high mortality. Additional endocrine abnormalities developed as the disease progressed, including diabetes insipidus, growth hormone deficiency, primary hypogonadism, adrenal insufficiency, and hypothyroidism. We identified growth hormone deficiency, central diabetes insipidus, and male hypogonadism as new features of PCSK1 insufficiency. Interestingly, despite early growth abnormalities, moderate obesity, associated with severe polyphagia, generally appears. CONCLUSIONS: In a study of 13 children with PC1/3 deficiency caused by disruption of PCSK1, failure of enteroendocrine cells to produce functional hormones resulted in generalized malabsorption. These findings indicate that PC1/3 is involved in the processing of one or more enteric hormones that are required for nutrient absorption.
Subject(s)
Diarrhea/etiology , Endocrine System Diseases/etiology , Malabsorption Syndromes/etiology , Obesity/complications , Proprotein Convertase 1/deficiency , Adolescent , Adrenocorticotropic Hormone/blood , Child , Child, Preschool , Cohort Studies , Endocrine System Diseases/complications , Endocrine System Diseases/congenital , Female , Humans , Infant , Male , Mutation , Obesity/congenital , Proprotein Convertase 1/geneticsABSTRACT
Remifentanil-induced hyperalgesia (RIH) is a common clinical phenomenon that limits the use of opioids in pain management. Esketamine, a non-competitive N-methyl-d-aspartate (NMDA) receptor antagonist, has been shown to prevent and treat RIH. However, the underlying effect mechanism of esketamine on RIH remains unclear. This study aimed to investigate the role and mechanism of esketamine in preventing and treating RIH based on the NMDA receptor-CaMKIIα pathway. In this study, an experimental animal model was used to determine the therapeutic effect of esketamine on pain elimination. Moreover, the mRNA transcription and protein expression levels of CaMKII and GluN2B were investigated to offer evidence of the protective capability of esketamine in ameliorating RIH. The results demonstrated that esketamine attenuated RIH by inhibiting CaMKII phosphorylation and downstream signaling pathways mediated by the NMDA receptor. Furthermore, ketamine reversed the upregulation of spinal CaMKII induced by remifentanil. These findings suggest that the NMDA receptor-CaMKII pathway plays a critical role in the development of RIH, and ketamine's effect on this pathway may provide a new therapeutic approach for the prevention and treatment of RIH.
ABSTRACT
Mix progenitors are short-lived multipotential cells formed as intestinal epithelial stem cells initiate a differentiation program. Clone dynamics indicates that various epithelial cell lineages arise from Mix via a sequence of progressively restricted progenitor states. Lateral inhibitory Notch signaling between the daughters of Mix (DOM) is thought to break their initial symmetry, thereby determining whether a DOM invokes a columnar (absorptive) or granulocytic (secretory) cell lineage program. This is supported by the absence of granulocytes following enforced Notch signaling or Atoh1 deletion. Conversely, granulocytes increase in frequency following inhibition of Notch signaling or Hes1 deletion. Thus reciprocal repression between Hes1 and Atoh1 is thought to implement a Notch signaling-driven cell-fate-determining binary switch in DOM. The brush (tuft) cells, a poorly understood chemosensory cell type, are not incorporated into this model. We report that brush cell numbers increase dramatically following conditional Atoh1-deletion, demonstrating that brush cell production, determination, differentiation and survival are Atoh1-independent. We also report that brush cells are derived from Gfi1b-expressing progenitors. These and related results suggest a model in which initially equivalent DOM progenitors have three metastable states defined by the transcription factors Hes1, Atoh1, and Gfi1b. Lateral inhibitory Notch signaling normally ensures that Hes1 dominates in one of the two DOMs, invoking a columnar lineage program, while either Atoh1 or Gfi1b dominates in the other DOM, invoking a granulocytic or brush cell lineage program, respectively, and thus implementing a cell fate-determining ternary switch.
Subject(s)
Cell Differentiation/physiology , Cell Lineage/physiology , Intestinal Mucosa/cytology , Microvilli/physiology , Multipotent Stem Cells/cytology , Signal Transduction/physiology , Animals , Basic Helix-Loop-Helix Transcription Factors/deficiency , Basic Helix-Loop-Helix Transcription Factors/metabolism , DNA Primers , Flow Cytometry , Homeodomain Proteins/metabolism , Mice , Mice, Knockout , Microscopy, Fluorescence , Polymerase Chain Reaction , Proto-Oncogene Proteins/metabolism , Receptors, Notch/metabolism , Repressor Proteins/metabolism , Transcription Factor HES-1ABSTRACT
Fluorescence-activated cell sorting (FACS) is an essential tool for studies requiring isolation of distinct intestinal epithelial cell populations. Inconsistent or lack of reporting of the critical parameters associated with FACS methodologies has complicated interpretation, comparison, and reproduction of important findings. To address this problem a comprehensive multicenter study was designed to develop guidelines that limit experimental and data reporting variability and provide a foundation for accurate comparison of data between studies. Common methodologies and data reporting protocols for tissue dissociation, cell yield, cell viability, FACS, and postsort purity were established. Seven centers tested the standardized methods by FACS-isolating a specific crypt-based epithelial population (EpCAM+/CD44+) from murine small intestine. Genetic biomarkers for stem/progenitor (Lgr5 and Atoh 1) and differentiated cell lineages (lysozyme, mucin2, chromogranin A, and sucrase isomaltase) were interrogated in target and control populations to assess intra- and intercenter variability. Wilcoxon's rank sum test on gene expression levels showed limited intracenter variability between biological replicates. Principal component analysis demonstrated significant intercenter reproducibility among four centers. Analysis of data collected by standardized cell isolation methods and data reporting requirements readily identified methodological problems, indicating that standard reporting parameters facilitate post hoc error identification. These results indicate that the complexity of FACS isolation of target intestinal epithelial populations can be highly reproducible between biological replicates and different institutions by adherence to common cell isolation methods and FACS gating strategies. This study can be considered a foundation for continued method development and a starting point for investigators that are developing cell isolation expertise to study physiology and pathophysiology of the intestinal epithelium.
Subject(s)
Epithelial Cells/physiology , Flow Cytometry/standards , Intestinal Mucosa/cytology , Animals , Cell Culture Techniques , Cell Survival , Gene Expression Regulation , Hyaluronan Receptors/genetics , Hyaluronan Receptors/metabolism , Male , Mice , Mice, Inbred C57BL , Observer Variation , Staining and LabelingABSTRACT
OBJECTIVES: Congenital diarrhea disorders are a group of genetically diverse and typically autosomal recessive disorders that have yet to be well characterized phenotypically or molecularly. Diagnostic assessments are generally limited to nutritional challenges and histologic evaluation, and many subjects eventually require a prolonged course of intravenous nutrition. Here we describe next-generation sequencing techniques to investigate a child with perplexing congenital malabsorptive diarrhea and other presumably unrelated clinical problems; this method provides an alternative approach to molecular diagnosis. METHODS: We screened the diploid genome of an affected individual, using exome sequencing, for uncommon variants that have observed protein-coding consequences. We assessed the functional activity of the mutant protein, as well as its lack of expression using immunohistochemistry. RESULTS: Among several rare variants detected was a homozygous nonsense mutation in the catalytic domain of the proprotein convertase subtilisin/kexin type 1 gene. The mutation abolishes prohormone convertase 1/3 endoprotease activity as well as expression in the intestine. These primary genetic findings prompted a careful endocrine reevaluation of the child at 4.5 years of age, and multiple significant problems were subsequently identified consistent with the known phenotypic consequences of proprotein convertase subtilisin/kexin type 1 (PCSK1) gene mutations. Based on the molecular diagnosis, alternate medical and dietary management was implemented for diabetes insipidus, polyphagia, and micropenis. CONCLUSIONS: Whole-exome sequencing provides a powerful diagnostic tool to clinicians managing rare genetic disorders with multiple perplexing clinical manifestations.
Subject(s)
Codon, Nonsense , Diabetes Insipidus/complications , Diarrhea/genetics , Exome , Malabsorption Syndromes/genetics , Proprotein Convertase 1/genetics , Diarrhea/congenital , Diarrhea/diagnosis , Homozygote , Humans , Infant, Newborn , Malabsorption Syndromes/congenital , Malabsorption Syndromes/diagnosis , Male , Proprotein Convertase 1/metabolism , Sequence Analysis, DNAABSTRACT
BACKGROUND: Clinical neurology is difficult for young residents. To familiarize with neurological emergencies as soon as possible for young doctors, the urgent inpatient neurologic consultations were analyzed. METHODS: A retrospective study was conducted on the urgent inpatient neurologic consultations in a large tertiary hospital for 4 consecutive years. RESULTS: A total of 1437 cases were included, and the annual consultation cases gradually decreased from 573 to 257, involving 29 clinical departments. The disorders of urgent inpatient neurologic consultations were divided into three categories: neurological disorders (77.8%), non-neurological disorders (10.4%), and undiagnosed disorders (11.8%), common causes in consultation were disturbance of consciousness (36.0%), convulsions/stiffness (13.6%), limb weakness (8%), and mental disorder (5.6%). Common neurological disorders included acute cerebrovascular disease (33.6%), epilepsy/status epilepticus (15.8%), and metabolic or infectious toxic encephalopathy (14.9%). CONCLUSION: Urgent inpatient neurologic consultations involve multidisciplinary critical diseases, mainly neurological diseases. The standardized training of residents may help to rapidly improve the comprehensive diagnosis and treatment ability of young residents and is suitable for use in hospitals at all levels.
Subject(s)
Epilepsy , Inpatients , Humans , Tertiary Care Centers , Retrospective Studies , Follow-Up Studies , Referral and Consultation , Epilepsy/diagnosis , Epilepsy/therapyABSTRACT
The intestinal muscle layers execute various gut wall movements to achieve controlled propulsion and mixing of intestinal content. Engineering intestinal muscle layers with complex contractile function is critical for developing bioartificial intestinal tissue to treat patients with short bowel syndrome. Here, the first demonstration of a living intestinal muscle patch capable of generating three distinct motility patterns and displaying multiple digesta manipulations is reported. Assessment of contractility, cellular morphology, and transcriptome profile reveals that successful generation of the contracting muscle patch relies on both biological factors in a serum-free medium and environmental cues from an elastic electrospun gelatin scaffold. By comparing gene-expression patterns among samples, it is shown that biological factors from the medium strongly affect ion-transport activities, while the scaffold unexpectedly regulates cell-cell communication. Analysis of ligandreceptor interactome identifies scaffold-driven changes in intercellular communication, and 78% of the upregulated ligand-receptor interactions are involved in the development and function of enteric neurons. The discoveries highlight the importance of combining biomolecular and biomaterial approaches for tissue engineering. The living intestinal muscle patch represents a pivotal advancement for building functional replacement intestinal tissue. It offers a more physiological model for studying GI motility and for preclinical drug discovery.
Subject(s)
Gastrointestinal Contents , Muscle, Smooth , Humans , Muscle, Smooth/physiology , Intestines , Tissue Engineering , Muscle Contraction , Biological FactorsABSTRACT
Background: Hemoporfin-mediated photodynamic therapy (PDT) is an effective treatment for port-wine stains (PWS), and pain is the main adverse effect of this therapy. General anesthesia is commonly used for pain management during PDT, but the effect of general anesthetics on the subsequent treatment efficacy of PDT in PWS has not been reported. Objectives: To assess the use of general anesthesia combined with PDT compared with PDT alone in 207 PWS patients, and to provide further safety and efficacy data on this combined therapy. Methods: Propensity score matching (PSM) was used at a 2:1 ratio to create a general anesthetic group (n = 138) and a highly comparable nonanesthetic group (n = 69). The clinical outcomes were evaluated, and the treatment reactions and adverse effects were recorded after one treatment with PDT. Results: After matching, there was no significant difference in the demographic data of the patients in the two groups (p > 0.05), while the treatment efficacy was significantly higher in the general anesthetic group than in the nonanesthetic group (76.81 vs. 56.52%, p < 0.05). Moreover, logistic regression analysis confirmed that patients receiving general anesthesia showed an association with a good response to PDT (OR = 3.06; 95% CI, 1.57-6.00; p = 0.0011). Purpura lasted longer in the general anesthetic group, but the other treatment reactions and adverse effects were similar in the two groups (p > 0.05). No serious systemic adverse reactions were observed. Conclusion: We recommend this combined therapy, which is associated with painless, as a high efficacy treatment option for PWS patients, especially for patients with a poor response to multiple PDT alone treatments.
ABSTRACT
Nanotechnology is used in a wide range of fields, including medicine, cosmetics, and new material development, and is one of the most popular technologies in the field of flexible electronic products. For the present work, the chemical reduction method with environmentally friendly reducing agents was used to synthesize copper nanoparticles (CuNPs) with good dispersibility. The CuNPs were characterized by transmission electron microscopy (TEM), X-ray diffraction (XRD), and ultraviolet-visible spectrophotometry (UV-vis). After the CuNPs were formed, the solvent, polymers, and additives were added to form copper ink. Finally, the prepared copper inks were applied to flexible polyethylene terephthalate (PET) substrate under low sintering temperature and the effects of sintering time and different concentrations of sintering agent on resistivity were investigated. The results show that the copper nanoparticles synthesized by secondary reduction were smaller, more uniform, and better dispersed than those formed by primary reduction. Ethylene glycol has reducing effects under high temperatures; therefore, the CuNPs formed using the mixed solvent were small and well dispersed. The copper ink was applied on the PET substrate, treated with a formic acid aqueous solution, and sintered at 130 °C for 60 min, and its resistivity was about 1.67 × 10-3 Ω cm. The proposed synthesizing method is expected to have potential applications in the flexible electronic products field.
ABSTRACT
Using the ground state dominance approximation and a variational theory, we study the encapsulation of a polyelectrolyte chain by an oppositely charged spherical surface. The electrostatic attraction between the polyelectrolyte and the surface and the entropy loss of the encapsulated polyelectrolyte chain dictate the optimum conditions for encapsulation. Two scenarios of encapsulation are identified: entropy-dominated and adsorption-dominated encapsulation. In the entropy-dominated encapsulation regime, the polyelectrolyte chain is delocalized, and the optimum radius of the encapsulating sphere decreases with increasing the attraction. In the adsorption-dominated encapsulation regime, the polyelectrolyte chain is strongly localized near the surface, and the optimum radius increases with increasing the attraction. After identifying a universal encapsulation parameter, the dependencies of the optimum radius on the salt concentration, surface charge density, polymer charge density, and polymer length are explored.
Subject(s)
Polymers/chemistry , Adsorption , Capsules/chemistry , Electrolytes/chemistry , Surface PropertiesABSTRACT
Memory effects of microphase segregation in diblock copolymer brushes and binary mixed homopolymer brushes exposed to solvents of different quality and selectivity are studied using Single-Chain-in-Mean-Field (SCMF) simulations. We gauge these memory effects by a fluctuation memory measure, reflecting the correlation between the quenched fluctuations of grafting points and the microphase-separated morphology, and a domain memory measure, quantifying the correlation between surface morphologies during cyclic exposure to different solvents. The fluctuation and domain memory measures are closely correlated, and both of them have their root in the broken translational symmetry of the distribution of grafting points. They become stronger upon increasing the fluctuations of the grafting points. The effects of solvent quality and selectivity, grafting density, and composition of brushes on the memory measures are discussed.
Subject(s)
Polymers/chemistry , Solvents/chemistryABSTRACT
OBJECTIVE: To analyze the clinical characteristics of patients with severe or critical coronavirus disease 2019 (COVID-19) receiving tracheal intubation. METHODS: We analyzed clinical characteristics of 18 severely or critically ill patients with COVID-19 undergoing tracheal intubation. The general demographic and clinical data of the patients including their age, gender, pre- intubation state of consciousness and the ventilation mode were recorded. The anesthesiologists performing the tracheal intubation procedure evaluated and recorded the tracheal intubation conditions of the patients. The changes in the vital signs of the patients before anesthesia induction and after intubation were recorded. RESULTS: The average ages of these patients were 70.39±8.02 years. Fifteen patients (83.33%) received non- invasive ventilation before tracheal intubation, and 13 patients (72.22%) were conscious before tracheal intubation. After induction of anesthesia, the blood pressure and heart rate of the patients decreased significantly (P < 0.05). Most of the patients (94.44%) were in excellent or good conditions for tracheal intubation, and the first-attempt success rate of tracheal intubation was 100%. Five patients died within 3 weeks following the intubation. Tracheotomy was performed in one patient. Twelve patients were still on endotracheal mechanical ventilation in the intensive care unit, and one of them received ECMO treatment due to poor oxygenation. A total of 16 experienced anesthesiologists participated in tracheal intubation, all with third-level protection during the operation, and no medical staff infection has been detected so far. CONCLUSIONS: For patients with severe and critical COVID-19 and indications of tracheal intubation, we recommend early intubation with invasive respiratory support to improve the treatment efficacy and reduce the mortality. Anesthetic agents should be used carefully during tracheal intubation to ensure patients' safety. The medical staff should have a high-level protection during the intubation to maximally ensure their safety.
Subject(s)
Coronavirus Infections/therapy , Intubation, Intratracheal , Pneumonia, Viral/therapy , Aged , Betacoronavirus , COVID-19 , Humans , Middle Aged , Pandemics , SARS-CoV-2ABSTRACT
Biallelic mutations of the gene encoding the transcription factor NEUROG3 are associated with a rare disorder that presents in neonates as generalized malabsorption - due to a complete absence of enteroendocrine cells - followed, in early childhood or beyond, by insulin-dependent diabetes mellitus (IDDM). The commonly delayed onset of IDDM suggests a differential requirement for NEUROG3 in endocrine cell generation in the human pancreas versus the intestine. However, previously identified human mutations were hypomorphic and, hence, may have had residual function in pancreas. We report 2 patients with biallelic functionally null variants of the NEUROG3 gene who nonetheless did not present with IDDM during infancy but instead developed permanent IDDM during middle childhood ages. The variants showed no evidence of function in traditional promoter-based assays of NEUROG3 function and also failed to exhibit function in a variety of potentially novel in vitro and in vivo molecular assays designed to discern residual NEUROG3 function. These findings imply that, unlike in mice, pancreatic endocrine cell generation in humans is not entirely dependent on NEUROG3 expression and, hence, suggest the presence of unidentified redundant in vivo pathways in human pancreas capable of yielding ß cell mass sufficient to maintain euglycemia until early childhood.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Diabetes Mellitus/genetics , Genetic Predisposition to Disease , Loss of Function Mutation , Nerve Tissue Proteins/genetics , Basic Helix-Loop-Helix Transcription Factors/metabolism , Cell Proliferation , Child , Diabetes Mellitus, Type 1 , Enteroendocrine Cells/metabolism , Female , Gene Expression Regulation , Helix-Loop-Helix Motifs/genetics , Humans , Insulin-Secreting Cells/metabolism , Islets of Langerhans , Malabsorption Syndromes , Male , Nerve Tissue Proteins/metabolism , Pancreas , Promoter Regions, GeneticABSTRACT
Human small intestinal crypts are the source of intestinal stem cells (ISCs) that are capable of undergoing self-renewal and differentiation to an epithelial layer. The development of methods to expand the ISCs has provided opportunities to model human intestinal epithelial disorders. Human crypt samples are usually obtained from either endoscopic or discarded surgical samples, and are thereby exposed to warm ischemia, which may impair their in vitro growth as three-dimensional culture as spheroids or enteroids. In this study we compared duodenal samples obtained from discarded surgical samples to those isolated from whole-body preserved cadaveric donors to generate in vitro cultures. We also examined the effect of storage solution (phosphate-buffered saline or University of Wisconsin [UW] solution) as well as multiple storage times on crypt isolation and growth in culture. We found that intestinal crypts were successfully isolated from cadaveric tissue stored for up to 144 h post-procurement and also were able to generate enteroids and spheroids in certain media conditions. Surgical samples stored in UW after procurement were sufficiently viable up to 24 h and also allowed the generation of enteroids and spheroids. We conclude that surgical samples stored for up to 24 h post-procurement in UW solution allowed for delayed crypt isolation and viable in vitro cultures. Furthermore, in situ, hypothermic preservation in cadaveric duodenal samples permitted crypt/ISC isolation, and successful culture of spheroids and enteroids from tissues held for up to 6 days post-procurement.
Subject(s)
Cell Culture Techniques/methods , Intestines/physiopathology , Cadaver , Cell Differentiation , HumansABSTRACT
BACKGROUND: Neurogenin-3 (NEUROG3) is expressed in endocrine progenitor cells and is required for endocrine-cell development in the pancreas and intestine. The NEUROG3 gene (NEUROG3) is therefore a candidate for the cause of a newly discovered autosomal recessive disorder characterized by generalized malabsorption and a paucity of enteroendocrine cells. METHODS: We screened genomic DNA from three unrelated patients with sparse enteroendocrine cells for mutations of NEUROG3. We then tested the ability of the observed mutations to alter NEUROG3 function, using in vitro and in vivo assays. RESULTS: The patients had few intestinal enteroendocrine cells positive for chromogranin A, but they had normal numbers of Paneth's, goblet, and absorptive cells. We identified two homozygous mutations in NEUROG3, both of which rendered the NEUROG3 protein unable to activate NEUROD1, a downstream target of NEUROG3, and compromised the ability of NEUROG3 to bind to an E-box element in the NEUROD1 promoter. The injection of wild-type but not mutant NEUROG3 messenger RNA into xenopus embryos induced NEUROD1 expression. CONCLUSIONS: A newly discovered disorder characterized by malabsorptive diarrhea and a lack of intestinal enteroendocrine cells is caused by loss-of-function mutations in NEUROG3.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/genetics , Diarrhea/congenital , Diarrhea/genetics , Intestine, Small/pathology , Malabsorption Syndromes/genetics , Mutation, Missense , Nerve Tissue Proteins/genetics , Amino Acid Sequence , Base Sequence , Basic Helix-Loop-Helix Transcription Factors/metabolism , Chronic Disease , Diarrhea/pathology , Enteroendocrine Cells/pathology , Fatal Outcome , Humans , Infant, Newborn , Malabsorption Syndromes/complications , Malabsorption Syndromes/pathology , Male , Molecular Sequence Data , Nerve Tissue Proteins/metabolism , Promoter Regions, GeneticABSTRACT
Microphase separation of binary mixed A/B polymer brushes exposed to different solvents is studied using Single-Chain-in-Mean-Field simulations. Effects of solvent quality and selectivity, grafting density, composition, and chain-length asymmetry are systematically investigated, and diagrams of morphologies in various solvents are constructed as a function of grafting density and composition or chain-length asymmetry. The structure of the microphase segregated morphologies lacks long-range periodic order, and it is analyzed quantitatively using Minkowski measures.
Subject(s)
Polymers/chemistry , Solvents/chemistryABSTRACT
We study the interplay between microphase assembly and macrophase separation in A/B/AB ternary polymer blends by examining the free energy of localized fluctuation structures (micelles or droplets), with emphasis on the thermodynamic relationship between swollen micelles (microemulsion) and the macrophase-separated state, using self-consistent field theory and an extended capillary model. Upon introducing homopolymer B into a micelle-forming binary polymer blend A/AB, micelles can be swollen by B. A small amount of component B (below the A-rich binodal of macrophase coexistence) will not affect the stability of the swollen micelles. A large excess of homopolymer, B, will induce a microemulsion failure and lead to a macrophase separation. Between the binodal and the microemulsion failure concentration, macrophase separation in A/B/AB occurs by a two-step nucleation mechanism via a metastable microemulsion droplet of finite size. Our results illustrate a recently proposed argument that the two-step nucleation via a metastable intermediate is a general phenomenon in systems involving short-range attraction and long-range repulsion.