The Challenging Diagnosis of Interstitial Lung Disease in Children-One Case Report and Literature Review.

Childhood interstitial lung disease (chILD) includes a heterogeneous spectrum of rare respiratory disorders in children associated with substantial morbi-mortality. Interstitial tissue, and other pulmonary structures, epithelium, blood vessels, or pleura are involved, resulting in a restrictive lung disfunction. Respiratory symptoms set in progressively and are often subtle, making thorough clinical history and physical examination fundamental. The etiology often is obscure. The clinical presentation mimics pneumonia or asthma, leading to a diagnostic delay. Challenging diagnosis may require genetic tests, bronchoalveolar lavage, or lung biopsy. Alongside general supportive therapeutic measures, anti-inflammatory, immunosuppressive or antifibrotic agents may be used, based on data derived from adult studies. However, if accurate diagnosis and treatment are delayed, irreversible chronic respiratory failure may ensue, impacting prognosis. The most frequent chILD is hypersensitivity pneumonitis (HP), although it is rare in children. HP is associated with exposure to an environmental antigen, resulting in inflammation of the airways. Detailed antigen exposure history and identification of the inciting trigger are the cornerstones of diagnostic. This article provides the current state of chILD, revealing specific features of HP, based on a clinical case report of a patient admitted in our clinic, requiring extensive investigations for diagnosis, with a favorable long-term outcome.

A PDX1 cistrome and single-cell transcriptome resource of the developing pancreas.

Pancreatic and duodenal homeobox 1 (PDX1) is crucial for pancreas organogenesis, yet the dynamic changes in PDX1 binding in human or mouse developing pancreas have not been examined. To address this knowledge gap, we performed PDX1 ChIP-seq and single-cell RNA-seq using fetal human pancreata. We integrated our datasets with published datasets and revealed the dynamics of PDX1 binding and potential cell lineage-specific PDX1-bound genes in the pancreas from fetal to adult stages. We identified a core set of developmentally conserved PDX1-bound genes that reveal the broad multifaceted role of PDX1 in pancreas development. Despite the well-known dramatic changes in PDX1 function and expression, we found that PDX1-bound genes are largely conserved from embryonic to adult stages. This points towards a dual role of PDX1 in regulating the expression of its targets at different ages, dependent on other functionally congruent or directly interacting partners. We also showed that PDX1 binding is largely conserved in mouse pancreas. Together, our study reveals PDX1 targets in the developing pancreas in vivo and provides an essential resource for future studies on pancreas development.

Advances in Understanding the Mechanism of Transitional Neonatal Hypoglycemia and Implications for Management.

Our lack of basic knowledge about the basic mechanisms of transitional hypoglycemia and other forms of hypoglycemia in newborns underlies the ongoing controversies over standards for managing these conditions. To address this deficiency, the authors evaluated regulation of insulin secretion in fetal, newborn, and adult rats. The results demonstrate that transitional hypoglycemia in normal neonates and persistent hypoglycemia in high-risk infants both reflect altered beta-cell insulin regulation. These findings provide a new foundation for improving detection and management and preventing hypoglycemic brain injury in normal neonates and, especially, in infants with persistent hypoglycemia and genetic forms of congenital hyperinsulinism.

Decreased KATP Channel Activity Contributes to the Low Glucose Threshold for Insulin Secretion of Rat Neonatal Islets.

Transitional hypoglycemia in normal newborns occurs in the first 3 days of life and has clinical features consistent with hyperinsulinism. We found a lower threshold for glucose-stimulated insulin secretion from freshly isolated embryonic day (E) 22 rat islets, which persisted into the first postnatal days. The threshold reached the adult level by postnatal day (P) 14. Culturing P14 islets also decreased the glucose threshold. Freshly isolated P1 rat islets had a lower threshold for insulin secretion in response to 2-aminobicyclo-(2, 2, 1)-heptane-2-carboxylic acid, a nonmetabolizable leucine analog, and diminished insulin release in response to tolbutamide, an inhibitor of ß-cell KATP channels. These findings suggested that decreased KATP channel function could be responsible for the lower glucose threshold for insulin secretion. Single-cell transcriptomic analysis did not reveal a lower expression of KATP subunit genes in E22 compared with P14 ß cells. The investigation of electrophysiological characteristics of dispersed ß cells showed that early neonatal and cultured cells had fewer functional KATP channels per unit membrane area. Our findings suggest that decreased surface density of KATP channels may contribute to the observed differences in glucose threshold for insulin release.

Measuring the predictability of life outcomes with a scientific mass collaboration.

How predictable are life trajectories? We investigated this question with a scientific mass collaboration using the common task method; 160 teams built predictive models for six life outcomes using data from the Fragile Families and Child Wellbeing Study, a high-quality birth cohort study. Despite using a rich dataset and applying machine-learning methods optimized for prediction, the best predictions were not very accurate and were only slightly better than those from a simple benchmark model. Within each outcome, prediction error was strongly associated with the family being predicted and weakly associated with the technique used to generate the prediction. Overall, these results suggest practical limits to the predictability of life outcomes in some settings and illustrate the value of mass collaborations in the social sciences.

Modeling Monogenic Diabetes using Human ESCs Reveals Developmental and Metabolic Deficiencies Caused by Mutations in HNF1A.

Human monogenic diabetes, caused by mutations in genes involved in beta cell development and function, has been a challenge to study because multiple mouse models have not fully recapitulated the human disease. Here, we use genome edited human embryonic stem cells to understand the most common form of monogenic diabetes, MODY3, caused by mutations in the transcription factor HNF1A. We found that HNF1A is necessary to repress an alpha cell gene expression signature, maintain endocrine cell function, and regulate cellular metabolism. In addition, we identified the human-specific long non-coding RNA, LINKA, as an HNF1A target necessary for normal mitochondrial respiration. These findings provide a possible explanation for the species difference in disease phenotypes observed with HNF1A mutations and offer mechanistic insights into how the HNF1A gene may also influence type 2 diabetes.

JUND regulates pancreatic ß cell survival during metabolic stress.

OBJECTIVE: In type 2 diabetes (T2D), oxidative stress contributes to the dysfunction and loss of pancreatic ß cells. A highly conserved feature of the cellular response to stress is the regulation of mRNA translation; however, the genes regulated at the level of translation are often overlooked due to the convenience of RNA sequencing technologies. Our goal is to investigate translational regulation in ß cells as a means to uncover novel factors and pathways pertinent to cellular adaptation and survival during T2D-associated conditions. METHODS: Translating ribosome affinity purification (TRAP) followed by RNA-seq or RT-qPCR was used to identify changes in the ribosome occupancy of mRNAs in Min6 cells. Gene depletion studies used lentiviral delivery of shRNAs to primary mouse islets or CRISPR-Cas9 to Min6 cells. Oxidative stress and apoptosis were measured in primary islets using cell-permeable dyes with fluorescence readouts of oxidation and activated cleaved caspase-3 and-7, respectively. Gene expression was assessed by RNA-seq, RT-qPCR, and western blot. ChIP-qPCR was used to determine chromatin enrichment. RESULTS: TRAP-seq in a PDX1-deficiency model of ß cell dysfunction uncovered a cohort of genes regulated at the level of mRNA translation, including the transcription factor JUND. Using a panel of diabetes-associated stressors, JUND was found to be upregulated in mouse islets cultured with high concentrations of glucose and free fatty acid, but not after treatment with hydrogen peroxide or thapsigargin. This induction of JUND could be attributed to increased mRNA translation. JUND was also upregulated in islets from diabetic db/db mice and in human islets treated with high glucose and free fatty acid. Depletion of JUND in primary islets reduced oxidative stress and apoptosis in ß cells during metabolic stress. Transcriptome assessment identified a cohort of genes, including pro-oxidant and pro-inflammatory genes, regulated by JUND that are commonly dysregulated in models of ß cell dysfunction, consistent with a maladaptive role for JUND in islets. CONCLUSIONS: A translation-centric approach uncovered JUND as a stress-responsive factor in ß cells that contributes to redox imbalance and apoptosis during pathophysiologically relevant stress.

CellBIC: bimodality-based top-down clustering of single-cell RNA sequencing data reveals hierarchical structure of the cell type.

Single-cell RNA sequencing (scRNA-seq) is a powerful tool to study heterogeneity and dynamic changes in cell populations. Clustering scRNA-seq is essential in identifying new cell types and studying their characteristics. We develop CellBIC (single Cell BImodal Clustering) to cluster scRNA-seq data based on modality in the gene expression distribution. Compared with classical bottom-up approaches that rely on a distance metric, CellBIC performs hierarchical clustering in a top-down manner. CellBIC outperformed the bottom-up hierarchical clustering approach and other recently developed clustering algorithms while maintaining the hierarchical structure of cells. Importantly, CellBIC identifies type 2 diabetes and age specific ß cell signatures characterized by SIX3 and CDH2, respectively.

Central Sleep Apnea - a Rare Cause for Acute Respiratory Insufficiency in Children. Case Report.

Central sleep apnea is characterized by frequent cessation of breathing during sleep, resulting in repetitive episodes of insufficient ventilation and abnormalities of acid-base balance. It may be primary or secondary, and it is uncommon in children, with limited data for this population. We present here the case of a five-year-old girl, known to have thoracolumbar myelomeningocele (for which she underwent a surgical procedure in infancy), secondary hydrocephalus (with a ventriculoperitoneal shunt) and flaccid paralysis, who was admitted in our hospital with prolonged fever syndrome, productive cough, severe dyspnea and perioral cyanosis. Following physical examination, laboratory investigations and thoracic radiography, we established the diagnosis of aspiration pneumonia with acute respiratory failure. Medical treatment with multiple systemic antibiotics, antifungal agents, systemic and inhaled bronchodilator, oxygen therapy and respiratory nursing were initiated, with favorable evolution. During the entire hospitalization, the patient showed nocturnal respiratory rhythm disorders, with sleep apnea crisis of approximately 20 seconds and desaturation, followed by severe hypercapnic respiratory acidosis, manifestations that persisted even after the remission of pulmonary infection, raising the suspicion of an apnea syndrome. After excluding the causes of obstructive apnea, a cerebral CT scan was performed, revealing isolated fourth ventricle compressing the brainstem. The patient underwent neurosurgical intervention and postoperatively, the evolution was favorable, with remission of apnea crisis.

GATA6 Plays an Important Role in the Induction of Human Definitive Endoderm, Development of the Pancreas, and Functionality of Pancreatic ß Cells.

Induced pluripotent stem cells were created from a pancreas agenesis patient with a mutation in GATA6. Using genome-editing technology, additional stem cell lines with mutations in both GATA6 alleles were generated and demonstrated a severe block in definitive endoderm induction, which could be rescued by re-expression of several different GATA family members. Using the endodermal progenitor stem cell culture system to bypass the developmental block at the endoderm stage, cell lines with mutations in one or both GATA6 alleles could be differentiated into ß-like cells but with reduced efficiency. Use of suboptimal doses of retinoic acid during pancreas specification revealed a more severe phenotype, more closely mimicking the patient's disease. GATA6 mutant ß-like cells fail to secrete insulin upon glucose stimulation and demonstrate defective insulin processing. These data show that GATA6 plays a critical role in endoderm and pancreas specification and ß-like cell functionality in humans.

Single cell transcriptomic profiling of mouse pancreatic progenitors.

The heterogeneity of the developing pancreatic epithelium and low abundance of endocrine progenitors limit the information derived from traditional expression studies. To identify genes that characterize early developmental tissues composed of multiple progenitor lineages, we applied single-cell RNA-Seq to embryonic day (e)13.5 mouse pancreata and performed integrative analysis with single cell data from mature pancreas. We identified subpopulations expressing macrophage or endothelial markers and new pancreatic progenitor markers. We also identified potential α-cell precursors expressing glucagon (Gcg) among the e13.5 pancreatic cells. Despite their high Gcg expression levels, these cells shared greater transcriptomic similarity with other e13.5 cells than with adult α-cells, indicating their immaturity. Comparative analysis identified the sodium-dependent neutral amino acid transporter, Slc38a5, as a characteristic gene expressed in α-cell precursors but not mature cells. By immunofluorescence analysis, we observed SLC38A5 expression in pancreatic progenitors, including in a subset of NEUROG3+ endocrine progenitors and MAFB+ cells and in all GCG+ cells. Expression declined in α-cells during late gestation and was absent in the adult islet. Our results suggest SLC38A5 as an early marker of α-cell lineage commitment.

Threshold-Dependent Cooperativity of Pdx1 and Oc1 in Pancreatic Progenitors Establishes Competency for Endocrine Differentiation and ß-Cell Function.

Pdx1 and Oc1 are co-expressed in multipotent pancreatic progenitors and regulate the pro-endocrine gene Neurog3. Their expression diverges in later organogenesis, with Oc1 absent from hormone+ cells and Pdx1 maintained in mature ß cells. In a classical genetic test for cooperative functional interactions, we derived mice with combined Pdx1 and Oc1 heterozygosity. Endocrine development in double-heterozygous pancreata was normal at embryonic day (E)13.5, but defects in specification and differentiation were apparent at E15.5, the height of the second wave of differentiation. Pancreata from double heterozygotes showed alterations in the expression of genes crucial for ß-cell development and function, decreased numbers and altered allocation of Neurog3-expressing endocrine progenitors, and defective endocrine differentiation. Defects in islet gene expression and ß-cell function persisted in double heterozygous neonates. These results suggest that Oc1 and Pdx1 cooperate prior to their divergence, in pancreatic progenitors, to allow for proper differentiation and functional maturation of ß cells.

A novel mutation in GATA6 causes pancreatic agenesis.

Heterozygous mutations in GATA6 have been linked to pancreatic agenesis and cardiac malformations. The aim of this study was to describe a new mutation in GATA6 in an infant with pancreatic agenesis, associated with truncus arteriosus and absent gallbladder. Clinical data were obtained from chart review. Gene sequencing was performed on genomic DNA. The patient was a female infant diagnosed shortly after birth with a severe cardiac malformation, absent gallbladder, anomalous hepatic blood flow, unilateral hydronephrosis and hydroureter, neonatal diabetes, and pancreatic exocrine insufficiency. Despite prolonged intensive management care, she died at 3 months of age because of cardiac complications. Analysis of her genomic DNA revealed a novel missense mutation of GATA6. The novel mutation described in this case extends the list of GATA6 mutations causing pancreatic agenesis and cardiac malformations.

Continuous subcutaneous IGF-1 therapy via insulin pump in a patient with Donohue syndrome.

Donohue syndrome (DS) is a severe form of congenital insulin resistance due to mutation(s) in the insulin receptor (INSR) gene. Given the similarities between insulin and insulin-like growth factor 1 (IGF-1) receptors, recombinant human IGF-1 (rhIGF-1) has been used to treat severe insulin resistance due to INSR mutation(s). Traditional subcutaneous therapy may be limited by the shortened IGF-1 half-life in these patients. We report the case of a female with molecularly confirmed DS treated with continuous rhIGF-1 therapy via an insulin pump. With treatment, the patient's hemoglobin A1c decreased from 9.8% to 8.8%, and her weight increased by 0.8 kg. Development of an ovarian tumor complicated her course, but it was unclear whether this was related to rhIGF-1 therapy. Limited treatment options exist for patients with DS. The use of continuous rhIGF-1 via an insulin pump may be a viable option, although further experience is needed to establish safety and efficacy.