A Quantitative Assay for Insulin-expressing Colony-forming Progenitors

The field of pancreatic stem and progenitor cell biology has been hampered by a lack of in vitro functional and quantitative assays that allow for the analysis of the single cell. Analyses of single progenitors are of critical importance because they provide definitive ways to unequivocally demonstrate the lineage potential of individual progenitors. Although methods have been devised to generate "pancreatospheres" in suspension culture from single cells, several limitations exist. First, it is time-consuming to perform single cell deposition for a large number of cells, which in turn commands large volumes of culture media and space. Second, numeration of the resulting pancreatospheres is labor-intensive, especially when the frequency of the pancreatosphere-initiating progenitors is low. Third, the pancreatosphere assay is not an efficient method to allow both the proliferation and differentiation of pancreatic progenitors in the same culture well, restricting the usefulness of the assay

Transformation and gene editing in the bioenergy grass \u3ci\u3eMiscanthus\u3c/i\u3e

Background: Miscanthus, a C4 member of Poaceae, is a promising perennial crop for bioenergy, renewable bioproducts, and carbon sequestration. Species of interest include nothospecies M. x giganteus and its parental species M. sacchariforus and M. sinensis. Use of biotechnology-based procedures to genetically improve Miscanthus, to date, have only included plant transformation procedures for introduction of exogenous genes into the host genome at random, non-targeted sites. Results: We developed gene editing procedures for Miscanthus using CRISPR/Cas9 that enabled the mutation of a specific (targeted) endogenous gene to knock out its function. Classified as paleo-allopolyploids (duplicated ancient Sorghum-like DNA plus chromosome fusion event), design of guide RNAs (gRNAs) for Miscanthus needed to target both homeologs and their alleles to account for functional redundancy. Prior research in Zea mays demonstrated that editing the lemon white1 (lw1) gene, involved in chlorophyll and carotenoid biosynthesis, via CRISPR/Cas9 yielded pale green/yellow, striped or white leaf phenotypes making lw1 a promising target for visual confirmation of editing in other species. Using sequence information from both Miscanthus and sorghum, orthologs of maize lw1 were identified; a multi-step screening approach was used to select three gRNAs that could target homeologs of lw1. Embryogenic calli of M. sacchariforus, M. sinensis and M. x giganteus were transformed via particle bombardment (biolistics) or Agrobacterium tumefaciens introducing the Cas9 gene and three gRNAs to edit lw1. Leaves on edited Miscanthus plants displayed the same phenotypes noted in maize. Sanger sequencing confirmed editing; deletions in lw1 ranged from 1 to 26 bp in length, and one deletion (433 bp) encompassed two target sites. Confocal microscopy verified lack of autofluorescence (chlorophyll) in edited leaves/sectors. Conclusions: We developed procedures for gene editing via CRISPR/Cas9 in Miscanthus and, to the best of our knowledge, are the first to do so. This included five genotypes representing three Miscanthus species. Designed gRNAs targeted all copies of lw1 (homeologous copies and their alleles); results also confirmed lw1 made a goo

Glucocorticoid signaling enhances expression of glucose-sensing molecules in immature pancreatic beta-like cells derived from murine embryonic stem cells in vitro

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. Here, we aim to identify small molecules that affect immature beta cells. A cell-based assay, employing pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an Insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative RT-PCR analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM D-glucose and stimulated by 17 mM D-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells

Glucocorticoid signaling enhances expression of glucose-sensing molecules in immature pancreatic beta-like cells derived from murine embryonic stem cells in vitro

Pluripotent stem cells may serve as an alternative source of beta-like cells for replacement therapy of type 1 diabetes; however, the beta-like cells generated in many differentiation protocols are immature. The maturation of endogenous beta cells involves an increase in insulin expression starting in late gestation and a gradual acquisition of the abilities to sense glucose and secrete insulin by week 2 after birth in mice; however, what molecules regulate these maturation processes are incompletely known. Here, we aim to identify small molecules that affect immature beta cells. A cell-based assay, employing pancreatic beta-like cells derived from murine embryonic stem (ES) cells harboring a transgene containing an Insulin 1-promoter driven enhanced green fluorescent protein reporter, was used to screen a compound library (NIH Clinical Collection-003). Cortisone, a glucocorticoid, was among five positive hit compounds. Quantitative RT-PCR analysis revealed that glucocorticoids enhance the gene expression of not only insulin 1 but also glucose transporter-2 (Glut2; Slc2a2) and glucokinase (Gck), two molecules important for glucose sensing. Mifepristone, a pharmacological inhibitor of glucocorticoid receptor (GR) signaling, reduced the effects of glucocorticoids on Glut2 and Gck expression. The effects of glucocorticoids on ES-derived cells were further validated in immature primary islets. Isolated islets from 1-week-old mice had an increased Glut2 and Gck expression in response to a 4-day treatment of exogenous hydrocortisone in vitro. Gene deletion of GR in beta cells using rat insulin 2 promoter-driven Cre crossed with GRflox/flox mice resulted in a reduced gene expression of Glut2, but not Gck, and an abrogation of insulin secretion when islets were incubated in 0.5 mM D-glucose and stimulated by 17 mM D-glucose in vitro. These results demonstrate that glucocorticoids positively regulate glucose sensors in immature murine beta-like cells

A species pair of Bivesicula Yamaguti, 1934 (Trematoda: Bivesiculidae) in unrelated Great Barrier Reef fishes: implications for the basis of speciation in coral reef fish trematodes

Combined morphological and molecular analysis shows that a species of Bivesicula Yamaguti, 1934 from four species of Apogonidae Günther [Nectamia fusca (Quoy & Gaimard), Ostorhinchus angustatus (Smith & Radcliffe), O. cookii (Macleay) and Taeniamia fucata (Cantor)] on the Great Barrier Reef is morphologically similar to, but clearly distinct from B. unexpecta Cribb, Bray & Barker, 1994 which infects a sympatric pomacentrid, Acanthochromis polyacanthus (Bleeker). Bivesicula neglecta n. sp. is proposed for the form from apogonids. Novel ITS2 rDNA sequences generated for the two species differ at just one consistent base position, implying that the two species are closely related. The combination of their close relationship, high but distinct specificity and co-occurrence suggests that speciation was driven by a recent host switching event enabled by similar dietary ecomorphology

Whole-body MR imaging in suspected physical child abuse: comparison with skeletal survey and bone scintigraphy findings from the PEDIMA prospective multicentre study

International audienceObjectives: To assess the contribution of whole-body magnetic resonance imaging (WBMRI) and bone scintigraphy (BS) in addition to skeletal survey (SS) in detecting traumatic bone lesions and soft-tissue injuries in suspected child abuse.Methods: In this prospective, multicentre, diagnostic accuracy study, children less than 3 years of age with suspected physical abuse were recruited. Each child underwent SS, BS and WBMRI. A blinded first review was performed in consensus by five paediatric radiologists and three nuclear medicine physicians. A second review investigated discrepancies reported between the modalities using a consensus result of all modalities as the reference standard. We calculated the sensitivity, specificity and corresponding 95% confidence interval for each imaging modality (SS, WBMRI and BS) and for the combinations [SS + WBMRI] and [SS + BS].Results: One hundred seventy children were included of which sixty-four had at least one lesion. In total, 146 lesions were included. The sensitivity and specificity of each examination were, respectively, as follows: 88.4% [95% CI, 82.0-93.1] and 99.7% [95% CI, 99.5-99.8] for the SS, 69.9% [95% CI, 61.7-77.2] and 99.5% [95% CI, 99.2-99.7] for WBMRI and 54.8% [95% CI, 46.4-63.0] and 99.7% [95% CI, 99.5-99.9] for BS. Sensitivity and specificity were, respectively, 95.9% [95% CI, 91.3-98.5] and 99.2% [95% CI, 98.9-99.4] for the combination SS + WBMRI and 95.2% [95% CI, 90.4-98.1] and 99.4% [95% CI, 99.2-99.6] for the combination SS + BS, with no statistically significant difference between them.Conclusion: SS was the most sensitive independent imaging modality; however, the additional combination of either WBMRI or BS examinations offered an increased accuracy.Key points: • SS in suspected infant abuse was the most sensitive independent imaging modality in this study, especially for detecting metaphyseal and rib lesions, and remains essential for evaluation. • The combination of either SS + BS or SS + WBMRI provides greater accuracy in diagnosing occult and equivocal bone injuries in the difficult setting of child abuse. • WBMRI is a free-radiation technique that allows additional diagnosis of soft-tissue and visceral injuries

Digestive Manifestations in Patients Hospitalized With Coronavirus Disease 2019

This article is made available for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.BACKGROUND & AIMS: The prevalence and significance of digestive manifestations in coronavirus disease 2019 (COVID-19) remain uncertain. We aimed to assess the prevalence, spectrum, severity, and significance of digestive manifestations in patients hospitalized with COVID-19. METHODS: Consecutive patients hospitalized with COVID-19 were identified across a geographically diverse alliance of medical centers in North America. Data pertaining to baseline characteristics, symptomatology, laboratory assessment, imaging, and endoscopic findings from the time of symptom onset until discharge or death were abstracted manually from electronic health records to characterize the prevalence, spectrum, and severity of digestive manifestations. Regression analyses were performed to evaluate the association between digestive manifestations and severe outcomes related to COVID-19. RESULTS: A total of 1992 patients across 36 centers met eligibility criteria and were included. Overall, 53% of patients experienced at least 1 gastrointestinal symptom at any time during their illness, most commonly diarrhea (34%), nausea (27%), vomiting (16%), and abdominal pain (11%). In 74% of cases, gastrointestinal symptoms were judged to be mild. In total, 35% of patients developed an abnormal alanine aminotransferase or total bilirubin level; these were increased to less than 5 times the upper limit of normal in 77% of cases. After adjusting for potential confounders, the presence of gastrointestinal symptoms at any time (odds ratio, 0.93; 95% CI, 0.76-1.15) or liver test abnormalities on admission (odds ratio, 1.31; 95% CI, 0.80-2.12) were not associated independently with mechanical ventilation or death. CONCLUSIONS: Among patients hospitalized with COVID-19, gastrointestinal symptoms and liver test abnormalities were common, but the majority were mild and their presence was not associated with a more severe clinical course