Fluorescent IGF-II analogues for FRET-based investigations into the binding of IGF-II to the IGF-1R

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. Material from this article can be used in other publications provided that the correct acknowledgement is given with the reproduced material.The interaction of IGF-II with the insulin receptor (IR) and type 1 insulin-like growth factor receptor (IGF-1R) has recently been identified as potential therapeutic target for the treatment of cancer. Understanding the interactions of IGF-II with these receptors is required for the development of potential anticancer therapeutics. This work describes an efficient convergent synthesis of native IGF-II and two nonnative IGF-II analogues with coumarin fluorescent probes incorporated at residues 19 and 28. These fluorescent analogues bind with nanomolar affinities to the IGF-1R and are suitable for use in fluorescence resonance energy transfer (FRET) studies. From these studies the F19Cou IGF-II and F28Cou IGF-II proteins were identified as good probes for investigating the binding interactions of IGF-II with the IGF-1R and its other high affinity binding partners

Characterizing a rare neurogenetic disease, SLC13A5 citrate transporter disorder, utilizing clinical data in a cloud-based medical record collection system

Introduction: SLC13A5 citrate transporter disorder is a rare autosomal recessive genetic disease that has a constellation of neurologic symptoms. To better characterize the neurologic and clinical laboratory phenotype, we utilized patient medical records collected by Ciitizen, an Invitae company, with support from the TESS Research Foundation.Methods: Medical records for 15 patients with a suspected genetic and clinical diagnosis of SLC13A5 citrate transporter disorder were collected by Ciitizen, an Invitae company. Genotype, clinical phenotypes, and laboratory data were extracted and analyzed.Results: The 15 patients reported all had epilepsy and global developmental delay. Patients continued to attain motor milestones, though much later than their typically developing peers. Clinical diagnoses support abnormalities in communication, and low or mixed tone with several movement disorders, including, ataxia and dystonia. Serum citrate was elevated in the 3 patients in whom it was measured; other routine laboratory studies assessing renal, liver and blood function had normal values or no consistent abnormalities. Many electroencephalograms (EEGs) were performed (1 to 35 per patient), and most but not all were abnormal, with slowing and/or epileptiform activity. Fourteen of the patients had one or more brain magnetic resonance imaging (MRI) reports: 7 patients had at least one normal brain MRI, but not with any consistent findings except white matter signal changes.Discussion: These results show that in addition to the epilepsy phenotype, SLC13A5 citrate transporter disorder impacts global development, with marked abnormalities in motor abilities, tone, coordination, and communication skills. Further, utilizing cloud-based medical records allows industry, academic, and patient advocacy group collaboration to provide preliminary characterization of a rare genetic disorder. Additional characterization of the neurologic phenotype will be critical to future study and developing treatment for this and related rare genetic disorders

FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion

BACKGROUND: Phycobilisomes (PBsomes) are the extrinsic antenna complexes upon the photosynthetic membranes in red algae and most cyanobacteria. The PBsomes in the cyanobacteria has been proposed to present high lateral mobility on the thylakoid membrane surface. In contrast, direct measurement of PBsome motility in red algae has been lacking so far. METHODOLOGY/PRINCIPAL FINDINGS: In this work, we investigated the dynamics of PBsomes in the unicellular red alga Porphyridium cruentum in vivo and in vitro, using fluorescence recovery after photobleaching (FRAP). We found that part of the fluorescence recovery could be detected in both partially- and wholly-bleached wild-type and mutant F11 (UTEX 637) cells. Such partial fluorescence recovery was also observed in glutaraldehyde-treated and betaine-treated cells in which PBsome diffusion should be restricted by cross-linking effect, as well as in isolated PBsomes immobilized on the glass slide. CONCLUSIONS/SIGNIFICANCE: On the basis of our previous structural results showing the PBsome crowding on the native photosynthetic membrane as well as the present FRAP data, we concluded that the fluorescence recovery observed during FRAP experiment in red algae is mainly ascribed to the intrinsic photoprocesses of the bleached PBsomes in situ, rather than the rapid diffusion of PBsomes on thylakoid membranes in vivo. Furthermore, direct observations of the fluorescence dynamics of phycoerythrins using FRAP demonstrated the energetic decoupling of phycoerythrins in PBsomes against strong excitation light in vivo, which is proposed as a photoprotective mechanism in red algae attributed by the PBsomes in response to excess light energy

Aneuploidy in pluripotent stem cells and implications for cancerous transformation

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation

Neuropeptidomics of the Supraoptic Rat Nucleus

The mammalian supraoptic nucleus (SON) is a neuroendocrine center in the brain regulating a variety of physiological functions. Within the SON, peptidergic magnocellular neurons that project to the neurohypophysis (posterior pituitary) are involved in controlling osmotic balance, lactation, and parturition, partly through secretion of signaling peptides such as oxytocin and vasopressin into the blood. An improved understanding of SON activity and function requires identification and characteriza-tion of the peptides used by the SON. Here, small-volume sample preparation approaches are optimized for neuropeptidomic studies of isolated SON samples ranging from entire nuclei down to single magnocellular neurons. Unlike most previous mammalian peptidome studies, tissues are not im-mediately heated or microwaved. SON samples are obtained from ex vivo brain slice preparations via tissue punch and the samples processed through sequential steps of peptide extraction. Analyses of the samples via liquid chromatography mass spectrometry and tandem mass spectrometry result in the identification of 85 peptides, including 20 unique peptides from known prohormones. As the sample size is further reduced, the depth of peptide coverage decreases; however, even from individually isolated magnocellular neuroendocrine cells, vasopressin and several other peptides are detected

Genetic and phenotypic spectrum associated with IFIH1 gain-of-function

IFIH1 gain‐of‐function has been reported as a cause of a type I interferonopathy encompassing a spectrum of autoinflammatory phenotypes including Aicardi–Goutières syndrome and Singleton Merten syndrome. Ascertaining patients through a European and North American collaboration, we set out to describe the molecular, clinical and interferon status of a cohort of individuals with pathogenic heterozygous mutations in IFIH1. We identified 74 individuals from 51 families segregating a total of 27 likely pathogenic mutations in IFIH1. Ten adult individuals, 13.5% of all mutation carriers, were clinically asymptomatic (with seven of these aged over 50 years). All mutations were associated with enhanced type I interferon signaling, including six variants (22%) which were predicted as benign according to multiple in silico pathogenicity programs. The identified mutations cluster close to the ATP binding region of the protein. These data confirm variable expression and nonpenetrance as important characteristics of the IFIH1 genotype, a consistent association with enhanced type I interferon signaling, and a common mutational mechanism involving increased RNA binding affinity or decreased efficiency of ATP hydrolysis and filament disassembly rate

Gene Expression Profile of Neuronal Progenitor Cells Derived from hESCs: Activation of Chromosome 11p15.5 and Comparison to Human Dopaminergic Neurons

BACKGROUND: We initiated differentiation of human embryonic stem cells (hESCs) into dopamine neurons, obtained a purified population of neuronal precursor cells by cell sorting, and determined patterns of gene transcription. METHODOLOGY: Dopaminergic differentiation of hESCs was initiated by culturing hESCs with a feeder layer of PA6 cells. Differentiating cells were then sorted to obtain a pure population of PSA-NCAM-expressing neuronal precursors, which were then analyzed for gene expression using Massive Parallel Signature Sequencing (MPSS). Individual genes as well as regions of the genome which were activated were determined. PRINCIPAL FINDINGS: A number of genes known to be involved in the specification of dopaminergic neurons, including MSX1, CDKN1C, Pitx1 and Pitx2, as well as several novel genes not previously associated with dopaminergic differentiation, were expressed. Notably, we found that a specific region of the genome located on chromosome 11p15.5 was highly activated. This region contains several genes which have previously been associated with the function of dopaminergic neurons, including the gene for tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine biosynthesis, IGF2, and CDKN1C, which cooperates with Nurr1 in directing the differentiation of dopaminergic neurons. Other genes in this region not previously recognized as being involved in the functions of dopaminergic neurons were also activated, including H19, TSSC4, and HBG2. IGF2 and CDKN1C were also found to be highly expressed in mature human TH-positive dopamine neurons isolated from human brain samples by laser capture. CONCLUSIONS: The present data suggest that the H19-IGF2 imprinting region on chromosome 11p15.5 is involved in the process through which undifferentiated cells are specified to become neuronal precursors and/or dopaminergic neurons