Employing Human Induced Pluripotent Stem Cells (hiPSCs) to Model NF1-Associated Low Grade Gliomas

From the Washington University Office of Undergraduate Research Digest (WUURD), Vol. 13, 05-01-2018. Published by the Office of Undergraduate Research. Joy Zalis Kiefer, Director of Undergraduate Research and Associate Dean in the College of Arts & Sciences; Lindsey Paunovich, Editor; Helen Human, Programs Manager and Assistant Dean in the College of Arts and Sciences Mentor(s): David Gutman

Isolation and Comparative Genomic Analysis of Final Third of Satis Genome

A highly novel Streptomyces phage, Satis, was isolated from a direct environmental sample collected from outside Danforth House on the Washington University campus. Satis infects bacterial species Streptomyces lividans producing pinpoint, cloudy plaques less than 1mm in diameter. Electron microscope data shows rare atypical physical features. Rather than the common octahedral capsid shape, Satis has a prolate head with visible cross-linked hexagonal protein structure and average measurements of 285 nm by 47 nm with a long, flexible tail measuring 268 nm. Upon sequencing, it was found that Satis contains the longest phage genome discovered to date through the SEA-PHAGE program at 186,702 base pairs. The genome is quite novel in sequence, as its closest genetic match, bacteriophage Chymera, is similar across only 15.9% of the genome. This means that Satis belongs to no known previously characterized cluster and is considered a Singleton phage. The genome contains 325 protein coding genes, of which our group analyzed Gene 230 to the end of the genome. The vast majority of the genes in this section run 3’ to 5’ and compared to the other two sections, these genes seem to be the most unique in primary, secondary, and tertiary structure. Due to the novelty of Satis, functional evidence from comparative genomic analysis is sparse. We are currently in the process of a more thurough comparative genomic analysis between Satis and other Streptomyces phages, particularly phage JustBecause, another Streptomyces phage isolated by Washington University in St. Louis students in 2016 with similar morphology to Satis

Patient-derived iPSC-cerebral organoid modeling of the 17q11.2 microdeletion syndrome establishes CRLF3 as a critical regulator of neurogenesis

Neurodevelopmental disorders are often caused by chromosomal microdeletions comprising numerous contiguous genes. A subset of neurofibromatosis type 1 (NF1) patients with severe developmental delays and intellectual disability harbors such a microdeletion event on chromosome 17q11.2, involving the NF1 gene and flanking regions (NF1 total gene deletion [NF1-TGD]). Using patient-derived human induced pluripotent stem cell (hiPSC)-forebrain cerebral organoids (hCOs), we identify both neural stem cell (NSC) proliferation and neuronal maturation abnormalities in NF1-TGD hCOs. While increased NSC proliferation results from decreased NF1/RAS regulation, the neuronal differentiation, survival, and maturation defects are caused by reduced cytokine receptor-like factor 3 (CRLF3) expression and impaired RhoA signaling. Furthermore, we demonstrate a higher autistic trait burden in NF1 patients harboring a deleterious germline mutation in the CRLF3 gene (c.1166T\u3eC, p.Leu389Pro). Collectively, these findings identify a causative gene within the NF1-TGD locus responsible for hCO neuronal abnormalities and autism in children with NF1

Human iPSC-derived neurons and cerebral organoids establish differential effects of germline NF1 gene mutations

Neurofibromatosis type 1 (NF1) is a common neurodevelopmental disorder caused by a spectrum of distinct germline NF1 gene mutations, traditionally viewed as equivalent loss-of-function alleles. To specifically address the issue of mutational equivalency in a disease with considerable clinical heterogeneity, we engineered seven isogenic human induced pluripotent stem cell lines, each with a different NF1 patient NF1 mutation, to identify potential differential effects of NF1 mutations on human central nervous system cells and tissues. Although all mutations increased proliferation and RAS activity in 2D neural progenitor cells (NPCs) and astrocytes, we observed striking differences between NF1 mutations on 2D NPC dopamine levels, and 3D NPC proliferation, apoptosis, and neuronal differentiation in developing cerebral organoids. Together, these findings demonstrate differential effects of NF1 gene mutations at the cellular and tissue levels, suggesting that the germline NF1 gene mutation is one factor that underlies clinical variability

Differential susceptibility of C57BL/6NCr and B6.Cg-Ptprca mice to commensal bacteria after whole body irradiation in translational bone marrow transplant studies

Abstract Background The mouse is an important and widely utilized animal model for bone marrow transplant (BMT) translational studies. Here, we document the course of an unexpected increase in mortality of congenic mice that underwent BMT. Methods Thirty five BMTs were analyzed for survival differences utilizing the Log Rank test. Affected animals were evaluated by physical examination, necropsy, histopathology, serology for antibodies to infectious disease, and bacterial cultures. Results Severe bacteremia was identified as the main cause of death. Gastrointestinal (GI) damage was observed in histopathology. The bacteremia was most likely caused by the translocation of bacteria from the GI tract and immunosuppression caused by the myeloablative irradiation. Variability in groups of animals affected was caused by increased levels of gamma and X-ray radiation and the differing sensitivity of the two nearly genetically identical mouse strains used in the studies. Conclusion Our retrospective analysis of thirty five murine BMTs performed in three different laboratories, identified C57BL/6NCr (Ly5.1) as being more radiation sensitive than B6.Cg-Ptprca/NCr (Ly5.2). This is the first report documenting a measurable difference in radiation sensitivity and its effects between an inbred strain of mice and its congenic counterpart eventually succumbing to sepsis after BMT.http://deepblue.lib.umich.edu/bitstream/2027.42/112743/1/12967_2007_Article_240.pd

Probing the embedded YSOs of the R CrA region through VLT-ISAAC spectroscopy

Near IR spectra obtained with ISAAC at VLT, have been used to pose constraints on the evolutionary state and accretion properties of a sample of five embedded YSOs located in the R CrA core. This sample includes three Class I sources (HH100 IR, IRS2 and IRS5), and two sources with NIR excesses (IRS6 and IRS3). Absorption lines have been detected in the medium resolution spectra of all the observed targets, together with emission lines likely originating in the disk-star-wind connected regions. We derived spectral types, veiling and stellar luminosity of the five observed sources, which in turn have been used to infer their mass and age adopting pre-main sequence evolutionary tracks. We find that in HH100 IR and IRS2 most of the bolometric luminosity is due to accretion, while the other three investigated sources, including the Class I object IRS5a, present a low accretion activity (L_{acc}/L_{bol} < 0.2). We observe a general correlation between the accretion luminosity, the IR veiling and the emission line activity of the sources. A correlation between the accretion activity and the spectral energy distribution slope is recognizable but with the notable exception of IRS5a. Our analysis therefore shows how the definition of the evolutionary stage of deeply embedded YSOs by means of IR colors needs to be more carefully refined.Comment: 17 pages, 13 figures, accepted on A&

Visual parameter optimisation for biomedical image processing

Background: Biomedical image processing methods require users to optimise input parameters to ensure high quality output. This presents two challenges. First, it is difficult to optimise multiple input parameters for multiple input images. Second, it is difficult to achieve an understanding of underlying algorithms, in particular, relationships between input and output. Results: We present a visualisation method that transforms users’ ability to understand algorithm behaviour by integrating input and output, and by supporting exploration of their relationships. We discuss its application to a colour deconvolution technique for stained histology images and show how it enabled a domain expert to identify suitable parameter values for the deconvolution of two types of images, and metrics to quantify deconvolution performance. It also enabled a breakthrough in understanding by invalidating an underlying assumption about the algorithm. Conclusions: The visualisation method presented here provides analysis capability for multiple inputs and outputs in biomedical image processing that is not supported by previous analysis software. The analysis supported by our method is not feasible with conventional trial-and-error approaches

POISSON project - II - A multi-wavelength spectroscopic and photometric survey of young protostars in L 1641

Characterising stellar and circumstellar properties of embedded young stellar objects (YSOs) is mandatory for understanding the early stages of the stellar evolution. This task requires the combination of both spectroscopy and photometry, covering the widest possible wavelength range, to disentangle the various protostellar components and activities. As part of the POISSON project, we present a multi-wavelength spectroscopic and photometric investigation of embedded YSOs in L1641, aimed to derive the stellar parameters and evolutionary stages and to infer their accretion properties. Our database includes low-resolution optical-IR spectra from the NTT and Spitzer (0.6-40 um) and photometric data covering a spectral range from 0.4 to 1100 um, which allow us to construct the YSOs spectral energy distributions (SEDs) and to infer the main stellar parameters. The SED analysis allows us to group our 27 YSOs into nine Class I, eleven Flat, and seven Class II objects. However, on the basis of the derived stellar properties, only six Class I YSOs have an age of ~10^5 yr, while the others are older 5x10^5-10^6 yr), and, among the Flat sources, three out of eleven are more evolved objects (5x10^6-10^7 yr), indicating that geometrical effects can significantly modify the SED shapes. Inferred mass accretion rates (Macc) show a wide range of values (3.6x10^-9 to 1.2x10^-5 M_sun yr^-1), which reflects the age spread observed in our sample. Average values of mass accretion rates, extinction, and spectral indices decrease with the YSO class. The youngest YSOs have the highest Macc, whereas the oldest YSOs do not show any detectable jet activity in either images and spectra. We also observe a clear correlation among the YSO Macc, M*, and age, consistent with mass accretion evolution in viscous disc models.Comment: 61 pages, 16 figures; A&A in pres

Irish women in the diaspora: exclusions and inclusions

Irish women have a long history of emigration which provides parallels with the experiences of women now moving to settle in Ireland. In both cases, women migrants have been needed to fill the massive deficit of paid domestic labor in rapidly industrialising economies. Over the last two centuries, these destinations for Irish women have included the USA, Britain and Australia, as well as Canada, New Zealand, South Africa and Argentina. Some of the complexities in the positioning of migrant Irish women within the “diaspora spaces” they occupy are explored in this article. I identify ongoing disadvantage for certain groups of Irish-born women, drawing on evidence primarily from Britain, which has the largest contemporary diasporic Irish population. Comparisons are made with Irish women's experiences in the USA and Australia, using Census and survey data generated by and for the 2002 Task Force on Policy regarding Emigrants. The concept of diaspora explicitly includes those identifying themselves as Irish over several generations. I use qualitative findings from the Irish 2 Project, a recent study of the large second-generation Irish population in Britain, to examine narratives of women living in Manchester who grew up in “Irish” households and are subsequently negotiating hybrid identities in adulthood. These offer insights into longitudinal dimensions of migrant experience and the continuing significance of ethnic difference