Authors: Hu P, Martinez AF, Kruszka P, Berger S, Roessler E, Muenke M
Journal: Genet Med. 2018 Sep 10. doi: 10.1038/s41436-018-0261-8. [Epub ahead of print]
PURPOSE: De novo variants (DNVs) represent an important fraction of the pathogenic variant burden in holoprosencephaly (HPE). However, unexpected recurrences can occur, as evidenced by multiple affected children harboring the same apparently DNV. This study was performed to estimate the rate of parental mosaicism in a cohort of patients with HPE.
METHODS: We developed a targeted capture next-generation sequencing (NGS) panel of 153 genes with potential implication in HPE. Sequencing data from a cohort of 136 HPE family trios were analyzed to identify probands with apparently DNVs. DNVs were examined in the proband and their parents to detect any deviations from the expected ~50/50 allele ratio of true heterozygosity. Selected variants were confirmed by Droplet Digital™ polymerase chain reaction (ddPCR).
RESULTS: We identified 28 high-confidence DNVs, 20 of which occurred in known HPE genes. Nineteen of the 20 variants (95%) were pathogenic or likely pathogenic. Sequence data analysis showed evidence of parental mosaicism in five cases, for an overall mosaicism rate of 26%. In addition, we found evidence for likely postzygotic events in four cases (50%).
CONCLUSIONS: High sensitivity methods, such as high-depth NGS and ddPCR, are essential to providing an accurate assessment of recurrence risk in HPE families with apparently DNVs.
Authors: Burks SR, Nagle ME, Bresler MN, Kim SJ, Star RA, Frank JA
Journal: J Cell Mol Med. 2018 Sep 14. doi: 10.1111/jcmm.13874. [Epub ahead of print]
Mesenchymal stromal cell (MSC) therapies combined with renal pulsed focused ultrasound (pFUS) pretreatment increase MSC homing and improve cisplatin-induced acute kidney injury (AKI) better than MSC alone. However, mechanisms underlying improved outcomes remain unknown. We hypothesize pFUS up-regulates renal interferon-γ (IFNγ) and stimulates MSC to produce interleukin-10 (IL-10) after migrating to kidneys. To demonstrate initially, MSC cultured with IFNγ up-regulated IL-10. More MSC-derived IL-10 was detected in kidneys when IFNγ-stimulated MSC were infused and they improved AKI better than unstimulated MSC. Next, IFNγ-knockout mice with AKI received pFUS+MSC, but MSC-derived IL-10 expression and AKI were similar to using MSC alone. AKI in wild-type mice receiving pFUS and IL-10-deficient MSC was also unimproved compared to administering IL-10-deficient MSC alone. Indoleamine 2,3-dioxygenase (IDO), an anti-inflammatory enzyme up-regulated in MSC by IFNγ, was up-regulated during AKI, but was not further elevated in MSC from pFUS-treated kidneys, suggesting that IDO is not involved in improved AKI healing by pFUS+MSC. These data suggest IFNγ is up-regulated by pFUS and after i.v.-infused MSC home to pFUS-treated kidneys, IFNγ stimulates additional IL-10 production by MSC to improve AKI. Analogous mechanisms of ultrasound-treated tissue microenvironments stimulating therapeutic MSC may exist in other pathologies where adjuvant ultrasound techniques are successful.
Authors: Qin H, Ishii K, Nguyen S, Su PP, Burk CR, Kim BH, Duncan BB, Tarun S, Shah NN, Kohler ME, Fry TJ
Journal: Blood. 2018 Sep 12. pii: blood-2017-12-815548. doi: 10.1182/blood-2017-12-815548. [Epub ahead of print]
Adoptive transfer of patient-derived T cells modified to express chimeric antigen receptors (CART) has demonstrated dramatic success in relapsed/refractory pre-B cell ALL but response and durability of remission requires exponential CART expansion and persistence. Tumors are known to affect T cell function but this has not been well studied in ALL and in the context of CAR expression. Using TCF3/PBX1 and MLL-AF4-driven murine ALL models, we assessed the impact of progressive ALL on T cell function in vivo. Vaccines protect against TCF3/PBX1.3 but were ineffective when administered after leukemia injection suggesting immunosuppression induced early during ALL progression. T cells from leukemia-bearing mice exhibited increased expression of inhibitory receptors including PD1, Tim3 and LAG3 and were dysfunctional following adoptive transfer in a model of TCR-dependent leukemia clearance. Although expression of inhibitory receptors has been linked to TCR signaling, pre-B ALL induced inhibitory receptor expression, at least in part, via a T cell receptor (TCR) independent manner. Finally, introduction of a CAR into T cells generated from leukemia-bearing mice failed to fully reverse poor in vivo function.
Authors: Martin-Martin I, Aryan A, Meneses C, Adelman ZN, Calvo E
Journal: PLoS Negl Trop Dis. 2018 Sep 4;12(9):e0006769. doi: 10.1371/journal.pntd.0006769. [Epub ahead of print]
BACKGROUND: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 technology has rapidly emerged as a very effective tool for gene editing. Although great advances on gene editing in the medical entomology field have arisen, no attempts of gene editing have been reported in sand flies, the vectors of Leishmaniasis.
METHODOLOGY/PRINCIPAL FINDINGS: Here, we described a detailed protocol for sand fly embryo microinjection taking into consideration the sand fly life cycle, and manipulation and oviposition requirements of this non-model organism. Following our microinjection protocol, a hatching rate of injected embryos of 11.90%-14.22% was achieved, a rate consistent with other non-model organism dipterans such as mosquitoes. Essential factors for the adaptation of CRISPR/Cas9 technology to the sand fly field were addressed including the selection of a target gene and the design and production of sgRNA. An in vitro cleavage assay was optimized to test the activity of each sgRNA and a protocol for Streptococcus pyogenes Cas9 (spCas9) protein expression and purification was described. Relevant considerations for a successful gene editing in the sand fly such as specifics of embryology and double-stranded break DNA repair mechanisms were discussed.
CONCLUSION AND SIGNIFICANCE: The step-by-step methodology reported in this article will be of significant use for setting up a sand fly embryo microinjection station for the incorporation of CRISPR/Cas9 technology in the sand fly field. Gene editing strategies used in mosquitoes and other model insects have been adapted to work with sand flies, providing the tools and relevant information for adapting gene editing techniques to the vectors of Leishmaniasis. Gene editing in sand flies will provide essential information on the biology of these vectors of medical and veterinary relevance and will rise a better understanding of vector-parasite-host interactions.
Authors: Anderson ED, Sastalla I, Earland NJ, Mahnaz M, Moore IN, Otaizo-Carrasquero F, Myers TG, Myles CA, Datta SK, Myles IA
Journal: PLoS One. 2018 Sep 12;13(9):e0198862. doi: 10.1371/journal.pone.0198862. eCollection 2018.
Keratinocytes are the most abundant cell type in the epidermis. They prevent desiccation and provide immunological and barrier defense against potential pathogens such as Staphylococcus aureus and Candida albicans. The study of this first line of immune defense may be hindered by invasive isolation methods and/or improper culture conditions to support stem cell maintenance and other potential mechanisms contributing to long-term subcultivation in vitro. Primary keratinocytes have been successfully isolated from blister roofs induced by negative pressure, which separates the epidermis from the dermis in vivo in human subjects. This method allows collection of pure epidermal cells without dermal contamination in a minimally invasive manner. However, the isolated keratinocytes differentiate and senesce when cultured in vitro beyond five passages. Here, we present evidence that the Rho kinase (ROCK) inhibitor Y-27632 can be used to effectively increase the proliferative capabilities of keratinocytes isolated using the suction blister method, similar to what has been previously reported for primary keratinocytes isolated using alternative methods. We show that the increase in passage number is directly correlated to delayed differentiation, and that cells passaged long term with the inhibitor retain their ability to stratify in organotypic raft cultures and respond to cytokine treatment; additionally, the late passage cells have a heterogeneous mix of differentiated and non-differentiated cells which may be predicted by a ratio of select differentiation markers. The described method presents a minimally invasive procedure for keratinocyte isolation and prolonged culture that allows analysis of keratinocyte function in both healthy volunteers and patients with dermatologic diseases.
Authors: Lignell A, Kerosuo L
Journal: Methods Mol Biol. 2018 Sep 8. doi: 10.1007/7651_2018_188. [Epub ahead of print]
Here, we present Spatial Genomic Analysis (SGA), a quantitative single-cell transcriptional profiling method that takes advantage of single-molecule imaging of individual transcripts for up to a hundred genes. SGA relies on a machine learning-based image analysis pipeline that performs cell segmentation and transcript counting in a robust way. SGA is suitable for various in situ applications and was originally developed to address heterogeneity in the neural crest, which is a transient embryonic stem cell population important for formation of various vertebrate body structures. After being specified as multipotent neural crest stem cells in the dorsal neural tube, they go through an epithelial to mesenchymal transition in order to migrate to different destinations around the body, and gradually turn from stem cells to progenitors prior to final commitment. The molecular details of this process remain largely unknown, and upon their emergence, the neural crest cells have been considered as a single homogeneous population. Technical limitations have restricted the possibility to parse the neural crest cell pool into subgroups according to multiplex gene expression properties. By using SGA, we were able to identify subgroups inside the neural crest niche in the dorsal neural tube. The high sensitivity of the method allows detection of low expression levels and we were able to determine factors not previously shown to be present in neural crest stem cells, such as pluripotency or lineage markers. Finally, SGA analysis also provides prediction of gene relationships within individual cells, and thus has broad utility for powerful transcriptome analyses in original biological contexts.
Authors: Matthews CE, Keadle SK, Saint-Maurice PF, Moore SC, Willis EA, Sampson JN, Berrigan D
Journal: Am J Prev Med. 2018 Sep;55(3):e61-e69. doi: 10.1016/j.amepre.2018.04.036. Epub 2018 Jul 19.
INTRODUCTION: The goal of this study was to describe differences in time use and energy expenditure associated with exercise, prolonged TV viewing, and work days in a longitudinal study of older adults.
METHODS: Participants were 1,020 adults who completed previous-day recalls that provided a profile of the use of time in sedentary and physical activity. Time use and physical activity energy expenditure were predicted for each type of day (exercise, prolonged TV, work) using linear mixed models, adjusting for age, sex, season of the year, and day of the week. Data were collected in 2012-2013; analysis was completed in 2017.
RESULTS: Exercise days had less sedentary time (-0.37 hours/day) and light activity (-0.29 hours/day), and less household, work, and shopping activities, such that the increase in total physical activity energy expenditure on exercise days (2.83 MET-hours/day) was only about half that expended during exercise (5.98 MET-hours/day). Prolonged TV viewing days had more total sedentary time (0.86 hours/days) and less light (-0.45 hours/day) and moderate-vigorous intensity activity (-0.41 hours/day), and thus lower total physical activity energy expenditure (-2.43 MET-hours/day). Work days had less sleep (-0.91 hours/day) and more total sedentary time (1.32 hours/day).
CONCLUSIONS: Exercise days had more physical activity energy expenditure, but because of reductions in other activities, only about half of the energy expended during exercise was added to total daily physical activity energy expenditure. Prolonged TV viewing days had less physical activity energy expenditure and less moderate-vigorous activity. These findings provide new insights into possible compensation associated with exercise, and suggest a strong link between TV viewing and physical inactivity.
Authors: Martin S, Chiramel AI, Schmidt ML, Chen YC, Whitt N, Watt A, Dunham EC, Shifflett K, Traeger S, Leske A, Buehler E, Martellaro C, Brandt J, Wendt L, Müller A, Peitsch S, Best SM, Stech J, Finke S, Römer-Oberdörfer A, Groseth A, Feldmann H, Hoenen T
Journal: Genome Med. 2018 Aug 7;10(1):58. doi: 10.1186/s13073-018-0570-1.
BACKGROUND: The 2014-2016 Ebola virus (EBOV) outbreak in West Africa highlighted the need for improved therapeutic options against this virus. Approaches targeting host factors/pathways essential for the virus are advantageous because they can potentially target a wide range of viruses, including newly emerging ones and because the development of resistance is less likely than when targeting the virus directly. However, systematic approaches for screening host factors important for EBOV have been hampered by the necessity to work with this virus at biosafety level 4 (BSL4).
METHODS: In order to identify host factors involved in the EBOV life cycle, we performed a genome-wide siRNA screen comprising 64,755 individual siRNAs against 21,566 human genes to assess their activity in EBOV genome replication and transcription. As a screening platform, we used reverse genetics-based life cycle modelling systems that recapitulate these processes without the need for a BSL4 laboratory.
RESULTS: Among others, we identified the de novo pyrimidine synthesis pathway as an essential host pathway for EBOV genome replication and transcription, and confirmed this using infectious EBOV under BSL4 conditions. An FDA-approved drug targeting this pathway showed antiviral activity against infectious EBOV, as well as other non-segmented negative-sense RNA viruses.
CONCLUSIONS: This study provides a minable data set for every human gene regarding its role in EBOV genome replication and transcription, shows that an FDA-approved drug targeting one of the identified pathways is highly efficacious in vitro, and demonstrates the power of life cycle modelling systems for conducting genome-wide host factor screens for BSL4 viruses.
Authors: Ji S, Samara NL, Revoredo L, Zhang L, Tran DT, Muirhead K, Tabak LA, Ten Hagen KG
Journal: Nat Commun. 2018 Aug 29;9(1):3508. doi: 10.1038/s41467-018-05978-9.
Regulated secretion is an essential process where molecules destined for export are directed to membranous secretory granules, where they undergo packaging and maturation. Here, we identify a gene (pgant9) that influences the structure and shape of secretory granules within the Drosophila salivary gland. Loss of pgant9, which encodes an O-glycosyltransferase, results in secretory granules with an irregular, shard-like morphology, and altered glycosylation of cargo. Interestingly, pgant9 undergoes a splicing event that acts as a molecular switch to alter the charge of a loop controlling access to the active site of the enzyme. The splice variant with the negatively charged loop glycosylates the positively charged secretory cargo and rescues secretory granule morphology. Our study highlights a mechanism for dictating substrate specificity within the O-glycosyltransferase enzyme family. Moreover, our in vitro and in vivo studies suggest that the glycosylation status of secretory cargo influences the morphology of maturing secretory granules.
Authors: Luk JW, Sita KR, Gilman SE, Goldstein RB, Haynie DL, Simons-Morton BG
Journal: J Adolesc Health. 2018 Aug 1. pii: S1054-139X(18)30214-3. doi: 10.1016/j.jadohealth.2018.05.027. [Epub ahead of print]
PURPOSE: To examine associations between adolescent sexual minority status and developmental transitions in school, work, residence, and transportation 5 years later.
METHOD: We analyzed data from Waves 2 (Mean age = 17.2) and 7 (Mean age = 22.6) of the NEXT Generation Health Study (n = 2,000). Relative risks were estimated using Poisson regressions.
RESULTS: Relative to heterosexual females, sexual minority females were more likely to report not attending school (relative risk [RR] = 1.27, 95% confidence interval [CI] = 1.02, 1.59), not anticipating college completion (RR = 1.60, 95% CI = 1.27, 2.01), and not having a driver's license (RR = 2.64, 95% CI 1.38, 5.05) at Wave 7. Relative to heterosexual males, sexual minority males were more likely to report living in three or more places in the past year (RR = 2.98, 95% CI = 1.31, 6.76).
CONCLUSIONS: Adolescent sexual minority status predicted worse educational outcomes among females and more unstable living environment among males.