Modeling of the human alveolar rhabdomyosarcoma Pax3-Foxo1 chromosome translocation in mouse myoblasts using CRISPR-Cas9 nuclease

Many recurrent chromosome translocations in cancer result in the generation of fusion genes that are directly implicated in the tumorigenic process. Precise modeling of the effects of cancer fusion genes in mice has been inaccurate, as constructs of fusion genes often completely or partially lack the correct regulatory sequences. The reciprocal t(2;13)(q36.1;q14.1) in human alveolar rhabdomyosarcoma (A-RMS) creates a pathognomonic PAX3-FOXO1 fusion gene. In vivo mimicking of this translocation in mice is complicated by the fact that Pax3 and Foxo1 are in opposite orientation on their respective chromosomes, precluding formation of a functional Pax3-Foxo1 fusion via a simple translocation. To circumvent this problem, we irreversibly inverted the orientation of a 4.9 Mb syntenic fragment on chromosome 3, encompassing Foxo1, by using Cre-mediated recombination of two pairs of unrelated oppositely oriented LoxP sites situated at the borders of the syntenic region. We tested if spatial proximity of the Pax3 and Foxo1 loci in myoblasts of mice homozygous for the inversion facilitated Pax3-Foxo1 fusion gene formation upon induction of targeted CRISPR-Cas9 nuclease-induced DNA double strand breaks in Pax3 and Foxo1. Fluorescent in situ hybridization indicated that fore limb myoblasts show a higher frequency of Pax3/Foxo1 co-localization than hind limb myoblasts. Indeed, more fusion genes were generated in fore limb myoblasts via a reciprocal t(1;3), which expressed correctly spliced Pax3-Foxo1 mRNA encoding Pax3-Foxo1 fusion protein. We conclude that locus proximity facilitates chromosome translocation upon induction of DNA double strand breaks. Given that the Pax3-Foxo1 fusion gene will contain all the regulatory sequences necessary for precise regulation of its expression, we propose that CRISPR-Cas9 provides a novel means to faithfully model human diseases caused by chromosome translocation in mice.[Author Summary]: Many cancers carry recurrent chromosome translocations, which often result in the formation of fusion genes that are directly involved in the tumorigenic process. Alveolar rhabdomyosarcoma, a muscle tumor in children, is typified by a translocation that fuses the PAX3 gene on chromosome 2 to the FOXO1 gene on chromosome 13. For translocation to occur both genes need to break and the disparate ends need to fuse via a process called non-homologous end joining. We determined that physical proximity of Pax3 and Foxo1 in mouse muscle progenitor cells (myoblasts) facilitates fusion gene formation. Because Pax3 and Foxo1 in the mouse are in an opposite orientation, we used a chromosome engineering strategy to invert the orientation of Foxo1 so that upon translocation a productive Pax3-Foxo1 fusion gene is created. Co-localization of the Pax3 and Foxo1 loci is higher in fore limb than in hind limb myoblasts. Simultaneous induction of a targeted double strand DNA break in each gene by CRISPR-Cas9 nuclease generated more fusion genes in fore limb than in hind limb myoblasts. Thus, gene proximity facilitates fusion gene formation. We propose that CRISPR-Cas9 nuclease can be used for the precise modeling of chromosome translocations of human cancer in mice.This work was supported by the Van Vleet foundation of Memphis and ALSAC of St Jude Children’s Research Hospital and the Cancer Center Core Grant 5P30CA021765-34. BVB and JJC were supported by grant C1178/A4520 from Cancer Research UK.Peer Reviewe

p27KIP1 Deletions in Childhood Acute Lymphoblastic Leukemia

AbstractThe p27KIP1 gene, which encodes a cyclin-dependent kinase (CDK) inhibitor, has been assigned to chromosome band 12p12, a region often affected by cytogenetically apparent deletions or translocations in childhood acute lymphoblastic leukemia (ALL). As described here, fluorescence in situ hybridization (FISH) analysis of 35 primary ALL samples with cytogenetic evidence of 12p abnormalities revealed hemizygous deletions of p27KIP1 in 29 cases. Further analysis of 19 of these cases with two additional gene-specific probes from the 12p region (hematopoietic cell phosphatase, HCP and cyclin D2, CCND2) showed that p27KIP1 is located more proximally on the short arm of chromosome 12 and is deleted more frequently than either HCP or CCND2. Of 16 of these cases with hemizygous deletion of p27KIP1, only eight showed loss of HCP or CCND2, whereas loss of either of the latter two loci was uniformly associated with loss of p27KIP1. Missense mutations or mutations leading to premature termination codons were not detected in the coding sequences of the retained p27KIP1 alleles in any of the 16 ALL cases examined, indicating a lack of homozygous inactivation. By Southern blot analysis, one case of primary T-cell ALL had hemizygous loss of a single p27KIP1 allele and a 34.5-kb deletion, including the second coding exon of the other allele. Despite homozygous inactivation of p27KIP1 in this case, our data suggest that haploinsufficiency for p27KIP1 is the primary consequence of 12p chromosomal deletions in childhood ALL. The oncogenic role of reduced, but not absent, levels of p27KIP1 is supported by recent studies in murine models and evidence that this protein not only inhibits the activity of complexes containing CDK2 and cyclin E, but also promotes the assembly and catalytic activity of CDK4 or CDK6 in complexes with cyclin D

The interview as narrative ethnography : seeking and shaping connections in qualitative research.

Acts of counter-subjectification in qualitative research are always present but are often submerged in accounts that seek to locate the power of subjectification entirely with the researcher. This is particularly so when talking to people about sensitive issues. Based on an interview-based study of infertility and reproductive disruption among British Pakistanis in Northeast England, we explore how we, as researchers, sought and were drawn into various kinds of connections with the study participants; connections that were actively and performatively constructed through time. The three of us that conducted interviews are all female academics with Ph.Ds in anthropology, but thereafter our backgrounds, life stories and experiences diverge in ways that intersected with those of our informants in complex and shifting ways. We describe how these processes shaped the production of narrative accounts and consider some of the associated analytical and ethical implications

Neural origins of human sickness in interoceptive responses to inflammation

BACKGROUND: Inflammation is associated with psychological, emotional, and behavioral disturbance, known as sickness behavior. Inflammatory cytokines are implicated in coordinating this central motivational reorientation accompanying peripheral immunologic responses to pathogens. Studies in rodents suggest an afferent interoceptive neural mechanism, although comparable data in humans are lacking. METHODS: In a double-blind, randomized crossover study, 16 healthy male volunteers received typhoid vaccination or saline (placebo) injection in two experimental sessions. Profile of Mood State questionnaires were completed at baseline and at 2 and 3 hours. Two hours after injection, participants performed a high-demand color word Stroop task during functional magnetic resonance imaging. Blood samples were performed at baseline and immediately after scanning. RESULTS: Typhoid but not placebo injection produced a robust inflammatory response indexed by increased circulating interleukin-6 accompanied by a significant increase in fatigue, confusion, and impaired concentration at 3 hours. Performance of the Stroop task under inflammation activated brain regions encoding representations of internal bodily state. Spatial and temporal characteristics of this response are consistent with interoceptive information flow via afferent autonomic fibers. During performance of this task, activity within interoceptive brain regions also predicted individual differences in inflammation-associated but not placebo-associated fatigue and confusion. Maintenance of cognitive performance, despite inflammation-associated fatigue, led to recruitment of additional prefrontal cortical regions. CONCLUSIONS: These findings suggest that peripheral infection selectively influences central nervous system function to generate core symptoms of sickness and reorient basic motivational states. PMID:19409533[PubMed - indexed for MEDLINE] PMCID: PMC2885492Free PMC Articl

Th-MYCN Mice with Caspase-8 Deficiency Develop Advanced Neuroblastoma with Bone Marrow Metastasis

Neuroblastoma, the most common extracranial pediatric solid tumor, is responsible for 15% of all childhood cancer deaths. Patients frequently present at diagnosis with metastatic disease, particularly to the bone marrow (BM). Advances in therapy and understanding of the metastatic process have been limited due in part, to the lack of animal models harboring BM disease. The widely employed transgenic model, the Th-MYCN mouse, exhibits limited metastasis to this site. Here we establish the first genetic immunocompetent mouse model for metastatic neuroblastoma with enhanced secondary tumors in the BM. This model recapitulates two frequent alterations in metastatic neuroblasoma, over-expression of MYCN and loss of caspase-8 expression. Mouse caspase-8 gene was deleted in neural crest lineage cells by crossing a Th-Cre transgenic mouse with a caspase-8 conditional knockout mouse. This mouse was then crossed with the neuroblastoma prone Th-MYCN mouse. While over-expression of MYCN by itself rarely caused bone marrow metastasis, combining MYCN overexpression and caspase-8 deletion significantly enhanced BM metastasis (37% incidence). Microarray expression studies of the primary tumors mRNAs and microRNAs revealed extracellular matrix (ECM) structural changes, increased expression of genes involved in epithelial to mesenchymal transition, inflammation and down-regulation of miR-7a and miR-29b. These molecular changes have been shown to be associated with tumor progression and activation of the cytokine transforming growth factor beta (TGF-β) pathway in various tumor models. Cytokine TGF-β can preferentially promote single cell motility and blood borne metastasis and therefore activation of this pathway may explain the enhanced BM metastasis observed in this animal model.Fil: Teitz, Tal. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Inoue, Madoka. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Valentine, Marcus B.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Zhu, Kejin. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados UnidosFil: Rehg, Jerold E.. St. Jude Children’s Research Hospital. Department of Pathology; Estados UnidosFil: Zhao, Wei. St. Jude Children’s Research Hospital. Department of Biostatistics; Estados UnidosFil: Finkelstein, David. St. Jude Children’s Research Hospital. Department of Computational Biology; Estados UnidosFil: Wang, Yong-Dong. St. Jude Children’s Research Hospital. Hartwell Center for Bioinformatics and Biotechnology; Estados UnidosFil: Johnson, Melissa D.. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Calabrese, Christopher. St. Jude Children’s Research Hospital. Animal Imaging Center; Estados UnidosFil: Rubinstein, Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular; ArgentinaFil: Hakem, Razqallah. University of Toronto. Ontario Cancer Institute. Department of Medical Biophysics; CanadáFil: Weiss, William A.. University of California. Departments of Neurology, Pediatrics and Neurological Surgery; Estados UnidosFil: Lahti, Jill M.. St. Jude Children’s Research Hospital. Department of Tumor Cell Biology; Estados Unido

Manipulating infrared photons using plasmons in transparent graphene superlattices

Superlattices are artificial periodic nanostructures which can control the flow of electrons. Their operation typically relies on the periodic modulation of the electric potential in the direction of electron wave propagation. Here we demonstrate transparent graphene superlattices which can manipulate infrared photons utilizing the collective oscillations of carriers, i.e., plasmons of the ensemble of multiple graphene layers. The superlattice is formed by depositing alternating wafer-scale graphene sheets and thin insulating layers, followed by patterning them all together into 3-dimensional photonic-crystal-like structures. We demonstrate experimentally that the collective oscillation of Dirac fermions in such graphene superlattices is unambiguously nonclassical: compared to doping single layer graphene, distributing carriers into multiple graphene layers strongly enhances the plasmonic resonance frequency and magnitude, which is fundamentally different from that in a conventional semiconductor superlattice. This property allows us to construct widely tunable far-infrared notch filters with 8.2 dB rejection ratio and terahertz linear polarizers with 9.5 dB extinction ratio, using a superlattice with merely five graphene atomic layers. Moreover, an unpatterned superlattice shields up to 97.5% of the electromagnetic radiations below 1.2 terahertz. This demonstration also opens an avenue for the realization of other transparent mid- and far-infrared photonic devices such as detectors, modulators, and 3-dimensional meta-material systems.Comment: under revie

Faecal particle size in free-ranging proboscis monkeys, Nasalis larvatus: variation between seasons

Reducing the size of food particles is crucial for herbivores. Seasonal dietary changes are known to influence animals’ chewing efficiency. Proboscis monkeys (Nasalis larvatus) are foregut fermenters, with a high chewing efficiency allowing them to achieve very fine faecal particles. In this study, we investigated how proboscis monkeys’ chewing efficiency varies between wet and dry seasons, hypothesising differences possibly related to diet change. Faecal particle size analysis is an established approach to estimate chewing efficiency in mammalian herbivores. We analysed 113 proboscis monkey faecal samples collected in the Lower Kinabatangan Wildlife Sanctuary, between 2015 and 2017. By following standard sieve analysis protocols, we measured a mean particle size MPS0.025–8 of 0.45 ± 0.14 mm, and confirmed a previous result that proboscis monkeys have a very low faecal MPS. This study highlights a seasonal influence on proboscis monkeys’ chewing efficiency, with smaller MPS (better chewing efficiency) during the wet season. During that time of the year, individuals may potentially change their diet, as all faecal samples contained intact seeds. Whether the seasonal MPS difference in proboscis monkeys is smaller than in other colobines due to their “rumination” strategy remains to be investigated

OptiJ: Open-source optical projection tomography of large organ samples

Abstract: The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples

OptiJ: Open-source optical projection tomography of large organ samples

The three-dimensional imaging of mesoscopic samples with Optical Projection Tomography (OPT) has become a powerful tool for biomedical phenotyping studies. OPT uses visible light to visualize the 3D morphology of large transparent samples. To enable a wider application of OPT, we present OptiJ, a low-cost, fully open-source OPT system capable of imaging large transparent specimens up to 13 mm tall and 8 mm deep with 50 µm resolution. OptiJ is based on off-the-shelf, easy-to-assemble optical components and an ImageJ plugin library for OPT data reconstruction. The software includes novel correction routines for uneven illumination and sample jitter in addition to CPU/GPU accelerated reconstruction for large datasets. We demonstrate the use of OptiJ to image and reconstruct cleared lung lobes from adult mice. We provide a detailed set of instructions to set up and use the OptiJ framework. Our hardware and software design are modular and easy to implement, allowing for further open microscopy developments for imaging large organ samples