261 research outputs found
CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq
No full textSingle-cell transcriptomics requires a method that is sensitive, accurate, and reproducible. Here, we present CEL-Seq2, a modified version of our CEL-Seq method, with threefold higher sensitivity, lower costs, and less hands-on time. We implemented CEL-Seq2 on Fluidigm’s C1 system, providing its first single-cell, on-chip barcoding method, and we detected gene expression changes accompanying the progression through the cell cycle in mouse fibroblast cells. We also compare with Smart-Seq to demonstrate CEL-Seq2’s increased sensitivity relative to other available methods. Collectively, the improvements make CEL-Seq2 uniquely suited to single-cell RNA-Seq analysis in terms of economics, resolution, and ease of use.Seventh Framework Programme (European Commission)Israel Science Foundatio
Qigong Sensory Training for Autism: Promising Effects on Sensory Processing, Self-Regulation, and Parenting Stress
No full textAbstract
Date Presented 4/1/2017
Preliminary results of an evidence-based pilot project with young children with or at risk for autism demonstrated that Qigong Sensory Training promoted participation in everyday occupations by improving sensory processing and self-regulation by 18% and decreasing parenting stress by 30%.
Primary Author and Speaker: Orit Tal-Atzili</jats:p
Interpersonal Social Responsibility Effects on Students’ Professional Development: Longitudinal Study
No full textAbstract
Date Presented 3/30/2017
This study shows the impact of an educational structured model that facilitates students’ engagement within the community to promote the development of professional identity. Based on longitudinal analysis assessment research, we suggest an effective educational model for first-year occupational therapy students.
Primary Author and Speaker: Orit Lahav
Additional Authors and Speakers: Shira Yalon-Chamovitz</jats:p
Abstract 3037: Dissecting treatment resistance in patients with ovarian cancer and PDX-models using single-cell RNA-sequencing
No full textAbstract
Background: Ovarian cancer (OvCa) is frequently associated with malignant effusions, which are complex ecosystems with heterogeneous populations of malignant cells and non-malignant cells. Bulk RNA-seq or whole-exome sequencing (WES) only reflect average cellular behavior and thereby mask intrinsic cell diversity with potential relevance for treatment resistance.
Approach: To overcome some of these barriers, we applied single-cell RNA-sequencing (scRNA-seq) to malignant and non-malignant cells isolated from patients with platinum treatment resistant disease. Furthermore, we used patient-derived xenograft (PDX) cohorts, in which we isolated cells for scRNA-seq from vehicle tumors (VEH), treated the other models with carboplatin, and harvested cells at the time of minimal residual disease (MRD) or disease progression (PROG).
Results: To date, we have profiled ~12000 single cells from 12 patients with treatment naïve (n=3) or platinum-resistant disease (n=9), including sequential sampling in 3 patients with resistant disease. We observed significant inter- and intra-individual transcriptional heterogeneity in malignant cells. A recurrent pattern across resistant patients was the differential expression of inflammatory pathways in a subset of cells. In a patient with three consecutive specimens, we observed increasing accumulation of cells expressing a cell state characterized by tumor necrosis factor alpha (TNF-a) signaling, Importantly, these cells were genetically identical to the entire population, supporting the hypothesis that non-encoded mechanisms conferred treatment resistance. In a BRCA-mutant patient, unbiased analysis identified a stemness program in a subpopulation of cells, which was genetically identical to other cells, indicating phenotypic conversion. To systemically interrogate mechanisms of resistance to platinum therapy, sequenced single cells isolated from PDX models at three time points (VEH, MRD and PROG). In a BRCA-WT PDX model, resistant cells isolated at MRD and PROG shared a transcriptional program that was dominated by expression of a STAT3 program. Ex vivo cultures from platinum-resistant patients were exquisitely sensitivity to JAK/STAT3-inhibitor. Live cell imaging revealed that STAT3-inhibition prevented spheroid formation, attachment and clearance through a mesothelial monolayer in vitro.
Conclusion: Our results indicate that non-encoded mechanisms play an important role in the development of treatment resistance in ovarian cancer. Our initial studies indicate an important role of inflammatory pathways in treatment resistance, in particular STAT3 signaling, which can be overcome with specific inhibitors at nanomolar concentrations. These data suggests that single-cell profiling can be performed on clinical ovarian cancer specimens and may yield novel therapeutic avenues for patients with treatment-resistant ovarian cancer.
Citation Format: Benjamin Izar, Itay Tirosh, Elizabeth Stover, Asaf Rotem, Parin Shah, Mike Cuoco, Chris Rodman, Joyce Liu, Ursula Matulonis, Orit Rozenblatt-Rosen, Levi Garraway, Aviv Regev. Dissecting treatment resistance in patients with ovarian cancer and PDX-models using single-cell RNA-sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3037. doi:10.1158/1538-7445.AM2017-3037</jats:p
The menin tumor suppressor protein is phosphorylated in response to DNA damage.
No full textMultiple endocrine neoplasia type 1 (MEN1) is a heritable cancer syndrome characterized by tumors of the pituitary, pancreas and parathyroid. Menin, the product of the MEN1 gene, is a tumor suppressor protein that functions in part through the regulation of transcription mediated by interactions with chromatin modifying enzymes.Here we show menin association with the 5' regions of DNA damage response genes increases after DNA damage and is correlated with RNA polymerase II association but not with changes in histone methylation. Furthermore, we were able to detect significant levels of menin at the 3' regions of CDKN1A and GADD45A under conditions of enhanced transcription following DNA damage. We also demonstrate that menin is specifically phosphorylated at Ser394 in response to several forms of DNA damage, Ser487 is dynamically phosphorylated and Ser543 is constitutively phosphorylated. Phosphorylation at these sites however does not influence the ability to interact with histone methyltransferase activity. In contrast, the interaction between menin and RNA polymerase II is influenced by phosphorylation, whereby a phospho-deficient mutant had a higher affinity for the elongating form of RNA polymerase compared to wild type. Additionally, a subset of MEN1-associated missense point mutants, fail to undergo DNA damage dependent phosphorylation.Together, our findings suggest that the menin tumor suppressor protein undergoes DNA damage induced phosphorylation and participates in the DNA damage transcriptional response
Abstract A10: A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins
No full textAbstract
Reversing the dysfunctional (also known as “exhausted”) T cell state that arises in cancer and chronic viral infections is the focus of therapeutic interventions; however, current therapies are effective in only some patients and for only some tumor types. To gain a deeper molecular understanding of the dysfunctional T cell state, we leveraged mouse models to analyze RNA profiles of CD8+ tumor-infiltrating lymphocytes (TILs) at various functional states. In a comparative study we identified metallothioneins, regulators of zinc metabolism, as drivers of T cell dysfunction and used genetic perturbation to identify, for the first time, a distinct gene module for T cell dysfunction that is uncoupled from T cell activation and associated with a CD8+ Treg signature. An analysis of TILs at single-cell resolution revealed that our dysfunction module negatively correlates with activation signatures also at the single-cell level, indicating that our uncoupled signatures for T cell dysfunction and activation can represent cell-intrinsic states. Clustering of the single-cells revealed a previously uncharacterized subpopulation of CD8+ TILs that was strongly enriched for our novel dysfunction signature, but not for activation signatures. This subpopulation was depleted of cells from a metallothionein KO mouse model in which T cell dysfunction was not observed, implying that we have indeed identified a phenotypically dysfunctional subpopulation. Using CRISPR/Cas9 genome editing we validate that Gata-3, a transcription factor expressed specifically in our identified subpopulation, is a driver of the dysfunctional phenotype in CD8+ TILs. Our results open novel avenues for specifically targeting the dysfunctional T cell state while leaving T cell activation programs intact.
Citation Format: Meromit Singer, Chao Wang, Le Cong, Nemanja D. Marjanovic, Monika S. Kowalczyk, Huiyuan Zhang, Jackson Nyman, Kaori Sakuishi, Sema Kurtulus, David Gennert, Junrong Xia, John YH Kwon, James Nevin, Rebecca H. Herbst, Itai Yanai, Orit Rozenblatt-Rosen, Vijay K. Kuchroo, Aviv Regev, Ana C. Anderson. A distinct gene module for T cell dysfunction uncoupled from T cell activation and controlled by metallothioneins. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr A10.</jats:p
Abstract AP19: SINGLE–CELL RNA–SEQUENCING OF PATIENT–DERIVED OVARIAN CANCER CELLS AND PATIENT–DERIVED XENOGRAFT MODELS
No full textAbstract
BACKGROUND AND PURPOSE: Genetic heterogeneity is a hallmark of ovarian cancer (OvCa) biology and underlies treatment resistance. Macroscopic and or treatment resistant microscopic residual disease (MRD) after debulking surgery and chemotherapy are the source for disease recurrence and death in these patients. Current profiling methods are unable adequately reflect heterogeneity, and transcriptional programs associated with treatment resistance and MRD remain elusive. To elucidate transcriptional heterogeneity and potential mechanisms of drug-resistance, we isolated OvCa cells from patients with malignant ascites using flow-cytometry. We applied single-cell RNA-sequencing (sc-RNA-seq) to ascites-derived cells from patients with OvCa. We picked OvCa spheroids and profiled these separately. To investigate MRD, we used three PDX-models stably expressing mCherry, treated with carboplatin, and harvested tumor cells for sc-RNA-seq at three time points (pre-treatment, at time of MRD as determined by bio-luminescence imaging, and disease relapse).
SUMMARY OF RESULTS: We successfully sequenced 770 single-cell transcriptomes from 6 individuals with treatment-resistant OvCa, including 3 patients with sequential samples. We mapped the landscape of chromosomal aberrations by inferred large-scale copy number variations (CNVs) at a single-cell level. We observed significant inter-tumor heterogeneity and started to deconstruct the genomic architecture of individual patients. Using experimentally validated gene sets, we determined the cell cycle state of individual cells and identified transcriptional programs related to the cell cycle as significant bias in publically available bulk RNA-sequencing data. Principal component analysis revealed the expression of a stem-ness signature, including CD133, ALDH1A and AXL, in a sub-set of non-cycling cells. An important driver of transcriptional heterogeneity common to patients included in this study was the expression of gene sets related to inflammatory pathways, such as the NFkB and JAK/STAT pathways. Hierarchical clustering of 42 spheroid profiles identified four major clusters, including a highly “inflamed” phenotype. Therapeutic inhibition of the STAT pathway abrogated the capacity of spheroid formation on an ultra-low attachment surface, indicating its importance for metastasis. We have successfully isolated thousands of individual cells for single-cell profiling from PDX models treated with carboplatin. These cells were collected at three time points, including at the MRD stage. We have successfully sequenced 100 cells from this collection and were able to generate whole-transcriptome data comparable to that of freshly isolated patient cells and thousands of single cells are currently undergoing sequencing.
CONCLUSION: We have successfully applied single-cell RNA-sequencing to patient-derived ovarian cancer cells and PDX-models. Single-cell transcriptomes enabled inference of genomic information, genetic and transcriptional heterogeneity, cell cycle state, and programs related to stem-ness and inflammation, providing a unique and comprehensive perspective on ovarian cancer cell states. Ongoing profiling of carboplatin-resistant cells captured at the minimal residual disease stage in PDX-models will provide a unique opportunity to understand treatment resistance which ultimately leads to cancer recurrence.
Citation Format: Benjamin Izar, Elizabeth Stover, Itay Tirosh, Asaf Rotem, Parin Shah, Chris Rodman, Sanjay Prakadan, Marc Wadsworth, Mei-Ju Su, Rachel Leeson, Sangeetha Palakurthi, Joyce Liu, Ursula Matulonis, Alex Shalek, Orit Rozenblatt-Rosen, Aviv Regev, Levi Garraway. SINGLE–CELL RNA–SEQUENCING OF PATIENT–DERIVED OVARIAN CANCER CELLS AND PATIENT–DERIVED XENOGRAFT MODELS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr AP19.</jats:p
Integrated scRNA-seq identifies human postnatal thymus seeding progenitors and regulatory dynamics of differentiating immature thymocytes
No full textDuring postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow-derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the nature of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both precursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics underlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signature of immature human thymocytes and provides a resource for the further study of human thymopoiesis
Single-cell transcriptomics to explore the immune system in health and disease
No full textThe immune system varies in cell types, states, and locations. The complex networks, interactions, and responses of immune cells produce diverse cellular ecosystems composed of multiple cell types, accompanied by genetic diversity in antigen receptors. Within this ecosystem, innate and adaptive immune cells maintain and protect tissue function, integrity, and homeostasis upon changes in functional demands and diverse insults. Characterizing this inherent complexity requires studies at single-cell resolution. Recent advances such as massively parallel single-cell RNA sequencing and sophisticated computational methods are catalyzing a revolution in our understanding of immunology. Here we provide an overview of the state of single-cell genomics methods and an outlook on the use of single-cell techniques to decipher the adaptive and innate components of immunity.National Institute of Allergy and Infectious Diseases (U.S.) (Grant U24AI118672)National Institute of Allergy and Infectious Diseases (U.S.) (Grant R24AI072073
Evidence for a Separate Burial Ground at the Submerged Pottery Neolithic Site of Neve-Yam, Israel
No full textSalvage excavations and surveys at Neve-Yam, a submerged Pottery-Neolithic (PN) Wadi Rabah settlement (second half of the 8th millennium cal. BP) off the Carmel coast revealed unique stone-built graves. They were concentrated in a specifi c and separate section of the site devoted to burial and associated activities. There were no dwellings in this burial area and no graves were found in the occupation area. The oval graves, oriented in an east-west direction, were built of undressed stones and some were covered by stone slabs. This is an early example for a clear division between a dwelling zone and a burial ground with stone-built graves in a Neolithic settlement. Three concentrations of charred seeds in the burial area are possibly associated with ritual activities. The separation of the living from the dead in this late PN site is notably different from earlier PN and Pre-Pottery Neolithic (PPN) burial practices and should be considered in light of the later Chalcolithic Ghassulian (starting in middle 7th millennium cal. BP) appearance of formal off-site cemeteries.Résumé : Des fouilles de sauvetage et des observations de terrain à Neve-Yam, un village de la culture Wadi Rabah (Néolithique céramique, 2nde moitié du VIIIe millénaire cal. BP), submergé de la côte du Carmel, ont révélé des tombes en pierre aux caractéristiques particulières. Elles étaient concentrées dans un secteur spécifique du site, voué aux rituels funéraires. Il n’y avait pas d’habitations dans cette partie destinée aux inhumations, et aucune tombe n’a été trouvée dans la zone d’habitation. Les tombes ovales, orientées est-ouest, étaient construites avec des pierres brutes et certaines étaient recouvertes par des dalles de pierre. Il s’agit de l’un des plus anciens villages néolithiques connus, où la séparation est évidente entre la zone d’habitation et la zone funéraire dans laquelle se trouvaient les tombes en pierre. Trois concentrations de graines brûlées dans la zone funéraire peuvent être associées à des activités rituelles. La séparation entre les vivants et les morts dans ce site du Néolithique céramique présente des différences notables avec des pratiques funéraires du Néolithique céramique antérieur et du Néolithique pré-céramique. Elle annonce les pratiques funéraires qui seront observées lors de la période suivante, au Ghassulien (Chalcolithique) qui commence au milieu du VIIe millénaire cal. BP, pendant laquelle les cimetières seront à l’écart des établissements.Galili Ehud, Eshed Vered, Rosen Baruch, Kislev Mordechai E., Simchoni Orit, Hershkovitz Israel, Gopher Avi. Evidence for a Separate Burial Ground at the Submerged Pottery Neolithic Site of Neve-Yam, Israel. In: Paléorient, 2009, vol. 35, n°1. pp. 31-46
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