ORCID Profile
0000-0002-9625-1183
Current Organisations
University of Adelaide
,
Harvard Medical School
Does something not look right? The information on this page has been harvested from data sources that may not be up to date. We continue to work with information providers to improve coverage and quality. To report an issue, use the Feedback Form.
Publisher: American Association for Cancer Research (AACR)
Date: 15-12-2016
DOI: 10.1158/0008-5472.CAN-16-0298
Abstract: Solid papillary carcinoma with reverse polarity (SPCRP) is a rare breast cancer subtype with an obscure etiology. In this study, we sought to describe its unique histopathologic features and to identify the genetic alterations that underpin SPCRP using massively parallel whole-exome and targeted sequencing. The morphologic and immunohistochemical features of SPCRP support the invasive nature of this subtype. Ten of 13 (77%) SPCRPs harbored hotspot mutations at R172 of the isocitrate dehydrogenase IDH2, of which 8 of 10 displayed concurrent pathogenic mutations affecting PIK3CA or PIK3R1. One of the IDH2 wild-type SPCRPs harbored a TET2 Q548* truncating mutation coupled with a PIK3CA H1047R hotspot mutation. Functional studies demonstrated that IDH2 and PIK3CA hotspot mutations are likely drivers of SPCRP, resulting in its reversed nuclear polarization phenotype. Our results offer a molecular definition of SPCRP as a distinct breast cancer subtype. Concurrent IDH2 and PIK3CA mutations may help diagnose SPCRP and possibly direct effective treatment. Cancer Res 76(24) 7118–29. ©2016 AACR.
Publisher: ZappyLab, Inc.
Date: 22-11-2019
DOI: 10.17504/PROTOCOLS.IO.9P8H5RW
Abstract: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas aragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Publisher: Springer New York
Date: 05-09-2014
Publisher: Wiley
Date: 24-05-2015
DOI: 10.1111/HIS.12712
Publisher: Springer Science and Business Media LLC
Date: 09-10-2011
DOI: 10.1038/NM.2473
Publisher: Elsevier BV
Date: 07-2005
Publisher: ZappyLab, Inc.
Date: 11-05-2021
DOI: 10.17504/PROTOCOLS.IO.BUXNNXME
Abstract: This is a protocol in development, which means it has not yet tested/challenged with multiple s les. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment.
Publisher: Elsevier BV
Date: 05-2007
Publisher: ZappyLab, Inc.
Date: 26-02-2021
DOI: 10.17504/PROTOCOLS.IO.BSTZNEP6
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (D.O.F means Daughter Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® continuous gradient to remove debris. It is the alternative protocol when FACS is not available. This protocol works better with smaller s les. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions.
Publisher: ZappyLab, Inc.
Date: 12-03-2021
DOI: 10.17504/PROTOCOLS.IO.BS99NH96
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (D.O.F means Daughter Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® continuous gradient to remove debris. It is the alternative protocol when FACS is not available. This protocol works better with smaller s les. After centrifugation the nuclei will form a pellet and contaminating membranes will float at the top. This continuous gradient can be applied for as many times as needed to the same prep to continue cleaning up your nuclei suspensions.
Publisher: Wiley
Date: 27-04-2017
DOI: 10.1002/PATH.4890
Publisher: ZappyLab, Inc.
Date: 24-01-2020
Publisher: Wiley
Date: 14-07-2015
DOI: 10.1002/PATH.4573
Publisher: ZappyLab, Inc.
Date: 24-01-2020
Publisher: ZappyLab, Inc.
Date: 05-02-2020
DOI: 10.17504/PROTOCOLS.IO.BB7MIRK6
Abstract: -Here, I will only include tips and notes related to snATAC workflow. Thus, I assume you are familiar with Frankenstein protocol below: - These are ONLY a collection of my lab notes. This is not a protocol. -These notes are derived from trial and error in order to get successful runs applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues using Chromium Platform (10x Genomics). -Be mindful these are just my notes and by no means are ground truth of how snATAC-Seq needs to be performed. I WELCOME to comments, suggestions and amendments.
Publisher: ZappyLab, Inc.
Date: 28-12-2020
DOI: 10.17504/PROTOCOLS.IO.BQ23MYGN
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other s les types. Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.
Publisher: ZappyLab, Inc.
Date: 06-03-2020
DOI: 10.17504/PROTOCOLS.IO.BDBAI2IE
Abstract: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas aragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Publisher: Springer Science and Business Media LLC
Date: 16-09-2020
DOI: 10.1038/S41586-020-2734-6
Abstract: The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development
Publisher: Elsevier BV
Date: 11-2016
Publisher: ZappyLab, Inc.
Date: 06-03-2020
DOI: 10.17504/PROTOCOLS.IO.BDA8I2HW
Abstract: -Here, I will only include tips and notes related to snATAC workflow. Thus, I assume you are familiar with Frankenstein protocol below: - These are ONLY a collection of my lab notes. This is not a protocol. -These notes are derived from trial and error in order to get successful runs applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues using Chromium Platform (10x Genomics). -Be mindful these are just my notes and by no means are ground truth of how snATAC-Seq needs to be performed. I WELCOME to comments, suggestions and amendments.
Publisher: ZappyLab, Inc.
Date: 09-03-2020
DOI: 10.17504/PROTOCOLS.IO.BDENI3DE
Abstract: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas aragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Publisher: American Society of Clinical Oncology (ASCO)
Date: 05-2022
DOI: 10.1200/PO.21.00365
Abstract: Mitogen-activated protein kinase pathway–activating mutations occur in the majority of colorectal cancer (CRC) cases and show mutual exclusivity. We identified 47 epidermal growth factor receptor/BRAF inhibitor-naive CRC patients with dual RAS hotspot/ BRAF V600E mutations (CRC-DD) from a cohort of 4,561 CRC patients with clinical next-generation sequencing results. We aimed to define the molecular phenotypes of the CRC-DD and to test if the dual RAS hotspot/BRAF V600E mutations coexist within the same cell. We developed a single-cell genotyping method with a mutation detection rate of 96.3% and a genotype prediction accuracy of 92.1%. Mutations in the CRC-DD cohort were analyzed for clonality, allelic imbalance, copy number, and overall survival. Application of single-cell genotyping to four CRC-DD revealed the co-occurrence of both mutations in the following percentages of cells per case: NRAS G13D/ KRAS G12C, 95% KRAS G12D/ NRAS G12V, 48% BRAF V600E/ KRAS G12D, 44% and KRAS G12D/ NRAS G13V, 14%, respectively. Allelic imbalance favoring the oncogenic allele was less frequent in CRC-DD (24 of 76, 31.5%, somatic mutations) compared with a curated cohort of CRC with a single-driver mutation (CRC-SD 119 of 232 mutations, 51.3% P = .013). Microsatellite instability–high status was enriched in CRC-DD compared with CRC-SD (23% v 11.4%, P = .028). Of the seven CRC-DD cases with multiregional sequencing, five retained both driver mutations throughout all sequenced tumor sites. Both CRC-DD cases with discordant multiregional sequencing were microsatellite instability–high. Our findings indicate that dual-driver mutations occur in a rare subset of CRC, often within the same tumor cells and across multiple tumor sites. Their presence and a lower rate of allelic imbalance may be related to dose-dependent signaling within the mitogen-activated protein kinase pathway.
Publisher: American Association for Cancer Research (AACR)
Date: 31-05-2011
DOI: 10.1158/0008-5472.CAN-10-3738
Abstract: Hedgehog (Hh) signaling plays an important role in several malignancies but its clinical significance in breast cancer is unclear. In a cohort of 279 patients with invasive ductal carcinoma of the breast, expression of Hh ligand was significantly associated with increased risk of metastasis, breast cancer-specific death, and a basal-like phenotype. A paracrine signature, encompassing high epithelial Hh ligand and high stromal Gli1, was an independent predictor for overall survival in multivariate analysis. In 2 independent histological progression series (n = 301), Hh expression increased with atypia. Hh ligand overexpression in a mouse model of basal breast cancer increased growth, induced a poorly differentiated phenotype, accelerated metastasis, and reduced survival. A stromal requirement for these effects was supported by the lack of similar Hh-mediated changes in vitro, and by stromal-specific expression of Hh target genes in vivo. Furthermore, inhibition of Hh ligand with a monoclonal antibody (5E1) inhibited tumor growth and metastasis. These data suggest that epithelial–stromal Hh signaling, driven by ligand expression in carcinoma cells, promotes breast cancer growth and metastasis. Blockade of Hh signaling to peritumoral stromal cells may represent a novel therapeutic approach in some basal-like breast cancers. Cancer Res 71(11) 4002–14. ©2011 AACR.
Publisher: ZappyLab, Inc.
Date: 24-01-2020
Publisher: ZappyLab, Inc.
Date: 13-09-2021
DOI: 10.17504/PROTOCOLS.IO.BX64PRGW
Abstract: This is a protocol in development, which means it has not yet tested/challenged with multiple s les. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment. In this version I labelled DTT as optional since after further testing I have not seen any difference with or without in snRNA-Seq workflow. Also, I recommend WRB1 for snRNA-Seq workflow only (although still works fine for Multiome) and WRB2 for both snRNA-Seq and Multiome workflows. Since WRB2 is 10x Genomics' recommendation for Multiome I have adopted this for this workflow. I include a discussion about cycling during cDNA . SaltyEz50 option: this has been particularly useful for s les where lysis in SaltyEz10 showed to be suboptimal. In my hands, those were breast, liver and pancreas.
Publisher: Cold Spring Harbor Laboratory
Date: 24-08-2022
DOI: 10.1101/2022.08.23.505054
Abstract: FFPE (formalin-fixed, paraffin-embedded) tissue archives are the largest repository of clinically annotated human specimens. Despite numerous advances in technology, current methods for sequencing of FFPE-fixed single-cells are slow, labour intensive, insufficiently sensitive and have a low resolution, making it difficult to fully exploit their enormous research and clinical potential. Here we introduce single nuclei pathology sequencing (snPATHO-Seq), a sensitive and efficient high-throughput platform to profile the transcriptome of single nuclei extracted from formalin-fixed paraffin-embedded (FFPE) s les. snPATHO-Seq combines an optimised nuclei extraction protocol from archival s les with 10x Genomics probe-based technology targeting the whole transcriptome. We performed direct comparison of the Fixed RNA Profiling (FRP) and established 3’ single cell RNA-Sequencing (scRNA-Seq) workflows through a comprehensive bioinformatics analysis of matched fresh and fixed s les derived from the LNCaP prostate cancer cell line. FRP detected 2.1 times more transcripts in the fixed s le than the 3’ kit did in the fresh s le. Low mitochondrial genes detection using the FRP was translated into 99.9 percent of cells passing the QC filters, compared to 81.6 percent of cells using the v3.1 chemistry. We then optimized snPATHO-Seq and applied it to a human breast cancer metastasis to the liver collected at autopsy and preserved in FFPE, a particularly challenging s le type. Remarkably, at 28,000 reads/cell snPATHO-Seq was able to detect a median of 1850 genes/cell and 3,216 UMI counts/cell. Comparison of snPATHO-Seq with spatial transcriptomics data (10x Genomics Visium FFPE v1) derived from an adjacent section of the same s le revealed a strong correlation, validating the accuracy of the snPATHO-Seq data. Gene expression data from snPATHO-Seq was used to predict cell type composition within each spatial transcriptomic location via deconvolution. Overall, snPATHO-Seq enables high quality and sensitivity snRNA-Seq from preserved tissue s les, unlocking the vast archives of FFPE tissues and thereby allowing extensive retrospective clinical genomic studies.
Publisher: Wiley
Date: 26-06-2014
Publisher: Elsevier BV
Date: 2014
DOI: 10.1016/J.JHEP.2013.08.012
Abstract: In vertebrates, canonical Hedgehog (Hh) pathway activation requires Smoothened (SMO) translocation to the primary cilium (Pc), followed by a GLI-mediated transcriptional response. In addition, a similar gene regulation occurs in response to growth factors/cytokines, although independently of SMO signalling. The Hh pathway plays a critical role in liver fibrosis/regeneration, however, the mechanism of activation in chronic liver injury is poorly understood. This study aimed to characterise Hh pathway activation upon thioacetamide (TAA)-induced chronic liver injury in vivo by defining Hh-responsive cells, namely cells harbouring Pc and Pc-localised SMO. C57BL/6 mice (wild-type or Ptc1(+/-)) were TAA-treated. Liver injury and Hh ligand athway mRNA and protein expression were assessed in vivo. SMO/GLI manipulation and SMO-dependent/independent activation of GLI-mediated transcriptional response in Pc-positive (Pc(+)) cells were studied in vitro. In vivo, Hh activation was progressively induced following TAA. At the epithelial-mesenchymal interface, injured hepatocytes produced Hh ligands. Progenitors, myofibroblasts, leukocytes and hepatocytes were GLI2(+). Pc(+) cells increased following TAA, but only EpCAM(+)/GLI2(+) progenitors were Pc(+)/SMO(+). In vitro, SMO knockdown/hGli3-R overexpression reduced proliferation/viability in Pc(+) progenitors, whilst increased proliferation occurred with hGli1 overexpression. HGF induced GLI transcriptional activity independently of Pc/SMO. Ptc1(+/-) mice exhibited increased progenitor, myofibroblast and fibrosis responses. In chronic liver injury, Pc(+) progenitors receive Hh ligand signals and process it through Pc/SMO-dependent activation of GLI-mediated transcriptional response. Pc/SMO-independent GLI activation likely occurs in Pc(-)/GLI2(+) cells. Increased fibrosis in Hh gain-of-function mice likely occurs by primary progenitor expansion roliferation and secondary fibrotic myofibroblast expansion, in close contact with progenitors.
Publisher: American Association for the Advancement of Science (AAAS)
Date: 19-02-2021
Abstract: A new technology, SUGAR-seq, enables simultaneous detection of surface glycans, epitopes, transcripts, and TCR repertoire.
Publisher: ZappyLab, Inc.
Date: 27-05-2019
DOI: 10.17504/PROTOCOLS.IO.3EQGJDW
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol is routinely used in the single-cell innovation lab for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and tumors.
Publisher: ZappyLab, Inc.
Date: 28-12-2020
DOI: 10.17504/PROTOCOLS.IO.BQ27MYHN
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other s les types. Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.
Publisher: Bioscientifica
Date: 08-2013
DOI: 10.1530/REP-13-0087
Abstract: Uterine fibroids are the most common benign tumour afflicting women of reproductive age. Despite the large healthcare burden caused by fibroids, there is only limited understanding of the molecular mechanisms that drive fibroid pathophysiology. Although a large number of genes are differentially expressed in fibroids compared with myometrium, it is likely that most of these differences are a consequence of the fibroid presence and are not causal. The aim of this study was to investigate the expression and regulation of NR2F2 and CTNNB1 based on their potential causal role in uterine fibroid pathophysiology. We used real-time quantitative RT-PCR, western blotting and immunohistochemistry to describe the expression of NR2F2 and CTNNB1 in matched human uterine fibroid and myometrial tissues. Primary myometrial and fibroid smooth muscle cell cultures were treated with progesterone and/or retinoic acid (RA) and sonic hedgehog (SHH) conditioned media to investigate regulatory pathways for these proteins. We showed that NR2F2 and CTNNB1 are aberrantly expressed in fibroid tissue compared with matched myometrium, with strong blood vessel-specific localisation. Although the SHH pathway was shown to be active in myometrial and fibroid primary cultures, it did not regulate NR2F2 or CTNNB1 mRNA expression. However, progesterone and RA combined regulated NR2F2 mRNA, but not CTNNB1 , in myometrial but not fibroid primary cultures. In conclusion, we demonstrate aberrant expression and regulation of NR2F2 and CTNNB1 in uterine fibroids compared with normal myometrium, consistent with the hypothesis that these factors may play a causal role uterine fibroid development.
Publisher: ZappyLab, Inc.
Date: 17-01-2021
DOI: 10.17504/PROTOCOLS.IO.BRIZM4F6
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.
Publisher: Wiley
Date: 25-02-2013
DOI: 10.1111/NEP.12022
Abstract: Renal primary cilia are microscopic sensory organelles found on the apical surface of epithelial cells of the nephron and collecting duct. They are based upon a microtubular cytoskeleton, bounded by a specialized membrane, and contain an array of proteins that facilitate their assembly, maintenance and function. Cilium-based signalling is important for the control of epithelial differentiation and has been implicated in the pathogenesis of various cystic kidney diseases and in renal repair. As such, visualizing renal primary cilia and understanding their composition has become an essential component of many studies of inherited kidney disease and mechanisms of epithelial regeneration. Primary cilia were initially identified in the kidney using electron microscopy and this remains a useful technique for the high resolution examination of these organelles. New reagents and techniques now also allow the structure and composition of primary cilia to be analysed in detail using fluorescence microscopy. Primary cilia can be imaged in situ in sections of kidney, and many renal-derived cell lines produce primary cilia in culture providing a simplified and accessible system in which to investigate these organelles. Here we outline microscopy-based techniques commonly used for studying renal primary cilia.
Publisher: Public Library of Science (PLoS)
Date: 26-09-2013
Publisher: Cold Spring Harbor Laboratory
Date: 16-06-2023
DOI: 10.1101/2023.06.16.545221
Abstract: The use of single-cell technologies for clinical applications requires disconnecting s ling from downstream processing steps. Early s le preservation can further increase robustness and reproducibility by avoiding artifacts introduced during specimen handling. We present FixNCut, a methodology for the reversible fixation of tissue followed by dissociation that overcomes current limitations. We applied FixNCut to human and mouse tissues to demonstrate the preservation of RNA integrity, sequencing library complexity, and cellular composition, while diminishing stress-related artifacts. Besides single-cell RNA sequencing, FixNCut is compatible with multiple single-cell and spatial technologies, making it a versatile tool for robust and flexible study designs.
Publisher: EMBO
Date: 23-07-2013
Abstract: Here we report that ILK localizes in the mouse primary cilium, a sensory organelle required for signalling by the Hedgehog (Hh) pathway. Genetic or pharmacological inhibition of ILK blocks ciliary accumulation of the Hh pathway effector smoothened (Smo) and suppresses the induction of Gli transcription factor mRNAs by SHh. Conditional deletion of ILK or Smo also inhibits SHh‐driven activation of Gli2 in the embryonic mouse cerebellum. ILK regulation of Hh signalling probably requires the physical interaction of ILK and Smo in the cilium, and we also show selective cilia‐associated interaction of ILK with β‐arrestin, a known mediator of Smo‐dependent signalling.
Publisher: Wiley
Date: 29-06-2015
DOI: 10.1111/HIS.12735
Publisher: Springer Science and Business Media LLC
Date: 06-02-2017
DOI: 10.1038/NM.4279
Publisher: American Association for the Advancement of Science (AAAS)
Date: 25-07-2018
DOI: 10.1126/SCITRANSLMED.AAT3504
Abstract: Inhibition of activin signaling enhances the efficacy and safety of platinum chemotherapy in lung adenocarcinoma models.
Publisher: EMBO
Date: 03-09-2013
Publisher: ZappyLab, Inc.
Date: 02-03-2021
DOI: 10.17504/PROTOCOLS.IO.BSXHNFJ6
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.
Publisher: Wiley
Date: 28-12-2016
DOI: 10.1002/PATH.4675
Publisher: ZappyLab, Inc.
Date: 05-08-2021
DOI: 10.17504/PROTOCOLS.IO.BW6QPHDW
Abstract: This is a protocol in development, which means it has not yet tested/challenged with multiple s les. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment. In this version I labelled DTT as optional since after further testing I have not seen any difference with or without in snRNA-Seq workflow. Also, I recommend WRB1 for snRNA-Seq workflow only (although still works fine for Multiome) and WRB2 for both snRNA-Seq and Multiome workflows. Since WRB2 is 10x Genomics' recommendation for Multiome I have adopted this for this workflow. I include a discussion about cycling during cDNA .
Publisher: American Association for Cancer Research (AACR)
Date: 14-08-2016
DOI: 10.1158/1078-0432.CCR-15-2840
Abstract: Purpose: Male breast cancer is rare, and its genomic landscape has yet to be fully characterized. Lacking studies in men, treatment of males with breast cancer is extrapolated from results in females with breast cancer. We sought to define whether male breast cancers harbor somatic genetic alterations in genes frequently altered in female breast cancers. Experimental Design: All male breast cancers were estrogen receptor–positive, and all but two were HER2-negative. Fifty-nine male breast cancers were subtyped by immunohistochemistry, and tumor–normal pairs were microdissected and subjected to massively parallel sequencing targeting all exons of 241 genes frequently mutated in female breast cancers or DNA-repair related. The repertoires of somatic mutations and copy number alterations of male breast cancers were compared with that of subtype-matched female breast cancers. Results: Twenty-nine percent and 71% of male breast cancers were immunohistochemically classified as luminal A–like or luminal B–like, respectively. Male breast cancers displayed a heterogeneous repertoire of somatic genetic alterations that to some extent recapitulated that of estrogen receptor (ER)-positive/HER2-negative female breast cancers, including recurrent mutations affecting PIK3CA (20%) and GATA3 (15%). ER-positive/HER2-negative male breast cancers, however, less frequently harbored 16q losses, and PIK3CA and TP53 mutations than ER-positive/HER2-negative female breast cancers. In addition, male breast cancers were found to be significantly enriched for mutations affecting DNA repair–related genes. Conclusions: Male breast cancers less frequently harbor somatic genetic alterations typical of ER-positive/HER2-negative female breast cancers, such as PIK3CA and TP53 mutations and losses of 16q, suggesting that at least a subset of male breast cancers are driven by a distinct repertoire of somatic changes. Given the genomic differences, caution may be needed in the application of biologic and therapeutic findings from studies of female breast cancers to male breast cancers. Clin Cancer Res 22(16) 4045–56. ©2016 AACR.
Publisher: Ovid Technologies (Wolters Kluwer Health)
Date: 07-2016
DOI: 10.1097/PGP.0000000000000243
Abstract: Endometrial carcinomas (ECs) are heterogeneous at the genetic level. Although TP53 mutations are highly recurrent in serous endometrial carcinomas (SECs), these are also present in a subset of endometrioid endometrial carcinomas (EECs). Here, we sought to define the frequency, pattern, distribution, and type of TP53 somatic mutations in ECs by performing a reanalysis of the publicly available data from The Cancer Genome Atlas (TCGA). A total of 228 EECs (n=186) and SECs (n=42) from the TCGA data set, for which an integrated genomic characterization was performed, were interrogated for the presence and type of TP53 mutations, and for mutations in genes frequently mutated in ECs. TP53 mutations were found in 15% of EECs and 88% of SECs, and in 91% of copy-number-high and 35% of polymerase (DNA directed), epsilon, catalytic subunit (POLE) integrative genomic subtypes. In addition to differences in prevalence, variations in the type and pattern of TP53 mutations were observed between histologic types and between integrative genomic subtypes. TP53 hotspot mutations were significantly more frequently found in SECs (46%) than in EECs (15%). TP53 -mutant EECs significantly more frequently harbored a co-occurring PTEN mutation than TP53 -mutant SECs. Finally, a subset of TP53 -mutant ECs (22%) was found to harbor frameshift or nonsense mutations. Given that nonsense and frameshift TP53 mutations result in distinct p53 immunohistochemical results that require careful interpretation, and that EECs and SECs display different patterns, types, and distributions of TP53 mutations, the use of the TP53/ p53 status alone for the differential diagnosis of EECs and SECs may not be sufficient.
Publisher: ZappyLab, Inc.
Date: 28-12-2020
DOI: 10.17504/PROTOCOLS.IO.BQ25MYG6
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other s les types. Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.
Publisher: Wiley
Date: 25-01-2016
DOI: 10.1002/PATH.4672
Publisher: ZappyLab, Inc.
Date: 22-06-2023
DOI: 10.17504/PROTOCOLS.IO.14EGN3XJQL5D/V1
Abstract: This protocol details reversible fixation for cells and tissues for subsequent use in sc/snRNA, sc/snATAC or Multiome. Spatial-Omics compatibility is being validated. For more information check this preprint: ontent/10.1101/2023.06.16.545221v2
Publisher: ZappyLab, Inc.
Date: 28-05-2019
DOI: 10.17504/PROTOCOLS.IO.3FKGJKW
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol is routinely used in the single-cell innovation lab for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and tumors.
Publisher: Elsevier BV
Date: 08-2007
DOI: 10.1016/J.JPLPH.2006.07.002
Abstract: Molecular markers were used to analyze the genomic structure of an euploid series of Eragrostis curvula, obtained after a tetraploid dihaploidization procedure followed by chromosome re-doubling with colchicine. Considerable levels of genome polymorphisms were detected between lines. Curiously, a significant number of molecular markers showed a revertant behavior following the successive changes of ploidy, suggesting that genome alterations were specific and conferred genetic structures characteristic of a given ploidy level. Genuine reversion was confirmed by sequencing. Cluster analysis demonstrated grouping of tetraploids while the diploid was more distantly related with respect to the rest of the plants. Polymorphic revertant sequences involved mostly non-coding regions, although some of them displayed sequence homology to known genes. A revertant sequence corresponding to a P-type adenosine triphosphatase was found to be differentially represented in cDNA libraries obtained from the diploid and a colchiploid, but was not found expressed in the original tetraploid. Transcriptome profiling of inflorescence followed by real-time polymerase chain reaction validation showed 0.34% polymorphic bands between apomictic tetraploid and sexual diploid plants. Several of the polymorphic sequences corresponded to known genes. Possible correlation between the results observed here and a recently reported genome-wide non-Mendelian inheritance mechanism in Arabidopsis thaliana are discussed.
Publisher: Wiley
Date: 04-01-2016
DOI: 10.1111/HIS.12883
Publisher: Springer Science and Business Media LLC
Date: 15-01-2008
DOI: 10.1007/S11103-007-9282-4
Abstract: Eragrostis curvula (Schrad.) Nees is a forage grass native to the semiarid regions of Southern Africa, which reproduces mainly by pseudogamous diplosporous apomixis. A collection of ESTs was generated from four cDNA libraries, three of them obtained from panicles of near-isogenic lines with different ploidy levels and reproductive modes, and one obtained from 12 days-old plant leaves. A total of 12,295 high-quality ESTs were clustered and assembled, rendering 8,864 unigenes, including 1,490 contigs and 7,394 singletons, with a genome coverage of 22%. A total of 7,029 (79.11%) unigenes were functionally categorized by BLASTX analysis against sequences deposited in public databases, but only 37.80% could be classified according to Gene Ontology. Sequence comparison against the cereals genes indexes (GI) revealed 50% significant hits. A total of 254 EST-SSRs were detected from 219 singletons and 35 from contigs. Di- and tri- motifs were similarly represented with percentages of 38.95 and 40.16%, respectively. In addition, 190 SNPs and Indels were detected in 18 contigs generated from 3 to 4 libraries. The ESTs and the molecular markers obtained in this study will provide valuable resources for a wide range of applications including gene identification, genetic mapping, cultivar identification, analysis of genetic ersity, phenotype mapping and marker assisted selection.
Publisher: Wiley
Date: 2000
DOI: 10.1002/1098-2795(200010)57:2<194::AID-MRD11>3.0.CO;2-0
Publisher: Informa UK Limited
Date: 30-08-2011
DOI: 10.3109/08977194.2011.610756
Abstract: The Hedgehog (Hh) pathway is a conserved signalling system essential for embryonic development and for the maintenance of self-renewal pathways in progenitor cells. Mutations that deregulate Hh signalling are directly implicated in basal cell carcinoma and medulloblastoma. The mechanisms of Hh pathway activation in cancers in which no pathway mutations have been identified are less clear, but of great translational significance. Small molecule inhibitors of the pathway, many of which are in early phase clinical trials, may shed further light on this question. Canonical Hh signalling promotes the expression of target genes through the Glioma-associated oncogene (GLI) transcription factors. There is now increasing evidence suggesting that 'non-canonical' Hh signalling mechanisms, some of which are independent of GLI-mediated transcription, may be important in cancer and development. The focus of this review is to summarise some of the known mechanisms of Hh signalling as well as its emerging role in cancer.
Publisher: ZappyLab, Inc.
Date: 09-03-2020
DOI: 10.17504/PROTOCOLS.IO.BDEAI3AE
Abstract: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas aragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Publisher: Cold Spring Harbor Laboratory
Date: 17-06-2022
DOI: 10.1101/2022.06.16.496499
Abstract: Meiotic crossovers are required for accurate chromosome segregation and to produce new allelic combinations. Meiotic crossover numbers are tightly regulated within a narrow range, despite an excess of initiating DNA double-strand breaks. Here, we describe the tumour suppressor FANCM as a meiotic anti-crossover factor in mammals. Crossover analyses with single-gamete and pedigree datasets both reveal a genome-wide increase in crossover frequencies in Fancm -deficient mice. Gametogenesis is heavily perturbed in Fancm loss of function mice, which is consistent with the reproductive defects reported in humans with biallelic FANCM mutations. A portion of the gametogenesis defects can be attributed to the cGAS-STING pathway. Despite the gametogenesis phenotypes in Fancm mutants both sexes were capable of producing offspring. We propose that the anti-crossover function and role in gametogenesis of Fancm are separable and will inform diagnostic pathways for human genomic instability disorders.
Publisher: Springer Science and Business Media LLC
Date: 15-05-2008
DOI: 10.1007/S11103-008-9341-5
Abstract: Apomixis is a route of asexual reproduction through seeds, that progresses in the absence of meiosis and fertilization to generate maternal clonal progenies. Gametophytic apomicts are usually polyploid and probably arose from sexual ancestors through a limited number of mutations in the female reproductive pathway. A differential display analysis was carried out on immature inflorescences of sexual and apomictic tetraploid genotypes of Paspalum notatum, in order to identify genes associated with the emergence of apospory. Analysis of approximately 10,000 transcripts led to the identification of 94 high-quality differentially expressed sequences. Assembling analysis, plus validation, rendered 65 candidate unigenes, organized as 14 contigs and 51 singletons. Thirty-four unigenes were isolated from apomictic plants and 31 from sexual ones. A total of 45 (69.2%) unigenes were functionally categorized. While several of the differentially expressed sequences appeared to be components of an extracellular receptor kinase (ERK) signal transduction cascade, others seemed to participate in a variety of central cellular processes like cell-cycle control, protein turnover, intercellular signalling, transposon activity, transcriptional regulation and endoplasmic reticulum-mediated biosynthesis. In silico mapping revealed that a particular group of five genes silenced in apomictic plants clustered in a rice genomic area syntenic with the region governing apospory in Paspalum notatum and Brachiaria brizantha. Two of these genes mapped within the set of apo-homologues in P. notatum. Four genes previously reported to be controlled by ploidy were identified among those expressed differentially between apomictic and sexual plants. In situ hybridization experiments were performed for selected clones.
Publisher: ZappyLab, Inc.
Date: 05-08-2021
DOI: 10.17504/PROTOCOLS.IO.BW52PG8E
Abstract: This protocol is an adaptation and extension of the 'Frankenstein' protocol – originally developed for nuclei isolation from fresh and frozen tissue for snRNA-Seq – in order to perform snATAC-Seq on the same nuclei prep. It has been successfully applied to fresh, snap/flash and cryopreserved frozen cell lines as well as to tissue derived from solid tumours and other tissues such as pancreas adenocarcinoma (PDAC), breast cancers, pheochromocytomas aragangliomas, normal paraganglia, brain organoids, PDAC organoids, ovary, fallopian tube, mouse brain and sperm using the Chromium Platform (10x Genomics).
Publisher: MDPI AG
Date: 25-05-2017
Publisher: Springer Science and Business Media LLC
Date: 17-07-2017
DOI: 10.1038/NM.4369
Publisher: ZappyLab, Inc.
Date: 03-04-2020
Publisher: ZappyLab, Inc.
Date: 22-01-2021
DOI: 10.17504/PROTOCOLS.IO.BRQVM5W6
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.
Publisher: ZappyLab, Inc.
Date: 21-12-2020
DOI: 10.17504/PROTOCOLS.IO.BQXYMXPW
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol is routinely used in the single-cell innovation lab for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and tumors.
Publisher: Springer Science and Business Media LLC
Date: 13-05-2021
DOI: 10.1038/S41467-021-23044-9
Abstract: Chronic stimulation of CD8 + T cells triggers exhaustion, a distinct differentiation state with diminished effector function. Exhausted cells exist in multiple differentiation states, from stem-like progenitors that are the key mediators of the response to checkpoint blockade, through to terminally exhausted cells. Due to its clinical relevance, there is substantial interest in defining the pathways that control differentiation and maintenance of these subsets. Here, we show that chronic antigen induces the anergy-associated transcription factor EGR2 selectively within progenitor exhausted cells in both chronic LCMV and tumours. EGR2 enables terminal exhaustion and stabilizes the exhausted transcriptional state by both direct EGR2-dependent control of key exhaustion-associated genes, and indirect maintenance of the exhausted epigenetic state. We show that EGR2 is a regulator of exhaustion that epigenetically and transcriptionally maintains the differentiation competency of progenitor exhausted cells.
Publisher: ZappyLab, Inc.
Date: 11-05-2021
DOI: 10.17504/PROTOCOLS.IO.BUXKNXKW
Abstract: This is a protocol in development, which means it has not yet tested/challenged with multiple s les. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment.
Publisher: ZappyLab, Inc.
Date: 12-01-2021
DOI: 10.17504/PROTOCOLS.IO.BRDCM22W
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, and is an extension of the Frankenstein (S.O.F means Son Of Frankenstein). Developed to prepare nuclei isolates from fresh and frozen material of small-to-large sizes. The good thing is that it does not uses FACS but OptiPrep® discontinuous gradient to remove debris. It is the alternative protocol when FACS is not available.
Publisher: Wiley
Date: 30-03-2017
DOI: 10.1002/PATH.4883
Publisher: Wiley
Date: 29-07-2015
DOI: 10.1002/PATH.4566
Publisher: ZappyLab, Inc.
Date: 28-12-2020
DOI: 10.17504/PROTOCOLS.IO.BQ24MYGW
Abstract: This protocol is the result of the combination of various nuclei isolation protocols for single cell RNA-seq experiments using droplet-based methods, hence the name Frankenstein. Developed to prepare nuclei isolates from small s le sizes (as little as a grain of rice), this protocol uses FACS to identify cell subpopulations based on ploidy (e.g. tumor versus stroma), to ensure that nuclei suspensions are not clumped, and to remove any debris, especially ambient RNA, to help reduce background. The reference protocols can be found in the following papers: Hu, et al., Habib, et al. (2016), Habib, et al. (2017), Lake, et al., and Lacar, et al. This protocol has been validated in the Single-Cell Innovation Lab (UMCCR) and other labs worldwide for single nuclei experiments using 10x Genomics technologies. The protocol has been demonstrated to work successfully with fresh, snap/flash frozen, cryopreserved cells, and cell lines, as well as various solid cancers: pancreas, pheochromocytomas, paragangliomas, breast cancer, lymphoma, xenografts and other s les types. Cardiomyocytes can be really difficult to prepare but we have successfully prepare this. Get in touch for some tips.
Publisher: Springer Science and Business Media LLC
Date: 10-2014
Publisher: Springer Science and Business Media LLC
Date: 22-05-2015
Publisher: Public Library of Science (PLoS)
Date: 10-02-2017
Publisher: ZappyLab, Inc.
Date: 15-07-2021
DOI: 10.17504/PROTOCOLS.IO.BWMBPC2N
Abstract: This is a protocol in development, which means it has not yet tested/challenged with multiple s les. So, please make sure you take it for a test drive before committing to it. Once you do please share your experience with me via email, Twitter or as a comment. In this version, DTT, Digitonin and Tween-20 were included in the buffers.
Publisher: Springer Science and Business Media LLC
Date: 20-05-2014
DOI: 10.1186/BCR3658
Location: United States of America
No related grants have been discovered for Luciano Martelotto.