This document describes a study investigating compartment-specific microRNA expression profiles in normal human colons and tumor counterparts. Tissue samples from normal colons were microdissected to enrich for different crypt compartments, including whole mucosa, top crypt, and basal crypt. High-throughput quantitative PCR was used to analyze the expression of 677 microRNAs across these samples. Statistical analysis identified microRNAs differentially expressed between compartments that are involved in intestinal epithelial development. Five candidate microRNAs were selected for further validation using individual quantitative PCR on formalin-fixed samples from 42 normal-tumor pairs and 16 top-basal crypt pairs from normal tissue. The aim is to identify microRNA markers that define colonic stem cell niches and
Dr. Talita Resende - Organoids as an invitro model for enteric diseasesJohn Blue
Organoids as an invitro model for enteric diseases - Dr. Talita Resende, from the 2018 Allen D. Leman Swine Conference, September 15-18, 2018, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2018-leman-swine-conference-material
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
The use of genetic engineering technology in animals has been associated with ethical issues, some of which relate to animal welfare. Discuss examples of genetically engineering animals and evaluate the ethical concerns of genetic engineering.
Dr. Talita Resende - Organoids as an invitro model for enteric diseasesJohn Blue
Organoids as an invitro model for enteric diseases - Dr. Talita Resende, from the 2018 Allen D. Leman Swine Conference, September 15-18, 2018, St. Paul, Minnesota, USA.
More presentations at http://www.swinecast.com/2018-leman-swine-conference-material
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
The use of genetic engineering technology in animals has been associated with ethical issues, some of which relate to animal welfare. Discuss examples of genetically engineering animals and evaluate the ethical concerns of genetic engineering.
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
Genes and Tissue Culture Assignment Presentation (Group 3)Lim Ke Wen
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant. Discuss the potential of using three dimensional cell cultures for anti-cancer drug screening.
Sox2 suppresses the invasiveness of breast cancer cells via a mechanism that ...Enrique Moreno Gonzalez
Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant. Discuss the potential of using three dimensional cell cultures for anti-cancer drug screening.
Characterizing the Microbiome of Neonates and Infants to explore associations...QIAGEN
This webinar slidedeck will focus on the acquisition and development of the preterm gut microbiome from birth and following discharge from intensive care. Specifically, the discussion will be around the association of the gut microbiome with necrotizing enterocolitis (NEC) and late onset sepsis (LOS), as well as the impact of birth mode. The other discussion points will be the analysis of multi-omic datasets, including the analysis of the airway microbiome and metabolome in infants hospitalized with bronchiolitis.
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal model. In comparison to 2D cultures, discuss the potential of using 3D in vitro models for drug efficiency testing.
Conferencia de la Dra. Ana María Roa, Bióloga Molecular, sobre Epigenética, impartida en la Universidad Popular Carmen de Michelena de Tres Cantos el 1 de marzo de 2013.
Más información en:
http://www.universidadpopularc3c.es/index.php/actividades/conferencias/event/448-conferencia-una-revision-de-los-conocimientos-fundamentales-de-la-biologia-de-la-celula-la-epigenetica
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Femoral Head Bone vs Acetabular Subchondral Bone: Selecting the Optimal Anat...remedypublications2
Mesenchymal Stromal Cells (MSC) have a great importance for the field of regenerative
medicine. However, there is high variability in existing protocols for MSC
in vitro
expansion, which
can lead to low reproducibility of pre-clinical studies and, even more critically, the reduced safety
of patients undergoing clinical trials. Although bone marrow is one of the most important sources
for the isolation and
in vitro
culture of MSC, the preferred anatomical location for obtaining bone
marrow is often unclear, and this information is relevant for the interpretation of results obtained
from preclinical and clinical trials.
A normal cell can be transformed into a cancerous cell. Discuss the therapeutic strategies that are employed to target the cellular transformation process for cancer prevention and treatment.
Genes and Tissue Culture Assignment Presentation (Group 3)Lim Ke Wen
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant. Discuss the potential of using three dimensional cell cultures for anti-cancer drug screening.
Sox2 suppresses the invasiveness of breast cancer cells via a mechanism that ...Enrique Moreno Gonzalez
Sox2, an embryonic stem cell marker, is aberrantly expressed in a subset of breast cancer (BC). While the aberrant expression of Sox2 has been shown to significantly correlate with a number of clinicopathologic parameters in BC, its biological significance in BC is incompletely understood.
The culture of cells in two dimensions does not reproduce the histological characteristics of a tissue for informative or useful study. Growing cells as three-dimensional (3D) models more analogous to their existence in vivo may be more clinically relevant. Discuss the potential of using three dimensional cell cultures for anti-cancer drug screening.
Characterizing the Microbiome of Neonates and Infants to explore associations...QIAGEN
This webinar slidedeck will focus on the acquisition and development of the preterm gut microbiome from birth and following discharge from intensive care. Specifically, the discussion will be around the association of the gut microbiome with necrotizing enterocolitis (NEC) and late onset sepsis (LOS), as well as the impact of birth mode. The other discussion points will be the analysis of multi-omic datasets, including the analysis of the airway microbiome and metabolome in infants hospitalized with bronchiolitis.
Osteoblasts remotely supply lung tumors with cancer-promoting SiglecFhigh neu...Gul Muneer
Systemic cross-talk between lung tumors and bones
Bone marrow–derived myeloid cells can accumulate within tumors and foster
cancer outgrowth. Local immune-neoplastic interactions have been intensively
investigated, but the contribution of the systemic host environment to tumor growth
remains poorly understood. Here, we show in mice and cancer patients (n = 70) that
lung adenocarcinomas increase bone stromal activity in the absence of bone
metastasis. Animal studies reveal that the cancer-induced bone phenotype involves
bone-resident osteocalcin-expressing (Ocn+) osteoblastic cells. These cells promote
cancer by remotely supplying a distinct subset of tumor-infiltrating SiglecFhigh
neutrophils, which exhibit cancer-promoting properties. Experimentally reducing
Ocn+ cell numbers suppresses the neutrophil response and lung tumor outgrowth.
These observations posit osteoblasts as remote regulators of lung cancer and
identify SiglecFhigh neutrophils as myeloid cell effectors of the osteoblast-driven
protumoral response
Development of cancer therapeutics is often carried out in 2D cultures prior to testing on animal model. In comparison to 2D cultures, discuss the potential of using 3D in vitro models for drug efficiency testing.
Conferencia de la Dra. Ana María Roa, Bióloga Molecular, sobre Epigenética, impartida en la Universidad Popular Carmen de Michelena de Tres Cantos el 1 de marzo de 2013.
Más información en:
http://www.universidadpopularc3c.es/index.php/actividades/conferencias/event/448-conferencia-una-revision-de-los-conocimientos-fundamentales-de-la-biologia-de-la-celula-la-epigenetica
BACKGROUND: Sequential Epstein-Barr virus (EBV)–positive B cell lymphoma to the initial diagnosis of angioimmunoblastic T cell lymphoma (AITL) is very rare, the exact mechanism and standard therapy of which is still being explored. CASE: A 50-year-old man was admitted to our hospital in January 2014 with a three-week history of enlargement of multiple lymph nodes. His initial pathological evaluation indicated AILT. The reactivation of EBV was observed during the immunosuppression therapy for AITL, accompanied by onset of subcutaneous nodules proven to be EBV-positive diffuse large B cell lymphoma (DLBCL) based on the pathological findings of rebiopsy. The patient was successfully treated with chidamide, a histone deacetylase (HDAC) inhibitor, and rituximab.
Conclusion: The sufficient surveillance for serum EBV and repeat biopsy is necessary for patients with AITL, and this treatment modality may become an active option.
Keywords: angioimmunoblastic T cell lymphoma, Epstein-Barr virus, HDAC inhibitor, non-Hodgkin lymphoma, peripheral T cell lymphoma
Femoral Head Bone vs Acetabular Subchondral Bone: Selecting the Optimal Anat...remedypublications2
Mesenchymal Stromal Cells (MSC) have a great importance for the field of regenerative
medicine. However, there is high variability in existing protocols for MSC
in vitro
expansion, which
can lead to low reproducibility of pre-clinical studies and, even more critically, the reduced safety
of patients undergoing clinical trials. Although bone marrow is one of the most important sources
for the isolation and
in vitro
culture of MSC, the preferred anatomical location for obtaining bone
marrow is often unclear, and this information is relevant for the interpretation of results obtained
from preclinical and clinical trials.
Proteogenomic analysis of human colon cancer reveals new therapeutic opportun...Gul Muneer
We performed the first proteogenomic study on a prospectively collected colon cancer cohort. Comparative proteomic and phosphoproteomic analysis of paired tumor and normal adjacent tissues produced a catalog of colon cancer-associated proteins and phosphosites, including known and putative new biomarkers, drug targets, and cancer/testis antigens. Proteogenomic integration not only prioritized genomically inferred targets, such as copy-number drivers and mutation-derived neoantigens, but also yielded novel findings. Phosphoproteomics data associated Rb phosphorylation with increased proliferation and decreased apoptosis in colon cancer, which explains why this classical tumor suppressor is amplified in colon tumors and suggests a rationale for targeting Rb phosphorylation in colon cancer. Proteomics identified an association between decreased CD8 T cell infiltration and increased glycolysis in microsatellite instability-high (MSI-H) tumors, suggesting glycolysis as a potential target to overcome the resistance of MSI-H tumors to immune checkpoint blockade. Proteogenomics presents new avenues for biological discoveries and therapeutic development.
El lunes 23 de octubre de 2017 celebramos una jornada en la Fundación Ramón Areces sobre Microbiota Intestinal: Implicaciones en la Salud y Enfermedad.
Comparative analysis of gene regulation in mouse rat and humanconstantina mylona
Which is the most suitable model mouse or rat even in the use of the latest gene editing tool-CRISPR/Cas 9 ?
Final Project presentaion on BSc Human Biology
A tale of scale & speed: How the US Navy is enabling software delivery from l...sonjaschweigert1
Rapid and secure feature delivery is a goal across every application team and every branch of the DoD. The Navy’s DevSecOps platform, Party Barge, has achieved:
- Reduction in onboarding time from 5 weeks to 1 day
- Improved developer experience and productivity through actionable findings and reduction of false positives
- Maintenance of superior security standards and inherent policy enforcement with Authorization to Operate (ATO)
Development teams can ship efficiently and ensure applications are cyber ready for Navy Authorizing Officials (AOs). In this webinar, Sigma Defense and Anchore will give attendees a look behind the scenes and demo secure pipeline automation and security artifacts that speed up application ATO and time to production.
We will cover:
- How to remove silos in DevSecOps
- How to build efficient development pipeline roles and component templates
- How to deliver security artifacts that matter for ATO’s (SBOMs, vulnerability reports, and policy evidence)
- How to streamline operations with automated policy checks on container images
LF Energy Webinar: Electrical Grid Modelling and Simulation Through PowSyBl -...DanBrown980551
Do you want to learn how to model and simulate an electrical network from scratch in under an hour?
Then welcome to this PowSyBl workshop, hosted by Rte, the French Transmission System Operator (TSO)!
During the webinar, you will discover the PowSyBl ecosystem as well as handle and study an electrical network through an interactive Python notebook.
PowSyBl is an open source project hosted by LF Energy, which offers a comprehensive set of features for electrical grid modelling and simulation. Among other advanced features, PowSyBl provides:
- A fully editable and extendable library for grid component modelling;
- Visualization tools to display your network;
- Grid simulation tools, such as power flows, security analyses (with or without remedial actions) and sensitivity analyses;
The framework is mostly written in Java, with a Python binding so that Python developers can access PowSyBl functionalities as well.
What you will learn during the webinar:
- For beginners: discover PowSyBl's functionalities through a quick general presentation and the notebook, without needing any expert coding skills;
- For advanced developers: master the skills to efficiently apply PowSyBl functionalities to your real-world scenarios.
SAP Sapphire 2024 - ASUG301 building better apps with SAP Fiori.pdfPeter Spielvogel
Building better applications for business users with SAP Fiori.
• What is SAP Fiori and why it matters to you
• How a better user experience drives measurable business benefits
• How to get started with SAP Fiori today
• How SAP Fiori elements accelerates application development
• How SAP Build Code includes SAP Fiori tools and other generative artificial intelligence capabilities
• How SAP Fiori paves the way for using AI in SAP apps
Encryption in Microsoft 365 - ExpertsLive Netherlands 2024Albert Hoitingh
In this session I delve into the encryption technology used in Microsoft 365 and Microsoft Purview. Including the concepts of Customer Key and Double Key Encryption.
Securing your Kubernetes cluster_ a step-by-step guide to success !KatiaHIMEUR1
Today, after several years of existence, an extremely active community and an ultra-dynamic ecosystem, Kubernetes has established itself as the de facto standard in container orchestration. Thanks to a wide range of managed services, it has never been so easy to set up a ready-to-use Kubernetes cluster.
However, this ease of use means that the subject of security in Kubernetes is often left for later, or even neglected. This exposes companies to significant risks.
In this talk, I'll show you step-by-step how to secure your Kubernetes cluster for greater peace of mind and reliability.
zkStudyClub - Reef: Fast Succinct Non-Interactive Zero-Knowledge Regex ProofsAlex Pruden
This paper presents Reef, a system for generating publicly verifiable succinct non-interactive zero-knowledge proofs that a committed document matches or does not match a regular expression. We describe applications such as proving the strength of passwords, the provenance of email despite redactions, the validity of oblivious DNS queries, and the existence of mutations in DNA. Reef supports the Perl Compatible Regular Expression syntax, including wildcards, alternation, ranges, capture groups, Kleene star, negations, and lookarounds. Reef introduces a new type of automata, Skipping Alternating Finite Automata (SAFA), that skips irrelevant parts of a document when producing proofs without undermining soundness, and instantiates SAFA with a lookup argument. Our experimental evaluation confirms that Reef can generate proofs for documents with 32M characters; the proofs are small and cheap to verify (under a second).
Paper: https://eprint.iacr.org/2023/1886
The Art of the Pitch: WordPress Relationships and SalesLaura Byrne
Clients don’t know what they don’t know. What web solutions are right for them? How does WordPress come into the picture? How do you make sure you understand scope and timeline? What do you do if sometime changes?
All these questions and more will be explored as we talk about matching clients’ needs with what your agency offers without pulling teeth or pulling your hair out. Practical tips, and strategies for successful relationship building that leads to closing the deal.
Epistemic Interaction - tuning interfaces to provide information for AI supportAlan Dix
Paper presented at SYNERGY workshop at AVI 2024, Genoa, Italy. 3rd June 2024
https://alandix.com/academic/papers/synergy2024-epistemic/
As machine learning integrates deeper into human-computer interactions, the concept of epistemic interaction emerges, aiming to refine these interactions to enhance system adaptability. This approach encourages minor, intentional adjustments in user behaviour to enrich the data available for system learning. This paper introduces epistemic interaction within the context of human-system communication, illustrating how deliberate interaction design can improve system understanding and adaptation. Through concrete examples, we demonstrate the potential of epistemic interaction to significantly advance human-computer interaction by leveraging intuitive human communication strategies to inform system design and functionality, offering a novel pathway for enriching user-system engagements.
UiPath Test Automation using UiPath Test Suite series, part 4DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 4. In this session, we will cover Test Manager overview along with SAP heatmap.
The UiPath Test Manager overview with SAP heatmap webinar offers a concise yet comprehensive exploration of the role of a Test Manager within SAP environments, coupled with the utilization of heatmaps for effective testing strategies.
Participants will gain insights into the responsibilities, challenges, and best practices associated with test management in SAP projects. Additionally, the webinar delves into the significance of heatmaps as a visual aid for identifying testing priorities, areas of risk, and resource allocation within SAP landscapes. Through this session, attendees can expect to enhance their understanding of test management principles while learning practical approaches to optimize testing processes in SAP environments using heatmap visualization techniques
What will you get from this session?
1. Insights into SAP testing best practices
2. Heatmap utilization for testing
3. Optimization of testing processes
4. Demo
Topics covered:
Execution from the test manager
Orchestrator execution result
Defect reporting
SAP heatmap example with demo
Speaker:
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Enhancing Performance with Globus and the Science DMZGlobus
ESnet has led the way in helping national facilities—and many other institutions in the research community—configure Science DMZs and troubleshoot network issues to maximize data transfer performance. In this talk we will present a summary of approaches and tips for getting the most out of your network infrastructure using Globus Connect Server.
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
State of ICS and IoT Cyber Threat Landscape Report 2024 previewPrayukth K V
The IoT and OT threat landscape report has been prepared by the Threat Research Team at Sectrio using data from Sectrio, cyber threat intelligence farming facilities spread across over 85 cities around the world. In addition, Sectrio also runs AI-based advanced threat and payload engagement facilities that serve as sinks to attract and engage sophisticated threat actors, and newer malware including new variants and latent threats that are at an earlier stage of development.
The latest edition of the OT/ICS and IoT security Threat Landscape Report 2024 also covers:
State of global ICS asset and network exposure
Sectoral targets and attacks as well as the cost of ransom
Global APT activity, AI usage, actor and tactic profiles, and implications
Rise in volumes of AI-powered cyberattacks
Major cyber events in 2024
Malware and malicious payload trends
Cyberattack types and targets
Vulnerability exploit attempts on CVEs
Attacks on counties – USA
Expansion of bot farms – how, where, and why
In-depth analysis of the cyber threat landscape across North America, South America, Europe, APAC, and the Middle East
Why are attacks on smart factories rising?
Cyber risk predictions
Axis of attacks – Europe
Systemic attacks in the Middle East
Download the full report from here:
https://sectrio.com/resources/ot-threat-landscape-reports/sectrio-releases-ot-ics-and-iot-security-threat-landscape-report-2024/
Climate Impact of Software Testing at Nordic Testing DaysKari Kakkonen
My slides at Nordic Testing Days 6.6.2024
Climate impact / sustainability of software testing discussed on the talk. ICT and testing must carry their part of global responsibility to help with the climat warming. We can minimize the carbon footprint but we can also have a carbon handprint, a positive impact on the climate. Quality characteristics can be added with sustainability, and then measured continuously. Test environments can be used less, and in smaller scale and on demand. Test techniques can be used in optimizing or minimizing number of tests. Test automation can be used to speed up testing.
DevOps and Testing slides at DASA ConnectKari Kakkonen
My and Rik Marselis slides at 30.5.2024 DASA Connect conference. We discuss about what is testing, then what is agile testing and finally what is Testing in DevOps. Finally we had lovely workshop with the participants trying to find out different ways to think about quality and testing in different parts of the DevOps infinity loop.
Pushing the limits of ePRTC: 100ns holdover for 100 daysAdtran
At WSTS 2024, Alon Stern explored the topic of parametric holdover and explained how recent research findings can be implemented in real-world PNT networks to achieve 100 nanoseconds of accuracy for up to 100 days.
Pushing the limits of ePRTC: 100ns holdover for 100 days
Compartment specific micro rna expression profiles (poster) poster
1. Compartment-specific MicroRNA Expression Profiles
in Colorectal Cancer
Supervisors: Dr. TL Chan, Prof. SY Leung
Department of Pathology,
The University of Hong Kong
Jackie Lau
2. Epithelial stem cells within human colon crypts
Confocal imaging of human colon crypts
Putative intestinal stem cell marker
Musashi-1 (green) expression
Brittan & Wright (2004) Gut
Epithelial stem cell-pericryptal
microenvironment (stem cell niche) interaction
is crucial for homeostatic balance between
epithelial cell self-renewal, cell division, cell
differentiation and lineage determination
Blanpain et al. (2007) Cell
Spradling et al. (2001) Nature
Modified from Spradling et al. (2001) Nature
3. Gene expression analysis of human colonic epithelial cell differentiation
Kosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology
Identified > 900 cDNA clones differentially expressed between top and basal crypts
in normal human colons
Many involved in molecular pathways common to intestinal epithelial cell
development and neoplastic transformation
Modified from Kosinski & Li et al. (2007) PNAS
4. Resemblance between stem cell and cancer
Immortality
Self-renewal, clonal and heterogeneous properties
Signals from surrounding microenvironment
Common signaling pathways for regulation:
Wnt, Bone morphogenetic protein (BMP), Notch, Hedgehog
Colorectal Cancer (CRC)
Tumor cells demonstrate “crypt-progenitor phenotype”, i.e.
phenotypic resemblance to basal crypt
Deregulation of multiple signaling pathways mediating normal
intestinal epithelial development
Li, V.S. Profiling of gene expression changes in human colon crypt maturation and study on their dysregulation
in tumorigenesis. PhD Thesis, The University of Hong Kong: Hong Kong, January 2008.
5. Project Aim
Based on the hypothesis that perturbation of normal colon
maturation during tumorigenesis is cell type-dependent, the
present study aims to investigate compartment-specific microRNA
(miRNA) expression in normal human colons with tumor
counterparts.
6. Experimental methodology I
Real-time quantitative reverse transcription (qRT)-PCR
TaqMan® Human MicroRNA Array Set v2.0 Low Density Array (LDA)
Global expressions of 677 human miRNAs (miRBase v10)
Duplicate quantification for each sample
Frozen colonic tissues
Tumors
Normal counterparts: microdissection-partitioned into
enrichments in whole mucosa, epithelium, stroma, top crypt
and basal crypt
7. Crypt isolation from Stromal fraction
Protocol modified by Dr. Helen Yan from our lab based on Strater et al. (1996)
Gastroenterology; Whitehead et al. (1999) Gastroenterology; Hofmann et al. (2007)
Gastroenterology
8. Microdissection into Samples enriched in: whole mucosa*, top crypt, basal crypt*
* Mucularis mucosae and muscles removed
Kosinski & Li et al. (2007) PNAS; Li et al. (2009) Gastroenterology
9. Top Crypt harvested Basal Crypt harvested
* Muscularis mucosa and muscles excluded
10. Experimental methodology II
Individual qRT-PCR validation of miRNAs differentially
expressed across different compartments
TaqMan® MicroRNA Assay, Human
Triplicate quantification for each sample
Formalin-fixed, paraffin-embedded (FFPE) tissues
Tumors
Normal counterparts: microdissection-partitioned into
enrichments in whole mucosa, top crypt and basal crypt
Note: Concordance between qRT-PCR data from frozen tissues vs
parallel FFPE samples in published reports and in-house data
11. Results
qRT-PCR detection for Purity of isolated Crypts and Stroma in Normal Colons
Relative quantification by Comparative Ct method Livak & Schmittgen (2001) Methods
Obtained “near-mesenchyme-free Crypts” &
“near-epithelium-free Stroma” fractions
Perreault & Beaulieu
(1998) Exp. Cell Res.
12. Reproducibility between LDA Duplicates
R² = 0.970
15
20
25
30
35
40
45
15 20 25 30 35 40 45
Replicate2Ct
Replicate 1 Ct
Ct values of all miRNAs on TaqMan® Human MicroRNA Array Set v2.0
13. Selection of Endogenous Control for Normalization
15
20
25
30
35
RNU6B
RNU48
RNU44
RNU43
RNU24
MammU6
Ct values of Endogenous Controls
AverageCt
Samples
6 TaqMan® MicroRNA Assay endogenous controls on each LDA set
RNU48
Lowest Ct variation across samples
Ct values within reasonable range (21 – 25)
14. Statistical Analysis of LDA data
TM4: MeV (MultiExperiment Viewer) v4.4 Saeed et al. (2003) Biotechniques
Multiple statistical and computational algorithms identified distinct miRNA
expression patterns across compartments
Citical p-value = 0.01
75% amongst 29 miRNAs described to be associated with CRC show
agreement in T:N expression ratio to published data
Sensitivity and reliability of high-throughput LDA detection
miRNA expression profiles
documented in CRC
Lu et al. (2005) Nature
Bandres et al. (2006) Mol. Cancer
Schetter et al. (2009) JAMA
Aslam et al. (2009) Br. J. Surg.
15. In silico Target Prediction and Functional Enrichment Analysis
28 miRNAs D.E. between Top vs Basal crypts
Pathways involved in intestinal epithelial development along colon crypt axis
16. Candidate miRNAs for Individual qRT-PCR validation
Results collectively suggest D.E. miRNA list
High concordance with published data
High involvement in intestinal epithelial development
and CRC tumorigenesis
Selected 5 candidate miRNAs with robustness across various statistical
and functional analyses
Formalin-fixed, paraffin-embedded (FFPE) tissues
42 Normal vs CRC Tumor pairs
16 Top vs Basal Crypt pairs (Normal)
17. Individual qRT-PCR validation of 5 candidate miRNAs
42 Normal vs Tumor pairs of FFPE Colonic tissues
miR-A miR-B miR-C miR-D miR-E
18. Individual qRT-PCR validation of 5 candidate miRNAs
16 Top vs Basal Crypt pairs (Normal)
* Log2 Fold Expression calibrated to Whole mucosa counterparts
miR-A miR-B miR-C miR-D miR-E
19. Conclusion
Protocols developed for isolation of compartments and high-
throughput detection effective, as evident in data reproducibility and
concordance with literature
Fundamental support for a hierarchy of miRNA expression along the
colonic proliferation-differentiation axis
Foundation to identify candidate marker miRNAs that define colonic
epithelial stem/progenitor cell niche
Based on resemblance between stem cell and cancer, such miRNA
markers may constitute targets for mutation and malignant
transformation, thus provide novel way to elucidate clonal origin of CRC
20. Current Work
Characterization of candidate miRNAs in CRC cell lines and a larger
number of clinical samples
Functional Studies in CRC lines
Knockdown/Overexpression of candidate miRNAs
To elucidate biological roles of miRNAs
Monitoring phenotypic changes (cell proliferation, cell
differentiation, apoptosis) in transfected cells
Validate bioinformatically predicted mRNA targets
21. Acknowledgement
Department of Pathology, HKU
Dr. Chris TL Chan
Prof. SY Leung
Dr. Helen HN Yan
Miss Annie SY Chan
Miss Grace Cheng
Dr. Vivian Li
Department of Pathology,
St. Paul’s Hospital
Prof. ST Yuen
Department of Surgery, HKU
Prof. WL Law
22. Large intestine: Mucosa and Musculature in Humans
Adopted from Encyclopedia Britannica (http://www.britannica.com/eb/art-68639)
23. Why miRNA?
Gene expression profile – done
Kosinski & Li et al. (2007) Gene expression patterns of human colon tops and basal crypts and BMP antagonists
as intestinal stem cell niche factors. PNAS, 104(39):15418-23.
Li et al. (2009) Frequent inactivation of axon guidance molecule RGMA in human colon cancer through genetic
and epigenetic mechanisms. Gastroenterology, 137:176-187.
Diagnostic and prognostic potential of miRNAs implicated in
pathogenesis of CRC, either as oncogenes or tumor suppressors.
Potential to regulate multiple target genes or potentially an entire
pathway; networks of several different miRNAs may act cooperatively
in the translational regulation of individual mRNAs to elicit a
biological phenotype
24. TaqMan® Human MicroRNA LDA
Fresh Tumors Flash freeze Cryosectioning
Fresh Normal counterparts
Crypt isolation from Stromal fraction
Flash freeze Cryosectioning Microdissection into
Samples enriched in: whole mucosa*, top crypt, basal crypt*
* Mucularis mucosae and muscles removed
Experimental methodology – Sample preparation
Editor's Notes
Confocal imaging of human colon cryptsEpithelial stem cell-pericryptal microenvironment (stem cell niche) interaction is crucial for homeostatic balance between epithelial cell self-renewal, cell division, cell differentiation and lineage determination
The second commonest cancer in Hong Kong
Comprehensive coverage of Sanger miRBase v10
Note: Concordance between qRT-PCR data generated using frozen tissues vs parallel FFPE samples demonstrated in published reports and in-house data
Isolation of epithelial lining from mesenchyme in Normal colonsRelative quantification by Comparative Ct method Livak & Schmittgen (2001) MethodsEndogenous Control: GAPDH Calibrator sample: whole mucosa counterparts
X Y Scatter plotCt values from Replicate 1 (x-axis) against Ct values from Replicate 2 (y-axis)A representative example: Minimal Ct variation for Ct < 30 Replicate 1 vs Replicate 2:
6 TaqMan® MicroRNA Assay endogenous controls on each LDA set6 small RNAs on each LDA set Lowest variation in endogenous expression across all samples – high stability Ct values within reasonable range (21 – 25 for RNU48)
Multiple statistical and computational algorithms identified distinct miRNA expression patterns across compartmentsAmong the 29 miRNAs described to be associated with CRC, 75% show directional agreement in T:N expressions between our data and published dataSensitivity and reliability of the high-throughput LDA detection approach
In silico Target Prediction and Functional Enrichment Analysisof predicted targets
Selection of candidate miRNAs for downstream workResults collectively suggest D.E. miRNA lists generated from LDA dataHigh potential involvement in intestinal epithelial development and CRC tumorigenesis – novel miRNAs worth looking into
To elucidate biological roles of miRNAs Monitoring phenotypic changes (cell proliferation, cell differentiation, apoptosis) in transfected cells
Department of Pathology, HKUDr. Chris TL ChanProf. SY LeungDr. Helen HN YanMiss Annie SY ChanDepartment of Pathology, St. Paul’s HospitalDr. ST YuenDepartment of Surgery, HKUProf. WL Law
; networks of several different miRNAs may act cooperatively in the translational regulation of individual mRNAs to elicit a biological phenotype
Crypt/Epithelial isolation from Stroma/Mesenchymal fractionPurity of isolated crypt and stromal portions determined by tissue-specific markers Perreault & Beaulieu (1998) Exp. Cell Res.Real-time qRT-PCR using TaqMan® Human MicroRNA LDA