WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Functional analysis by mRNA knockdown using siRNAs is now routine in many molecular biology labs. However, many RNAi experiments fail due to diversion from simple, good practices. This webinar will review the steps leading to successful siRNA experiments, including:
- Understanding the target transcript
- siRNA selection
- Choosing the cell type
- Validating the assay
- Including appropriate biological controls
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
WDR7 up-regulation upon knocking down of neighboring noncoding RNA using siRN...Vahid Erfani-Moghadam
Objective(s): Breast cancer is the second leading cause of cancer death in females. Understanding molecular mechanisms in cancer cells compared with normal cells is crucial for diagnostic and therapeutic strategies. Long intergenic non-protein coding RNA, a regulator of reprogramming (lincRNA-RoR) is a noncoding RNA which initially was detected in induced pluripotent stem cells, and it has an important role in cell reprogramming and highly expressed in breast cancer cells. A key point in successful gene silencing is the usage of siRNA delivery system that is safe and efficient. Materials and Methods: In this study, the fifth-generation of PAMAM dendrimer is used as a nanocarrier for entering siRNA molecules for gene silencing of lincRNA-RoR. WDR7 is the gene encoding adjacent of lincRNA-RoR, which has an important role in apoptosis and cell cycle. Gel retardation assay was used to find the best Negative/Positive (N/P) molar charge ratio of siRNA- PAMAM transfected into MDA-MB 231 cells. MTT assay was performed 24 hr after transfection revealed the IC50 value (half maximal inhibitory concentrations) about 100 nanomolar for lincRNA-ROR siRNA. Results: The lincRNA-RoR and WDR7 gene expression changes were evaluated by real-time PCR after siRNA treatment and showed an increase in the gene expression of WDR7. Conclusion: This study showed that PAMAM dendrimer G5/ siRNA could be a useful system delivery for future gene therapy approaches.
Functional analysis by mRNA knockdown using siRNAs is now routine in many molecular biology labs. However, many RNAi experiments fail due to diversion from simple, good practices. This webinar will review the steps leading to successful siRNA experiments, including:
- Understanding the target transcript
- siRNA selection
- Choosing the cell type
- Validating the assay
- Including appropriate biological controls
Los días 20 y 21 de octubre de 2016, la Fundacion Ramón Areces organizó un simposio internacional para analizar las 'Enfermedades raras de la piel: de la clínica al gen y viceversa'. El doctor Fernando Larcher Laguzzi, del CIEMAT-Universidad Carlos III de Madrid-IIS Fundación Jiménez Díaz, ejerció de coordinador.
Making genome edits in mammalian cellsChris Thorne
Looking at the kind of modifications that can be made in mammalian cells, and how at Horizon moving to a haploid model system has significantly improved efficiency of both editing and validation
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
El martes 26 de septiembre del 2017 organizamos en la Fundación Ramón Areces un Simposio Internacional sobre nuevas perspectivas en la investigación sobre el cáncer. En colaboración con el Centro Nacional de Investigaciones Oncológicas (CNIO) y Weizmann Institute for Science.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. In a recent webinar, "New RNA Tools for Optimized CRISPR/Cas 9 Genome Editing", we presented how we developed the Alt-R™ CRISPR-Cas9 System for genome editing. Here, we take a look at designing your target sequences and ordering them as Alt-R CRISPR crRNA. We review the other components of the system and walk through the experimental process step by step, from design to evaluation of editing potency. We also discuss challenges and potential pitfalls and provide tips and guidance towards successful genome editing experiments. Learn more: http://www.idtdna.com/crispr
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
Co-amplification at lower denaturation temperature PCR specifically used for precise detection of mutation in DNA samples.It also improves precision of downstream techniques used for mutation detection
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
El martes 26 de septiembre del 2017 organizamos en la Fundación Ramón Areces un Simposio Internacional sobre nuevas perspectivas en la investigación sobre el cáncer. En colaboración con el Centro Nacional de Investigaciones Oncológicas (CNIO) y Weizmann Institute for Science.
miRNA profiling from blood challenges and recommendations - Download the articleQIAGEN
The discovery of stable miRNA species circulating in blood has led to increased research focus on disease-related variations in serum and plasma miRNA expression and the possibility that such variations could serve as noninvasive biomarkers for disease. Working with serum and plasma miRNA presents various challenges in purification and characterization. In this paper, we outline QIAGEN recommendations for robust purification and quantification, as well as reliable data normalization and analysis.
Technical Guide to Qiagen PCR Arrays - Download the GuideQIAGEN
Total RNA discovery with RT2 and miScript PCR Arrays : Explore the RNA universe - Whatever your destination within the RNA universe, QIAGEN will help you get there. The miRNeasy kits deliver pure, high-quality total RNA from a broad range of samples. The RT2 and miScript PCR arrays are a complete solution both for focused analysis of gene and microRNA expression and for validation of microarray and RNA sequencing experiments. Together with the powerful analytics tools of GeneGlobe® and QIAGEN Ingenuity® Pathway Analysis, these products give you a smooth path from your sample to high-quality results.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. In a recent webinar, "New RNA Tools for Optimized CRISPR/Cas 9 Genome Editing", we presented how we developed the Alt-R™ CRISPR-Cas9 System for genome editing. Here, we take a look at designing your target sequences and ordering them as Alt-R CRISPR crRNA. We review the other components of the system and walk through the experimental process step by step, from design to evaluation of editing potency. We also discuss challenges and potential pitfalls and provide tips and guidance towards successful genome editing experiments. Learn more: http://www.idtdna.com/crispr
Maximizing PCR and RT-PCR Success - Download the BrochureQIAGEN
The invention of the polymerase chain reaction (PCR) by K. Mullis and coworkers in 1985 revolutionized molecular biology and molecular medicine. Major research areas, such as biomarker discovery, gene regulation and cancer research are challenging today’s PCR technologies with more demanding requirements. These include the need for increased throughput while reducing costs, higher assay sensitivity and reliable data normalization. Assay development and evaluation, reproducibility of data and time to result are still major problems encountered by researchers.
Meeting today’s challenges in PCR requires advances in all methods of the workflow that starts with sample collection, sample stabilization, and nucleic acid purification, and ends with amplification and detection. The following pages focus on the importance of amplification in meeting these challenges.
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
Hot-start DNA polymerases are commonly used in PCR for genotyping, sequencing, molecular diagnostics, and high-throughput applications. In this presentation, PCR performance of Invitrogen™ Platinum II Taq Hot-Start DNA Polymerase and Invitrogen™ AccuPrime Taq DNA Polymerase is compared in the following areas:
• PCR run time for targets of different lengths
• Amplification of AT-rich and GC-rich sequences
• Tolerance to PCR inhibitors
• Sensitivity in target detection
• Universal protocol for PCR targets of different lengths
• Multiplex PCR of 15 targets
• Product format for direct gel loading
Request a sample of Platinum II Taq enzyme at http://bit.ly/2M4U9cw
Find other PCR enzymes at http://bit.ly/2JIPrzj
Learn more about PCR at http://bit.ly/2y2aSVo
#PCR #PCREducation #Invitrogen #InvitrogenSchoolofMolBio
Co-amplification at lower denaturation temperature PCR specifically used for precise detection of mutation in DNA samples.It also improves precision of downstream techniques used for mutation detection
Streamlined next generation sequencing assay development using a highly multi...Thermo Fisher Scientific
Next generation sequencing (NGS) assay development for solid tumor sequencing requires characterization of variant calling directly from formalin-fixed paraffin embedded (FFPE) tissue samples. However, cell line based FFPE and human FFPE samples only contain 2 to 20 variants, which require laboratories to invest significant resources in sample sourcing and preparation when developing assays to detect 100+ variants
Learn the latest eqigenetic techniques including: discriminating epigenetically inactive chromatin from active chromatin, discriminating between aberrant and Monoallelic DNA methylation, predicting gene expression levels via chromatin structure assay and analyzing how DNA methylation affects promoter activity.
The Functional and Pathway Analysis talk given in March 2010 at the CRUK CRI. Cambridge UK.
It was designed to introduce wet-lab researchers to using web-based tools for doing functional analysis of gene lists, such as from microarray experiments.
Characterization of Novel ctDNA Reference Materials Developed using the Genom...Thermo Fisher Scientific
Liquid biopsy diagnostic technologies have revolutionized cancer testing and therapeutic monitoring. Non-invasive sample collection removes the need for invasive and dangerous biopsies to diagnose cancer and monitor therapeutic efficacy. As liquid biopsy technologies become more sensitive, screening for early detection of cancer DNA using a blood test could become routine clinical practice. However, such technologies cannot be developed without high quality reference materials. In this study, ctDNA reference materials using the NIST Genome in a Bottle GM24385 cell line DNA were developed in a human plasma-EDTA matrix. The allelic frequency (AF), size and stability of the materials were analyzed.
RAS is one of the most frequently mutated oncogenes in human cancer. KRAS is the isoform most frequently mutated, which constitutes about 85% of RAS mutations. As the most frequently mutated RAS isoform, KRAS is intensively studied in the past years.
In the formulation of KRAS integrated research plan, Medicilon has in-depth communication with customers. The backbone of scientific research has combined the characteristics of each case with years of practical experience and technical accumulation, and carefully submitted high-quality experimental plans and results to customers. Medicilon provides KRAS-targeted drug discovery, CMC research (API + formulation), pharmacodynamics research, PK study, safety evaluation and other services.https://www.medicilon.com/platform/kras/
RNase H2-dependent PCR (rhPCR) is a powerful method for increasing PCR specificity and eliminating primer-dimers by using blocked primers and a thermostable RNase H2 from Pyrococcus abyssi (P. abyssi). Primers will only support extension and replication after the blocked portion is cleaved. Cleavage by the RNase H2 enzyme occurs only when primers are bound to their complementary target sequence, thus providing increased specificity. Also, the thermostability of P. abyssi RNase H2 provides a “hot start” capability to the reaction. In this presentation, Dr Joseph Dobosy (senior research scientist in the molecular genetics research division of IDT) gives a detailed explanation of the rhPCR mechanism, offer tips on how to design assays using this powerful technology, and discuss examples of applications that benefit from rhPCR.
Neuro-symbolic is not enough, we need neuro-*semantic*Frank van Harmelen
Neuro-symbolic (NeSy) AI is on the rise. However, simply machine learning on just any symbolic structure is not sufficient to really harvest the gains of NeSy. These will only be gained when the symbolic structures have an actual semantics. I give an operational definition of semantics as “predictable inference”.
All of this illustrated with link prediction over knowledge graphs, but the argument is general.
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.
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.
Software Delivery At the Speed of AI: Inflectra Invests In AI-Powered QualityInflectra
In this insightful webinar, Inflectra explores how artificial intelligence (AI) is transforming software development and testing. Discover how AI-powered tools are revolutionizing every stage of the software development lifecycle (SDLC), from design and prototyping to testing, deployment, and monitoring.
Learn about:
• The Future of Testing: How AI is shifting testing towards verification, analysis, and higher-level skills, while reducing repetitive tasks.
• Test Automation: How AI-powered test case generation, optimization, and self-healing tests are making testing more efficient and effective.
• Visual Testing: Explore the emerging capabilities of AI in visual testing and how it's set to revolutionize UI verification.
• Inflectra's AI Solutions: See demonstrations of Inflectra's cutting-edge AI tools like the ChatGPT plugin and Azure Open AI platform, designed to streamline your testing process.
Whether you're a developer, tester, or QA professional, this webinar will give you valuable insights into how AI is shaping the future of software delivery.
UiPath Test Automation using UiPath Test Suite series, part 3DianaGray10
Welcome to UiPath Test Automation using UiPath Test Suite series part 3. In this session, we will cover desktop automation along with UI automation.
Topics covered:
UI automation Introduction,
UI automation Sample
Desktop automation flow
Pradeep Chinnala, Senior Consultant Automation Developer @WonderBotz and UiPath MVP
Deepak Rai, Automation Practice Lead, Boundaryless Group and UiPath MVP
Smart TV Buyer Insights Survey 2024 by 91mobiles.pdf91mobiles
91mobiles recently conducted a Smart TV Buyer Insights Survey in which we asked over 3,000 respondents about the TV they own, aspects they look at on a new TV, and their TV buying preferences.
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.
Elevating Tactical DDD Patterns Through Object CalisthenicsDorra BARTAGUIZ
After immersing yourself in the blue book and its red counterpart, attending DDD-focused conferences, and applying tactical patterns, you're left with a crucial question: How do I ensure my design is effective? Tactical patterns within Domain-Driven Design (DDD) serve as guiding principles for creating clear and manageable domain models. However, achieving success with these patterns requires additional guidance. Interestingly, we've observed that a set of constraints initially designed for training purposes remarkably aligns with effective pattern implementation, offering a more ‘mechanical’ approach. Let's explore together how Object Calisthenics can elevate the design of your tactical DDD patterns, offering concrete help for those venturing into DDD for the first time!
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.
FIDO Alliance Osaka Seminar: Passkeys and the Road Ahead.pdf
Семинар ДНК 16/05/2014 Сибэнзим
1. New epigenetic tools for
cancer diagnostics
Gonchar D.A., Kuznetsov V.V.,
Akishev A.G., Abdurashitov M.A.,
Degtyarev S.Kh.
1
2. DNA methylation in mammalians genomes is mostly
DNA methylation of CG dinucleotides with formation
of 5-methylcytosine (5mC) in both DNA strands.
Mammalian DNA-methyltransferases DNMT1,
DNMT3a and DNMT3b catalyze a reaction of DNA
methylation.
DNMT1 maintains DNA methylation pattern in vivo
modifying a new strand after replication.
DNMT3a and DNMT3b are responsible for DNA
methylation de novo. This modification in regulation
region (promotor and first exon) of gene results in the
gene silencing.
2
3. At present time 5mC is determined mostly by a
chemical treatment of DNA with sodium bisulphite,
which results in cytosine transformation into uracil,
whereas 5mC is resistant against this modification.
A subsequent analysis of modified and native DNA
allows to locate positions of methylated cytosines in
studied DNA.
Method of bisulphite conversion is quite
sophisticated and often results in obtaining false
positive data.
3
4. There is another approach — enzymatic methods of
determination of DNA methylation. Among enzymatic
methods of 5mC determination, so called methyl-
sensitive PCR assay (MS PCR) is
the most popular. Determination of DNA methylation by
MS PCR proceeds in two steps:
DNA hydrolysis with site-specific DNA endonuclease
(e.g., restriction enzyme) followed
by PCR with primers located upsteam and downstream
DNA region of interest.
4
5. This method is based on inability of restriction
enzymes, which contain CG dinucleotide in the
recognition site, to cut this site if 5mC is present in the
dinucleotide.
A subsequent PCR from primers, which are located
around a chosen recognition site, produces a
corresponding DNA fragment if there is a methylated
CG-dinucleotide within this site. On the contrary,
DNA fragment is not produced in PCR if there is
no methylated CG-dinucleotide in a recognition
sequence of restriction enzyme.
5
6. HpaII (recognition site CCGG) cleaves DNA
sequence CCGG, but doesn't cut C(5mC)GG.
Singer-Sam et.al.(Mol. Cell Biol. (1990) Vol.
10, 4987-4989) called a method of methyl-
sensitive PCR with HpaII as HpaII-PCR assay.
HpaII-PCR assay includes DNA hydrolysis with
HpaII followed by PCR with primers located
upsteam and downstream DNA region of interest.
6
7. Application of methyl-sensitive PCR assays similar to
HpaII-PCR assay is limited by a very short list of
recognition sequences of corresponding restriction
endonucleases.
7
8. Study of DNMT3a and DNMT3b substrate
specificity has shown that both enzymes
methylate CG-dinucleotide mostly in DNA
sequence PuCGPy. This is a reason why
restriction enzymes with recognition sites
ACGT and GCGC (MaeII and HhaI,
respectively) are widely used in methyl-
sensitive PCR study of de novo DNA
methylation.
8
9. • DNMT3 is the main enzyme responsible for de novo cytosine
modification and epigenetic regulation of human and mammalian
genes activity.
• DNMT3 recognizes and methylates a tetranucleotide RCGY in
DNA as follows:
5’- Pu C G Py -3’ 5’- Pu(5mC) G Py -3’
3’- Py G C Pu -5’ 3’- Py G(5mC) Pu -5’
9
10. New enzymes
BlsI and GlaI
belong to a new type of 5-methylcytosine-directed site-specific DNA
endonucleases that cleave only methylated DNA.
10
12. Substrate specificity of DNMT3a, DNMT3b and GlaI
PuCGPy Pu( )GPy
PyGCPu PyG( )Pu
5mC
5mC
DNMT
AdoMet
Pu( )GPy Pu( ) G Py
PyG( )Pu Py G ( )Pu
↓
↑
5mC 5mC
5mC 5mC
GlaI
12
13. BlsI
Cleaves a recognition site
5’-PuPyN↓PuPy-3’
3’-PyPu↑NPyPu-5’
carrying at least one 5-methylcytosine (N is not
considering) in each DNA strand.
Two sites methylated by Dnmt3 and separated by N form
BlsI cleavage site
5’ - Pu (5mC) G Py N Pu (5mC) G Py - 3’
3’ - Py G (5mC) Pu N Py G (5mC) Pu - 5’
BlsI recognition site
13
15. BlsI- и GlaI-PCR assays include DNA hydrolysis
with BlsI or GlaI, respectively, followed by PCR
with primers located upsteam and downstream
DNA region of interest.
15
16. • a promoter region of CEPBD (CCAAT/enhancer binding protein, delta);
• a promoter region of DAPK1 (death-associated protein kinase 1);
• a promoter and first exon region of RASSF1A (Ras association domain
family 1A);
• a promoter and first exon region of SEPT9b (septin 9b);
• a promoter and first exon region of MGMT (O6-methylguanine DNA
methyltransferase);
• a promoter and first exon region of RARB (retinoic acid receptor, beta);
• a promoter and first exon region of IGFBP3 (insulin-like growth factor
binding protein 3).
Studied DNА regions of human genome
16
18. DNA preparations from five human cell lines:
L-68 (control, lung fibroblast), HeLa (cerbix adenocarcinoma),
Raji (Burkitt’s lymphoma), U-937 (histiocystic lymphoma)
and Jurkat (acute T-cell leukemia) have been treated separately with:
1) Restriction enzyme with recognition site in studied region
(HaeIII for CEPBD, RASSF1A and SEPT9b; FatI for RARB), positive
control;
2) GlaI (recognizes 5'-Pu(5mC)GPy-3' [2]);
3) BlsI (recognizes 5'-GCNGC-3' if at least two 5-methylcytosines (N isn't
considering) are present in both DNA strands [3]);
4) no added enzyme, negative control.
After incubation 4 reaction mixtures have been used as a DNA template for
PCR. DNA from Drosophila melanogaster at the same concentration has
been used as a negative PCR control.
Protocol of BlsI- and GlaI- PCR assay
18
25. Fig. . BlsI- and GlaI- PCR assay of promoter
region (fragment d2-r2, 173 bp in length).
9 Real time DAPK1
U-937
JurkatL-68
HeLa
Raji
Pretreated DNA
With GlaI
With BlsI
With HaeIII
Methylation: Raji - >99%;
L-68, HeLa, U-937, Jurkat - <1%
25
27. Comparison of bisulphite conversion and BlsI- and GlaI- PCR
assay
A quantity of DNA for analysis:
2-5 DNA molecules for BlsI- and GlaI- PCR assay
Fidelity of BlsI- and GlaI- PCR assay – 2%
Bisulphite conversion – 15%
BlsI- and GlaI- PCR assay analyzes DNA fragments from
100 to 10000 b.p., while bisulphite conversion only 150-
200 b.p.
27
29. Introduction to GLAD-PCR assay
29
There is one vital disadvantage of BlsI- and GlaI- PCR
assay – it is good for epigenetic typing of cancer cell lines
and is hardly ever may be applied in clinical practice
because the studied DNA samples include unmethylated
DNA from stroma, blood cells, etc.
A new GLAD-PCR assay we have developed recently
allows to determine minimal quantities of methylated
sites in presence of excess of unmethylated DNA.
GLAD-PCR assay may find a wide application in routine
clinical practice
31. GLAD-PCR assay
31
GlaI hydrolysis and Ligation Adapter Dependent
PCR (GLAD-PCR) is the novel method to determine
R(5mC)GY sites produced by methylation with
DNMT3A and DNMT3B. GLAD PCR analysis is
performed in one tube and includes 3 steps: DNA
hydrolysis with site-specific methyl-directed DNA
endonuclease GlaI, universal adapter ligation and
Real-time PCR with
Taqman probe.
One primer is designed for DNA region of interest,
structure of another primer is based on an adapter
sequence.
32. Studied genes
1 — transcription start. H — position of hybrid primer.
32
34. GLAD PCR analysis of DNA methylation in regulatory region
of tumor suppressor genes
Amplification chart of GLAD PCR assay of 15 ng DNA per reaction using Bio-
Rad CFX96. We accept Raji DNA methylation to be 100%.
CEBPD RARB
34
35. GLAD PCR analysis of DNA methylation in regulatory region
of tumor suppressor genes
Sensitivity determination of the GLAD PCR assay
CEBPD RARB
35
37. Conclusions
A new method of GLAD PCR assay has been developed to study
DNA methylation. Method includes GlaI hydrolysis of studied
DNA, the universal adapter ligation and subsequent real-time
PCR of the studied RCGY site. Method is performed in one tube,
takes about four hours and allows to determine several copies of
methylated DNA.
GLAD PCR assay has been applied to study aberrant
methylation of selected RCGY site in regulatory regions of tumor
suppressor genes. GLAD PCR assay has revealed different
patterns of RCGY sites methylation in four malignant cell lines.
All studied RCGY sites are highly methylated in Raji cells and
unmethylated in control fibroblast line.
GLAD PCR assay may be used for determination of methylation
status of particular RCGY site and for a rapid epigenetic
characterization of malignant cells.
37