Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs), presented by Dr Mark Behlke, Chief Scientific Officer at Integrated DNA Technologies
Pharmacogenetics and individual variation of drug responseNarasimha Kumar G V
This document discusses pharmacogenetics and individual variation in drug response. It begins by outlining how individuals can metabolize and respond to drugs differently due to genetic factors like polymorphisms. Three key discoveries in the 1950s helped establish the field of pharmacogenetics by showing how genetic differences can impact drug metabolism and efficacy. The document then examines specific genetic variations that influence drug pharmacokinetics and pharmacodynamics, such as differences in metabolizing enzymes and receptors. Understanding these genetic factors can help optimize drug therapy for each individual patient.
This document discusses several newer antiarrhythmic drugs, including ranolazine, vernakalant, ivabradine, celivarone, budiodarone, and tecadenoson. It provides details on the mechanisms of action, clinical trials, efficacy, and safety profiles of these drugs. Ranolazine, vernakalant, and budiodarone have shown efficacy in cardioversion or rate control of atrial fibrillation, while ivabradine reduces heart rate without affecting contractility. Celivarone and tecadenoson are being investigated for maintaining sinus rhythm and terminating supraventricular tachycardias, respectively.
This document provides information on beta lactam antibiotics and cell wall synthesis inhibitors. It discusses the classification, properties, mechanisms of action and resistance, kinetics, adverse effects and individual agents of penicillins and cephalosporins. Penicillins are derivatives of benzylpenicillin and inhibit the last step of peptidoglycan synthesis in bacterial cell walls. Cephalosporins are similar to penicillins in their mechanism of action but have greater antimicrobial spectra due to modifications at the C7 and C3 positions. Both classes face resistance from bacterial production of beta-lactamases.
The document discusses several new agents for the treatment of plasma cell disorders like multiple myeloma. It provides information on newer proteasome inhibitors like carfilzomib and ixazomib, immunomodulatory drugs like pomalidomide, HDAC inhibitors like panobinostat, and monoclonal antibodies like daratumumab and elotuzumab. It also mentions some investigational drugs in development and discusses the importance of combination therapies and addressing issues like cost of these new treatments.
Caco-2 cells are the most widely used cell line for predicting intestinal drug absorption as they form polarized monolayers with tight junctions and microvilli like intestinal cells. Other cell lines used include MDCK, HT29-MTX, 2/4/A1, TC-7, LLC-PK1, IEC-18 and T84, but they have limitations such as lack of transporters, unstable monolayers, or poor differentiation. While cell models show good correlation with passive drug permeability, correlations with active transport are more variable. Extensive studies are still needed to fully characterize intestinal drug transport mechanisms in these models.
This document discusses pharmacogenomics and how genetic differences can influence individual responses to drugs. It provides examples of how single nucleotide polymorphisms and other genetic variations can affect drug targets, metabolizing enzymes, transporters, and ultimately impact pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Specifically, it examines cases of polymorphisms in cytochrome P450 drug metabolizing enzymes like CYP2C9, CYP2C19, and CYP2D6 that can lead to differences in drug metabolism and clearance between fast, normal, and poor metabolizers. The goals of pharmacogenomics are to maximize drug efficacy, minimize toxicity, and aid precision medicine by predicting who will respond to certain drugs. Widespread application
ANTISENSE MOLECULES AND APTAMERS AS DRUGS OF FUTURE.pptxSyedaYameen2
This document provides information on antisense molecules and aptamers. It discusses how antisense oligonucleotides work by hybridizing with mRNA in a sequence-specific manner, leading to mRNA degradation or translational arrest. Several antisense drugs have been approved to treat diseases like cytomegalovirus retinitis and spinal muscular atrophy. The document also introduces aptamers, which are nucleic acid or peptide molecules that can bind to proteins. Aptamers are identified through a process called SELEX and have potential for therapeutic use.
Pharmacogenetics and individual variation of drug responseNarasimha Kumar G V
This document discusses pharmacogenetics and individual variation in drug response. It begins by outlining how individuals can metabolize and respond to drugs differently due to genetic factors like polymorphisms. Three key discoveries in the 1950s helped establish the field of pharmacogenetics by showing how genetic differences can impact drug metabolism and efficacy. The document then examines specific genetic variations that influence drug pharmacokinetics and pharmacodynamics, such as differences in metabolizing enzymes and receptors. Understanding these genetic factors can help optimize drug therapy for each individual patient.
This document discusses several newer antiarrhythmic drugs, including ranolazine, vernakalant, ivabradine, celivarone, budiodarone, and tecadenoson. It provides details on the mechanisms of action, clinical trials, efficacy, and safety profiles of these drugs. Ranolazine, vernakalant, and budiodarone have shown efficacy in cardioversion or rate control of atrial fibrillation, while ivabradine reduces heart rate without affecting contractility. Celivarone and tecadenoson are being investigated for maintaining sinus rhythm and terminating supraventricular tachycardias, respectively.
This document provides information on beta lactam antibiotics and cell wall synthesis inhibitors. It discusses the classification, properties, mechanisms of action and resistance, kinetics, adverse effects and individual agents of penicillins and cephalosporins. Penicillins are derivatives of benzylpenicillin and inhibit the last step of peptidoglycan synthesis in bacterial cell walls. Cephalosporins are similar to penicillins in their mechanism of action but have greater antimicrobial spectra due to modifications at the C7 and C3 positions. Both classes face resistance from bacterial production of beta-lactamases.
The document discusses several new agents for the treatment of plasma cell disorders like multiple myeloma. It provides information on newer proteasome inhibitors like carfilzomib and ixazomib, immunomodulatory drugs like pomalidomide, HDAC inhibitors like panobinostat, and monoclonal antibodies like daratumumab and elotuzumab. It also mentions some investigational drugs in development and discusses the importance of combination therapies and addressing issues like cost of these new treatments.
Caco-2 cells are the most widely used cell line for predicting intestinal drug absorption as they form polarized monolayers with tight junctions and microvilli like intestinal cells. Other cell lines used include MDCK, HT29-MTX, 2/4/A1, TC-7, LLC-PK1, IEC-18 and T84, but they have limitations such as lack of transporters, unstable monolayers, or poor differentiation. While cell models show good correlation with passive drug permeability, correlations with active transport are more variable. Extensive studies are still needed to fully characterize intestinal drug transport mechanisms in these models.
This document discusses pharmacogenomics and how genetic differences can influence individual responses to drugs. It provides examples of how single nucleotide polymorphisms and other genetic variations can affect drug targets, metabolizing enzymes, transporters, and ultimately impact pharmacokinetics, pharmacodynamics, efficacy, and toxicity. Specifically, it examines cases of polymorphisms in cytochrome P450 drug metabolizing enzymes like CYP2C9, CYP2C19, and CYP2D6 that can lead to differences in drug metabolism and clearance between fast, normal, and poor metabolizers. The goals of pharmacogenomics are to maximize drug efficacy, minimize toxicity, and aid precision medicine by predicting who will respond to certain drugs. Widespread application
ANTISENSE MOLECULES AND APTAMERS AS DRUGS OF FUTURE.pptxSyedaYameen2
This document provides information on antisense molecules and aptamers. It discusses how antisense oligonucleotides work by hybridizing with mRNA in a sequence-specific manner, leading to mRNA degradation or translational arrest. Several antisense drugs have been approved to treat diseases like cytomegalovirus retinitis and spinal muscular atrophy. The document also introduces aptamers, which are nucleic acid or peptide molecules that can bind to proteins. Aptamers are identified through a process called SELEX and have potential for therapeutic use.
This document provides guidance on planning and executing successful siRNA experiments through following good practices. It reviews key steps including understanding the target transcript through identifying variants and structures, selecting effective siRNAs using design tools and rules, choosing a cell line with appropriate expression profiles, optimizing experimental conditions through controls and pilot experiments, and validating the assay. Following these steps can help achieve optimized gene knockdown.
This document provides guidance on designing quantitative PCR (qPCR) assays for specific applications, including species-specific, strain-specific, and copy number variation (CNV) assays. It outlines general design strategies and considerations, including using sequence alignments to identify unique target regions and primers that avoid nonspecific amplification. Examples are provided for designing assays to distinguish similar genes in Arabidopsis thaliana and related viral strains. Design of CNV assays is also discussed, highlighting the importance of a single-copy reference gene.
This document discusses sources of variation in oligonucleotide optical density (OD) measurements and yields. It explains that OD and yield values can vary between instruments due to differences in spectrophotometers used. The document also describes how sequence composition, base stacking interactions, and calculation methods all influence the molar extinction coefficient value used to determine OD and nmol/OD for a given oligonucleotide. Users are advised not to be alarmed by variations in OD values between their measurements and the manufacturer's, and to contact technical support if variations are very large.
IDT provides oligonucleotides and panels for targeted sequencing including stocked and custom gene panels. Their panels include 264 genes for acute myeloid leukemia, 127 genes for pan-cancer analysis, and 4503 genes for inherited diseases. IDT probes are individually synthesized and quality controlled before being pooled. Universal blockers improve on-target rates by blocking adapter participation. Additional services include custom barcoded adapters and gBlocks fragments for quality control.
The document describes xGen Predesigned Gene Capture Pools, which are pools of xGen Lockdown Probes designed to target specific genes. The pools offer advantages over custom panels such as better customization ability, decreased optimization time, and lower cost. They can be used to build custom gene panels, supplement existing panels, and share targets between researchers studying different diseases. Data shows the pools provide high on-target performance, consistent deep coverage, and can supplement existing panels without affecting coverage.
In addition to a standard gene synthesis service, IDT offers a novel, rapid, and reliable method to build and clone the genes you need at a fraction of the cost of full gene synthesis services. gBlocks® Gene Fragments are double-stranded, sequence-verified DNA blocks of length 125–750 bp. Their high sequence fidelity and rapid delivery time make gBlocks Gene Fragments ideal for a large range of synthetic biology applications. In this presentation, Dr Adam Clore reviews a variety of uses of gBlocks fragments, including CRISPR-based genome modification, qPCR and HRM controls, and the assembly of gene fragments using the Gibson Assembly® Method.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
In this joint slidedeck, oligonucleotide synthesis specialists Integrated DNA Technologies (IDT) join with bioconjugation experts Innova Biosciences to explain the importance of using top quality reagents when preparing oligonucleotide-antibody conjugates. Antibody-oligonucleotide conjugates are the next generation of tools in biomarker detection, overcoming sensitivity and linear range issues often encountered with standard antibody labels.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
This document summarizes a presentation about RNase H2 PCR (rhPCR), a new molecular technology that uses an RNA residue in PCR primers and a thermostable RNase H2 enzyme. It describes how rhPCR works, the advantages it provides over traditional PCR including reduced primer-dimer formation and improved specificity for rare allele detection. Two generations of cleavable primer designs - GEN1 and GEN2 - are discussed, along with their different applications. Examples are provided that demonstrate how rhPCR can improve assays for SNP genotyping, multiplex PCR, and detection in complex backgrounds.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
This document summarizes a presentation given by Dr. Jo Vandesompele on state-of-the-art normalization of RT-qPCR data. It discusses the importance of normalization to remove experimental variation and introduces the geNorm algorithm for determining the optimal number and combination of reference genes for normalization. GeNorm has become the standard method for reference gene validation and normalization and has improved qPCR data analysis. The document also proposes a novel global mean normalization strategy for large-scale gene expression studies.
The document discusses using NCBI databases to design quantitative PCR (qPCR) assays. It describes several NCBI tools that can be used:
1) The NCBI Nucleotide and Gene databases to obtain sequence information for the gene of interest.
2) NCBI BLAST to perform sequence searches and check primer specificity against relevant databases.
3) NCBI dbSNP to search for single nucleotide polymorphisms (SNPs) in the primer binding sites that could affect assay performance.
The document provides guidance on how to use these NCBI tools at various steps of the qPCR assay design process.
IDT provides a range of solutions for targeted next generation sequencing. Labs processing hundreds to thousands of samples can create highly uniform, custom panels using xGen® Lockdown Probes. The new xGen Acute Myeloid Leukemia (AML) panel is a predesigned set of Lockdown Probes that captures 260 genes identified by whole genome and exome sequencing of 200 patient samples. The AML panel can be used as stand-alone or customized with additional probes to detect other targets of interest.
This document describes PrimeTime® qPCR products for gene expression analysis, including probe- and primer-based assays for human, mouse, and rat sequences as well as custom assays. It provides details on master mixes, probes, controls, and an assay design and ordering process to ensure specific and efficient assays. The assays are guaranteed to have high efficiency and sensitivity for accurate quantification of gene expression.
xGen® target capture products from IDT enable greater sensitivity and higher sample throughput for targeted next generation sequencing (NGS). NGS researchers can now benefit from the IDT high throughput, quality controlled manufacturing process to order gene panels that have the greatest level of uniformity. These panels can be easily optimized with the addition of more probes.
Learn more at http://www.idtdna.com/xgen
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.
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.
Precision medicine for oncology requires accurate and sensitive molecular characterization. However, sample degradation, polymerase errors, and sequencing errors reduce accuracy for sequencing genetic variants. By incorporating molecular tagged adapters in target enrichment, and using DNA probes that deliver extremely even and deep coverage, we are able to demonstrate a 300-fold reduction in false positives at or above 0.25% variant frequency. In this presentation, Dr Mirna Jarosz discusses these methods and how they can significantly reduce error rates in your sequencing data.
Gene silencing is a process that downregulates gene expression without altering the DNA sequence. It can occur at the transcriptional or post-transcriptional level through various methods like RNA interference, DNA methylation, and histone modifications. Gene silencing is important for normal development and cellular differentiation, but can also contribute to diseases if aberrantly silencing critical genes. It is currently being explored as a tool for crop improvement, drug discovery, and antiviral therapies.
Gene silencing is a process that down regulates gene expression without altering the DNA sequence. It can occur at the transcriptional or post-transcriptional level. Common methods of gene silencing include RNA interference technologies like siRNA, shRNA, and miRNA, which induce degradation of mRNA transcripts. Gene silencing is used for both basic research and applications like crop improvement, as it allows precise down regulation of gene expression.
This document provides guidance on planning and executing successful siRNA experiments through following good practices. It reviews key steps including understanding the target transcript through identifying variants and structures, selecting effective siRNAs using design tools and rules, choosing a cell line with appropriate expression profiles, optimizing experimental conditions through controls and pilot experiments, and validating the assay. Following these steps can help achieve optimized gene knockdown.
This document provides guidance on designing quantitative PCR (qPCR) assays for specific applications, including species-specific, strain-specific, and copy number variation (CNV) assays. It outlines general design strategies and considerations, including using sequence alignments to identify unique target regions and primers that avoid nonspecific amplification. Examples are provided for designing assays to distinguish similar genes in Arabidopsis thaliana and related viral strains. Design of CNV assays is also discussed, highlighting the importance of a single-copy reference gene.
This document discusses sources of variation in oligonucleotide optical density (OD) measurements and yields. It explains that OD and yield values can vary between instruments due to differences in spectrophotometers used. The document also describes how sequence composition, base stacking interactions, and calculation methods all influence the molar extinction coefficient value used to determine OD and nmol/OD for a given oligonucleotide. Users are advised not to be alarmed by variations in OD values between their measurements and the manufacturer's, and to contact technical support if variations are very large.
IDT provides oligonucleotides and panels for targeted sequencing including stocked and custom gene panels. Their panels include 264 genes for acute myeloid leukemia, 127 genes for pan-cancer analysis, and 4503 genes for inherited diseases. IDT probes are individually synthesized and quality controlled before being pooled. Universal blockers improve on-target rates by blocking adapter participation. Additional services include custom barcoded adapters and gBlocks fragments for quality control.
The document describes xGen Predesigned Gene Capture Pools, which are pools of xGen Lockdown Probes designed to target specific genes. The pools offer advantages over custom panels such as better customization ability, decreased optimization time, and lower cost. They can be used to build custom gene panels, supplement existing panels, and share targets between researchers studying different diseases. Data shows the pools provide high on-target performance, consistent deep coverage, and can supplement existing panels without affecting coverage.
In addition to a standard gene synthesis service, IDT offers a novel, rapid, and reliable method to build and clone the genes you need at a fraction of the cost of full gene synthesis services. gBlocks® Gene Fragments are double-stranded, sequence-verified DNA blocks of length 125–750 bp. Their high sequence fidelity and rapid delivery time make gBlocks Gene Fragments ideal for a large range of synthetic biology applications. In this presentation, Dr Adam Clore reviews a variety of uses of gBlocks fragments, including CRISPR-based genome modification, qPCR and HRM controls, and the assembly of gene fragments using the Gibson Assembly® Method.
Real-Time quantitative PCR (qPCR) is a mainstream method that is used in research and diagnostic applications for quantification of gene expression. IDT has developed a robust and affordable qPCR master mix for use with probe-based qPCR in single and multiplex assays. In this presentation, we explore a variety of applications of PrimeTime® Gene Expression Master Mix. We cover the use of PrimeTime master mix with probe based assays from IDT. We also look at the use of PrimeTime master mix in multiplex applications without the loss of sensitivity that is commonly observed. Finally, we demonstrate the unmatched stability of PrimeTime master mix under ambient temperatures, saving your research money and minimizing on shipping delays.
In this joint slidedeck, oligonucleotide synthesis specialists Integrated DNA Technologies (IDT) join with bioconjugation experts Innova Biosciences to explain the importance of using top quality reagents when preparing oligonucleotide-antibody conjugates. Antibody-oligonucleotide conjugates are the next generation of tools in biomarker detection, overcoming sensitivity and linear range issues often encountered with standard antibody labels.
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth.
This document summarizes a presentation about RNase H2 PCR (rhPCR), a new molecular technology that uses an RNA residue in PCR primers and a thermostable RNase H2 enzyme. It describes how rhPCR works, the advantages it provides over traditional PCR including reduced primer-dimer formation and improved specificity for rare allele detection. Two generations of cleavable primer designs - GEN1 and GEN2 - are discussed, along with their different applications. Examples are provided that demonstrate how rhPCR can improve assays for SNP genotyping, multiplex PCR, and detection in complex backgrounds.
Real-time quantitative PCR (qPCR) is a preferred platform for high throughput gene expression profiling, where large numbers of samples are characterized for hundreds of expression markers. Technically, the qPCR measurements are performed in the same way as when classical qPCR is used to analyze only a few targets per sample, but the higher throughput introduces additional sources of potential confounding variation that must be controlled for. In this presentation, Dr Kubista describes how high throughput qPCR profiling studies are designed. He covers assay optimization and validation, sample quality testing, and how to merge multi-plate measurements into a common analysis. Dr Kubista also discusses how to cost-effectively measure and compensate for background due to genomic DNA.
This document summarizes a presentation given by Dr. Jo Vandesompele on state-of-the-art normalization of RT-qPCR data. It discusses the importance of normalization to remove experimental variation and introduces the geNorm algorithm for determining the optimal number and combination of reference genes for normalization. GeNorm has become the standard method for reference gene validation and normalization and has improved qPCR data analysis. The document also proposes a novel global mean normalization strategy for large-scale gene expression studies.
The document discusses using NCBI databases to design quantitative PCR (qPCR) assays. It describes several NCBI tools that can be used:
1) The NCBI Nucleotide and Gene databases to obtain sequence information for the gene of interest.
2) NCBI BLAST to perform sequence searches and check primer specificity against relevant databases.
3) NCBI dbSNP to search for single nucleotide polymorphisms (SNPs) in the primer binding sites that could affect assay performance.
The document provides guidance on how to use these NCBI tools at various steps of the qPCR assay design process.
IDT provides a range of solutions for targeted next generation sequencing. Labs processing hundreds to thousands of samples can create highly uniform, custom panels using xGen® Lockdown Probes. The new xGen Acute Myeloid Leukemia (AML) panel is a predesigned set of Lockdown Probes that captures 260 genes identified by whole genome and exome sequencing of 200 patient samples. The AML panel can be used as stand-alone or customized with additional probes to detect other targets of interest.
This document describes PrimeTime® qPCR products for gene expression analysis, including probe- and primer-based assays for human, mouse, and rat sequences as well as custom assays. It provides details on master mixes, probes, controls, and an assay design and ordering process to ensure specific and efficient assays. The assays are guaranteed to have high efficiency and sensitivity for accurate quantification of gene expression.
xGen® target capture products from IDT enable greater sensitivity and higher sample throughput for targeted next generation sequencing (NGS). NGS researchers can now benefit from the IDT high throughput, quality controlled manufacturing process to order gene panels that have the greatest level of uniformity. These panels can be easily optimized with the addition of more probes.
Learn more at http://www.idtdna.com/xgen
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.
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.
Precision medicine for oncology requires accurate and sensitive molecular characterization. However, sample degradation, polymerase errors, and sequencing errors reduce accuracy for sequencing genetic variants. By incorporating molecular tagged adapters in target enrichment, and using DNA probes that deliver extremely even and deep coverage, we are able to demonstrate a 300-fold reduction in false positives at or above 0.25% variant frequency. In this presentation, Dr Mirna Jarosz discusses these methods and how they can significantly reduce error rates in your sequencing data.
Gene silencing is a process that downregulates gene expression without altering the DNA sequence. It can occur at the transcriptional or post-transcriptional level through various methods like RNA interference, DNA methylation, and histone modifications. Gene silencing is important for normal development and cellular differentiation, but can also contribute to diseases if aberrantly silencing critical genes. It is currently being explored as a tool for crop improvement, drug discovery, and antiviral therapies.
Gene silencing is a process that down regulates gene expression without altering the DNA sequence. It can occur at the transcriptional or post-transcriptional level. Common methods of gene silencing include RNA interference technologies like siRNA, shRNA, and miRNA, which induce degradation of mRNA transcripts. Gene silencing is used for both basic research and applications like crop improvement, as it allows precise down regulation of gene expression.
mRNA stability and localization.RNA is critical at many stages of gene expression. How frequently it will be translated, how long it is likely to survive, and where in the cell it will be translated. RNA cis-elements & associated proteins
CRISPR is a powerful new tool for genome editing that allows targeted modifications to genes. It utilizes the Cas9 enzyme to cut DNA at a specific site guided by a short RNA molecule. This summary will discuss the history and mechanisms of CRISPR/Cas9 and its applications in biotechnology and agriculture. CRISPR represents a major breakthrough that will revolutionize genetic engineering by enabling precise edits to genomes. However, further refinement is needed to address issues such as off-target effects. Overall, CRISPR technology holds tremendous promise for developing improved crop traits.
1. The document discusses a study on microRNAs (miRNAs) in Oryza rufipogon, a wild rice species, under drought stress conditions.
2. Small RNA sequencing was performed on shoot and root tissues with or without drought treatment. Differentially expressed miRNAs were identified, including 31 miRNAs regulated in both tissues.
3. The study identified 200 drought-responsive miRNAs and 97 novel miRNAs. Hierarchical clustering analysis showed different expression patterns of miRNAs between shoot and root tissues under drought.
Jin-Soo Kim, a molecular biologist at Seoul National University, used gene-editing technology to create super-muscly pigs by introducing a mutation in the myostatin (MSTN) gene. He used a technology called TALENs, which uses a DNA-cutting enzyme attached to a DNA-binding protein to cut the MSTN gene. The cell's repair system stitches the DNA back together but sometimes deletes or adds base pairs, rendering the gene dysfunctional. The team is now doing the same experiment with CRISPR/Cas9 technology. CRISPR/Cas9 uses CRISPR sequences and a Cas9 enzyme to target and cut specific DNA sequences.
1. The document discusses RNA synthesis and processing, including the different types of RNA (mRNA, rRNA, tRNA), the process of transcription, initiation, elongation, and termination.
2. It also covers RNA processing after transcription, including 5' capping, polyadenylation, splicing, and modifications to tRNA, rRNA and other non-coding RNAs.
3. The clinical applications of understanding RNA synthesis and processing are discussed, such as targets for antibiotics, implications for genetic diseases, and miRNA roles in various human health conditions.
RNA interference (RNAi) is a mechanism that inhibits gene expression through degradation of mRNA. It was discovered in 1998 when researchers found that injecting double-stranded RNA into worms caused specific gene silencing. The mechanism involves dicer enzymes cutting double-stranded RNA into small interfering RNAs (siRNAs) which are incorporated into the RNA-induced silencing complex (RISC) and guide it to degrade complementary mRNA targets. siRNAs can be designed to target specific genes and various delivery methods exist to introduce siRNAs into cells and organisms. RNAi has applications in research, therapeutics, and agriculture by allowing targeted gene silencing.
TAPPING THE RNA WORLD FOR THERAPEUTICSHasnat Tariq
ASO drugs, Si-RNA, Delivery of si-RNA based drugs, CRISPR-Cas gene editing, mRNA based drugs, Aptamer based therapeutics, RNAI PATHWAY, Aptamer based delivery.
Gene silencing is a mechanism of gene regulation that switches off genes without genetic modification. It can occur at the transcriptional or post-transcriptional level. Post-transcriptional gene silencing is achieved through antisense technology or RNA interference (RNAi). Antisense technology uses synthetic nucleic acid molecules that are complementary to mRNA to block translation into proteins. RNAi involves introducing double-stranded RNA that is processed into small interfering RNAs (siRNAs) that guide the RNA-induced silencing complex (RISC) to degrade mRNAs with complementary sequences, thereby silencing genes. Both antisense technology and RNAi have applications for treating diseases and studying gene function and regulation.
This document provides an overview of the origins and mechanisms of microRNAs (miRNAs) and small interfering RNAs (siRNAs). It discusses how double-stranded RNAs are cut by the enzyme Dicer into short RNA fragments that then base pair with mRNAs to induce degradation or transcriptional silencing. Key players in this RNA interference (RNAi) pathway include Dicer, Argonaute proteins, and the RNA-induced silencing complex (RISC). The document contrasts siRNAs, which originate from long double-stranded RNA, and miRNAs, which are encoded from single-stranded RNA precursors that form hairpin structures. It examines the processing steps and roles of various proteins in mediating the effects of si
This lecture discusses post-transcriptional regulation of gene expression through alternative splicing and RNA interference. Alternative splicing allows a single gene to produce different protein variants by including or excluding exons during mRNA processing in the nucleus. RNA interference regulates gene expression by introducing small RNA molecules like miRNAs and siRNAs that bind mRNA in the cytoplasm to inhibit translation or promote degradation. Both mechanisms allow for flexible and precise control of protein production from a gene in a cell type-specific manner.
Gene silencing refers to epigenetic regulation of gene expression.
General Techniques are:
Transcriptional gene silencing
Genomic Imprinting
Paramutation
Transposon silencing
Transcriptional gene silencing
Position effect
RNA-directed DNA methylation
Used in research
Antisense oligonucleotides
Ribozymes
RNA interference
This document discusses gene silencing, which is the suppression or interruption of gene expression at the transcriptional or translational level. It regulates gene expression and prevents certain genes from being expressed. There are several types of gene silencing, including transcriptional silencing through genomic imprinting, paramutation, and transposon silencing. Post-transcriptional gene silencing occurs through RNA interference pathways using small interfering RNA, microRNA, Dicer, and RISC complex. Research methods for gene silencing include antisense oligonucleotides, ribozymes, siRNA, and microRNA. Gene silencing has applications in biotechnology and medicine, with advantages such as being cost effective and having high specificity, though it also has disadvantages like potential toxicity.
1. Primary mRNA transcripts in eukaryotes undergo extensive post-transcriptional processing to become mature mRNA in the nucleus.
2. This processing includes intron removal, exon joining, 5' capping, poly-A tailing, and other modifications.
3. Introns are removed through endonuclease cleavage and splicing, which joins the exons to create a continuous coding sequence in the mature mRNA.
Riboswitches and RNA interference (RNAi)JanmoniBorah1
Riboswitches are the control buttons of mRNAs. They control the expression of gene by regulating transcription and translation.
Gene silencing by RNA interference is a mechanism of post transcriptional regulation of gene expression that involves mainly siRNA and miRNA.
1) The document discusses various mechanisms of gene regulation in bacteria and eukaryotes including operons, DNA methylation, histone modifications, and factors that control transcription, splicing, translation, and mRNA/protein stability.
2) It provides examples of operon regulation in bacteria like the Lac and Trp operons.
3) In eukaryotes, gene expression is controlled at the DNA, transcription, splicing, translation, and protein levels through a variety of mechanisms like chromatin remodeling, transcription factors, hormone signaling, and ubiquitination.
This document contains information about molecular biology techniques including DNA replication, restriction enzyme digestion of DNA, agarose gel electrophoresis, polymerase chain reaction, DNA sequencing, and cloning of recombinant DNA. It includes diagrams of these processes and concepts such as restriction sites, sticky and blunt ends, plasmids, cloning vectors, and the production of recombinant organisms through transformation. The document also discusses gene regulation examples including operons, transcription factors, epigenetics, and RNA interference.
In my report on the cell cycle and its checkpoints, I delve into the phases of cell division and the regulatory mechanisms that ensure accurate replication. By examining these checkpoints, I uncover the essential processes that safeguard genomic integrity and maintain organismal function.
The spliceosome is a large molecular machine that catalyzes pre-mRNA splicing. It is composed of five small nuclear ribonucleoproteins (snRNPs) and over 300 proteins. Mutations that affect splicing can cause diseases like Duchenne muscular dystrophy and myelodysplastic syndrome (MDS). In DMD, splice-switching oligonucleotides induce exon skipping to restore the reading frame. In MDS, mutations in splicing factors SF3B1 and U2AF1 promote aberrant splicing and contribute to pathogenesis. Alternative splicing of VEGF pre-mRNA produces pro-angiogenic and anti-angiogenic isoforms impacting cancer. Detection methods like minigene assays and RT-PCR analyze
Similar to Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs) (20)
Use of CRISPR-Cas9 has revolutionized targeted genome editing. However, rapid design of high-quality guide RNA (gRNA) sequences with high on-target and low off-target editing remains challenging. We implemented a machine learning algorithm to design high-quality gRNA sequences in 5 commonly used species (human, mouse, rat, zebrafish, and nematode). Our tool also designs gRNA sequences against custom targets, and can check existing gRNA designs for quality. In this webinar, we review our data illustrating this tool's performance and demonstrate its use in predicting and designing improved gRNAs for genome editing.
Advances in next generation sequencing enable the detection of variants at exceptionally low frequencies. The accurate detection of low-frequency variants is challenging due in part to errors that are introduced during sample preparation, target enrichment, and sequencing. After tagging individual DNA library molecules with adapters containing unique molecular identifiers (UMIs), bioinformatic filters can be applied to identify and correct errors introduced during the sequencing workflow. In this presentation, we walk through the analytical workflows developed at IDT for processing data containing UMIs. We highlight methods to extract UMI information, correct errors, and build consensus among multiple observations of an original source molecule.
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.
Next generation sequencing (NGS) of circulating tumor DNA (ctDNA) from patient plasma is becoming more widespread in oncology clinical trials. The noninvasive nature of acquiring these samples is particularly important when resection of representative tumor samples is not advised or not possible. However, profiling of ctDNA has challenges to overcome, such as low concentration of ctDNA shed from the tumor and a low signal:noise ratio caused by somatic alterations with less than 1% variant allele fraction. Improving the sensitivity of these assays to detect low allele frequency events with high confidence requires robust sequencing of low input libraries while employing error correction to reduce background noise. To overcome these challenges, we have incorporated unique molecular identifiers (UMIs) into our NGS workflow. Using these novel adapters paired with our proprietary bioinformatics pipeline (AstraZeneca), the number of false positive variants reported for allele fractions less than 0.5% was reduced tenfold. We also refined our curation based on the mapping quality and strand bias in the vicinity of each variant to further reduce the background noise. The use of xGen® Dual Index UMI Adapters—Tech Access (Integrated DNA Technologies) has enabled us to sequence thousands of plasma samples from diverse tumor indications and at differing time points during our trials. The generated data are highly informative with the potential to answer critical questions relating to individual response or resistance to experimental therapies. During this webinar, we discuss our current NGS ctDNA workflow and our future plans to increase our sequencing sensitivity with these novel UMI adapters.
Single-nucleotide polymorphisms (SNPs) provide important information about the biology and evolution of different organisms. SNPs may also help predict an individual’s response to certain drugs, susceptibility to environmental factors, and risk of developing particular diseases providing valuable insight into pathophysiology of the human condition. As a result, SNPs with important functional roles often become subjects for high-throughput experiments.
In this webinar, Daniel Tsang provides an overview of genotyping using real-time PCR (qPCR) technology, including challenges and ways to overcome these challenges. He presents a novel qPCR-based genotyping solution, the rhAmp™ SNP Genotyping System, along with its advantages in genotyping, details on cluster separation, as well as solutions to improve the calling accuracy and confidence of making genotype calls.
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
The rapid increase in throughput of next generation sequencing (NGS) platforms is changing the genomics landscape. Typically, adapters containing sample indexes are added during library construction to allow multiple samples to be sequenced in parallel. Some strategies also introduce a unique molecular identifier (UMI) within the adapter to correct for PCR and sequencing errors. When a UMI is added, reads are assigned to each sample based on their associated sample index, and the UMI is used for error correction during data analysis. For simplicity, a single adapter that is suitable for a variety of applications would be ideal.
xGen® Dual Index UMI Adapters take the guesswork out of adapter design and ordering. These adapters, created for Illumina sequencers, are compatible with standard library preparation methods and may be sequenced in different modes depending on your application. In addition to unique, dual indexes, the adapters contain a molecular barcode in an optional read position. We will discuss how unique, dual indexes mitigate sample index hopping for multiplexed sequencing and demonstrate how UMIs reduce false positives to improve detection of low-frequency variants.
Alzheimer’s disease (AD) is a devastating neurodegenerative disease that is genetically complex. Although great progress has been made in identifying fully penetrant mutations in genes that cause early-onset AD, these still represent a very small percentage of AD cases. Large-scale, genome-wide association studies (GWAS) have identified at least 20 additional genetic risk loci for the more common form: late-onset AD. However, the identified SNPs are typically not the actual risk variants, but are in linkage disequilibrium with the presumed causative variants [1].
To help identify causative genetic variants, we have combined highly accurate, long-read sequencing with hybrid-capture technology. In this collaborative webinar*, we present this method and show how combining IDT xGen® Lockdown® Probes with PacBio SMRT® Sequencing allows targeting and sequencing of candidate genes from genomic DNA and corresponding transcripts from cDNA. Using a panel of target capture probes for 35 AD candidate genes, we demonstrate the power of this approach by looking at data for two individuals with AD. Some additional benefits of this method include the ability to leverage long reads, phase heterozygous variants, and link corresponding transcript isoforms to their respective alleles.
Reference: 1. Van Cauwenberghe C, Van Broeckhoven C, Sleegers K. (2016) The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet Med, 18(5):421–430.
* This presentation represents a collaboration between Pacific Biosciences and Integrated DNA Technologies. The individual opinions expressed may not reflect shared opinions of Pacific Biosciences and Integrated DNA Technologies.
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.
Genomics research and discovery has led to a large increase of reported single nucleotide polymorphisms (SNPs). From 2006 to 2017, the number of refSNPs in the NCBI dbSNP database has increased 13-fold. Many polymorphisms can be linked to disease susceptibility and responses to chemical therapies. Other polymorphisms are used as trait identifiers in livestock and plants. Being able to inexpensively and accurately determine the genotype in high-throughput fashion, with low sample input is a critical need in current, large-scale screening efforts. In this presentation, we present a novel, probe-based, PCR genotyping solution that possesses the universal cycling conditions, strong signal generation, and benchtop reaction stability needed for high-throughput screening. We also present the mechanism and unique technical advantages of using the rhAmp SNP Genotyping System, and we will illustrate how easy it is to generate high quality genotyping data.
The increasing throughput of NGS platforms has fueled the demand to sequence many samples in parallel, also referred to as multiplex sequencing. During multiplex sequencing, the identity of each sample library within a pool is maintained using index sequences that are subsequently separated in a process called demultiplexing during data analysis. Historically, a relatively small number of unique sequences (8 x i5 and 12 x i7) were used to create index combinations to multiplex samples. Unfortunately, with this combinatorial approach, a single index swap may cause a read to be mis-assigned to a different sample causing cross-talk. In this presentation, we discuss some sources of sample cross-talk, including index hopping during cluster amplification or multiplexed capture, and how index sequencing errors may lead to demultiplexing mistakes. We discuss how sample cross-talk causes demultiplexing errors and present a method for increasing the accuracy of sample identification using unique, dual-matched index adapters.
The document discusses exome sequencing and compares the performance of the xGen Exome Research Panel to other commercial exome sequencing panels. Key points:
1) An independent study directly compared the xGen panel to 3 other commercial exome panels and found that the xGen panel had a higher on-target rate and more uniform coverage than the other panels.
2) When deeply sequenced, the xGen panel was able to achieve greater than 20x coverage of over 94% of bases in the RefSeq database with only 40 million reads, which is 2.5-4 times fewer reads than the other panels tested.
3) The coverage profile produced by the xGen panel more closely resembled whole genome sequencing
The document provides an overview of gene knockout and homology-directed repair using CRISPR. It discusses designing guide RNAs and comparing delivery methods like lipofection, electroporation, and microinjection. It also covers designing repair templates for homology-directed repair to insert or change DNA sequences. Optimization of guide RNAs, delivery method, and repair template design can improve genome editing efficiency.
The CRISPR-Cpf1 nuclease is the best alternative to the commonly used Cas9 for genome editing. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence of TTTV, which differs from the Cas9 PAM sequence, NGG. Having Cpf1 as a second option increases the likelihood of editing as close as possible to your desired target site. In this presentation, Dr Rolf Turk introduces the optimized Alt-R™ CRISPR-Cpf1 System and explains how it can be used as a powerful new tool for your genome editing research. Dr Turk presents the basics of the system, as well as protocols for getting started in genome editing.
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.
The National Center for Biotechnology Information (NCBI) provides one of the most extensive sets of web-based tools for biological research. The tools are indispensable when planning genomics experiments, including for qPCR, NGS, and CRISPR. In this presentation, Dr Matt McNeill takes a practical look at getting started with the wealth of NCBI tools, and shares some relevant tips to help you sift through the tools and options that we regularly use. In particular, he focuses on commonly adjusted parameters that will allow you to more effectively use the powerful Basic Local Alignment Algorithm Tool (BLAST) to identify off-target hybridization/annealing events. Dr McNeill also covers practical examples using NCBI tools to design assays.
Adam Clore presents on IDT's gBlocks Gene Fragments, which are synthetic linear DNA fragments up to 2000 base pairs in length that are high fidelity and sequence verified. Some key applications of gBlocks Gene Fragments include gene and vector construction, metabolic engineering, viral and vaccine development, protein evolution, next generation sequencing controls, antibody expression, PCR controls, CRISPR/Cas9 genome editing, and homology directed repair. IDT offers design support and online tools to assist customers with ordering and designing gBlocks Gene Fragments.
Struggling with low editing efficiency or delivery problems? IDT has developed a simple and affordable CRISPR-Cas9 solution that outperforms other methods. In this presentation we present the advantages of using a Cas9:tracrRNA:crRNA ribonucleoprotein (RNP) complex in genome editing experiments, and explain why it is the most efficient driver for genome editing compared to alternative methods, such as expression plasmids or the use of sgRNAs. We also review RNP delivery using cationic lipids and electroporation, and provide tips for optimized transfection in your system.
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
Dr Adam Clore discusses uses for gBlocks® Gene Fragments in the context of the 2015 iGEM competition. Dr Clore also describes how iGEM teams can register to receive 20 kb of free gBlocks Gene Fragments for their projects.
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
• Evidence-based strategies to address health misinformation effectively
• Building trust with communities online and offline
• Equipping health professionals to address questions, concerns and health misinformation
• Assessing risk and mitigating harm from adverse health narratives in communities, health workforce and health system
Cell Therapy Expansion and Challenges in Autoimmune DiseaseHealth Advances
There is increasing confidence that cell therapies will soon play a role in the treatment of autoimmune disorders, but the extent of this impact remains to be seen. Early readouts on autologous CAR-Ts in lupus are encouraging, but manufacturing and cost limitations are likely to restrict access to highly refractory patients. Allogeneic CAR-Ts have the potential to broaden access to earlier lines of treatment due to their inherent cost benefits, however they will need to demonstrate comparable or improved efficacy to established modalities.
In addition to infrastructure and capacity constraints, CAR-Ts face a very different risk-benefit dynamic in autoimmune compared to oncology, highlighting the need for tolerable therapies with low adverse event risk. CAR-NK and Treg-based therapies are also being developed in certain autoimmune disorders and may demonstrate favorable safety profiles. Several novel non-cell therapies such as bispecific antibodies, nanobodies, and RNAi drugs, may also offer future alternative competitive solutions with variable value propositions.
Widespread adoption of cell therapies will not only require strong efficacy and safety data, but also adapted pricing and access strategies. At oncology-based price points, CAR-Ts are unlikely to achieve broad market access in autoimmune disorders, with eligible patient populations that are potentially orders of magnitude greater than the number of currently addressable cancer patients. Developers have made strides towards reducing cell therapy COGS while improving manufacturing efficiency, but payors will inevitably restrict access until more sustainable pricing is achieved.
Despite these headwinds, industry leaders and investors remain confident that cell therapies are poised to address significant unmet need in patients suffering from autoimmune disorders. However, the extent of this impact on the treatment landscape remains to be seen, as the industry rapidly approaches an inflection point.
These lecture slides, by Dr Sidra Arshad, offer a quick overview of the physiological basis of a normal electrocardiogram.
Learning objectives:
1. Define an electrocardiogram (ECG) and electrocardiography
2. Describe how dipoles generated by the heart produce the waveforms of the ECG
3. Describe the components of a normal electrocardiogram of a typical bipolar lead (limb II)
4. Differentiate between intervals and segments
5. Enlist some common indications for obtaining an ECG
6. Describe the flow of current around the heart during the cardiac cycle
7. Discuss the placement and polarity of the leads of electrocardiograph
8. Describe the normal electrocardiograms recorded from the limb leads and explain the physiological basis of the different records that are obtained
9. Define mean electrical vector (axis) of the heart and give the normal range
10. Define the mean QRS vector
11. Describe the axes of leads (hexagonal reference system)
12. Comprehend the vectorial analysis of the normal ECG
13. Determine the mean electrical axis of the ventricular QRS and appreciate the mean axis deviation
14. Explain the concepts of current of injury, J point, and their significance
Study Resources:
1. Chapter 11, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 9, Human Physiology - From Cells to Systems, Lauralee Sherwood, 9th edition
3. Chapter 29, Ganong’s Review of Medical Physiology, 26th edition
4. Electrocardiogram, StatPearls - https://www.ncbi.nlm.nih.gov/books/NBK549803/
5. ECG in Medical Practice by ABM Abdullah, 4th edition
6. Chapter 3, Cardiology Explained, https://www.ncbi.nlm.nih.gov/books/NBK2214/
7. ECG Basics, http://www.nataliescasebook.com/tag/e-c-g-basics
Recomendações da OMS sobre cuidados maternos e neonatais para uma experiência pós-natal positiva.
Em consonância com os ODS – Objetivos do Desenvolvimento Sustentável e a Estratégia Global para a Saúde das Mulheres, Crianças e Adolescentes, e aplicando uma abordagem baseada nos direitos humanos, os esforços de cuidados pós-natais devem expandir-se para além da cobertura e da simples sobrevivência, de modo a incluir cuidados de qualidade.
Estas diretrizes visam melhorar a qualidade dos cuidados pós-natais essenciais e de rotina prestados às mulheres e aos recém-nascidos, com o objetivo final de melhorar a saúde e o bem-estar materno e neonatal.
Uma “experiência pós-natal positiva” é um resultado importante para todas as mulheres que dão à luz e para os seus recém-nascidos, estabelecendo as bases para a melhoria da saúde e do bem-estar a curto e longo prazo. Uma experiência pós-natal positiva é definida como aquela em que as mulheres, pessoas que gestam, os recém-nascidos, os casais, os pais, os cuidadores e as famílias recebem informação consistente, garantia e apoio de profissionais de saúde motivados; e onde um sistema de saúde flexível e com recursos reconheça as necessidades das mulheres e dos bebês e respeite o seu contexto cultural.
Estas diretrizes consolidadas apresentam algumas recomendações novas e já bem fundamentadas sobre cuidados pós-natais de rotina para mulheres e neonatos que recebem cuidados no pós-parto em unidades de saúde ou na comunidade, independentemente dos recursos disponíveis.
É fornecido um conjunto abrangente de recomendações para cuidados durante o período puerperal, com ênfase nos cuidados essenciais que todas as mulheres e recém-nascidos devem receber, e com a devida atenção à qualidade dos cuidados; isto é, a entrega e a experiência do cuidado recebido. Estas diretrizes atualizam e ampliam as recomendações da OMS de 2014 sobre cuidados pós-natais da mãe e do recém-nascido e complementam as atuais diretrizes da OMS sobre a gestão de complicações pós-natais.
O estabelecimento da amamentação e o manejo das principais intercorrências é contemplada.
Recomendamos muito.
Vamos discutir essas recomendações no nosso curso de pós-graduação em Aleitamento no Instituto Ciclos.
Esta publicação só está disponível em inglês até o momento.
Prof. Marcus Renato de Carvalho
www.agostodourado.com
Basavarajeeyam is an important text for ayurvedic physician belonging to andhra pradehs. It is a popular compendium in various parts of our country as well as in andhra pradesh. The content of the text was presented in sanskrit and telugu language (Bilingual). One of the most famous book in ayurvedic pharmaceutics and therapeutics. This book contains 25 chapters called as prakaranas. Many rasaoushadis were explained, pioneer of dhatu druti, nadi pareeksha, mutra pareeksha etc. Belongs to the period of 15-16 century. New diseases like upadamsha, phiranga rogas are explained.
Osteoporosis - Definition , Evaluation and Management .pdfJim Jacob Roy
Osteoporosis is an increasing cause of morbidity among the elderly.
In this document , a brief outline of osteoporosis is given , including the risk factors of osteoporosis fractures , the indications for testing bone mineral density and the management of osteoporosis
8 Surprising Reasons To Meditate 40 Minutes A Day That Can Change Your Life.pptxHolistified Wellness
We’re talking about Vedic Meditation, a form of meditation that has been around for at least 5,000 years. Back then, the people who lived in the Indus Valley, now known as India and Pakistan, practised meditation as a fundamental part of daily life. This knowledge that has given us yoga and Ayurveda, was known as Veda, hence the name Vedic. And though there are some written records, the practice has been passed down verbally from generation to generation.
Rasamanikya is a excellent preparation in the field of Rasashastra, it is used in various Kushtha Roga, Shwasa, Vicharchika, Bhagandara, Vatarakta, and Phiranga Roga. In this article Preparation& Comparative analytical profile for both Formulationon i.e Rasamanikya prepared by Kushmanda swarasa & Churnodhaka Shodita Haratala. The study aims to provide insights into the comparative efficacy and analytical aspects of these formulations for enhanced therapeutic outcomes.
Basavarajeeyam is a Sreshta Sangraha grantha (Compiled book ), written by Neelkanta kotturu Basavaraja Virachita. It contains 25 Prakaranas, First 24 Chapters related to Rogas& 25th to Rasadravyas.
Improved Anti-miRNA (AMOs) and Splice-Switching Oligonucleotides (SSOs)
1. Integrated DNA Technologies
Improved Anti-miRNA (AMOs) and Splice-
Switching Oligonucleotides (SSOs)
Mark Behlke MD, PhD Chief Scientific Officer
Biopolis, Singapore July 26, 2013
2. Inhibition of miRNAs by Antisense Oligonucleotides
RISC
RNA Induced
Silencing Complex
Target mRNA
Inhibit translation, mRNA cleavage …
AAAAA
Transfect AMO
miRNA
Steric blocking
Eventual Degradation?
2
3. Role of Chemical Modifications
1. Nuclease Stabilization
2. Increased binding affinity
a. Tighter binding greater potency
b. Tighter binding decreased specificity
3. Compatible with invasion of RISC?
4. Assist with delivery?
3
6. Newer AMO designs
Original “antagomir”
M*M*MMMMMMMMMMMMMMMMMM*M*M*M-Chol
M = 2’OMe
m = 2’MOE
F = 2’F
D = DNA
L = LNA
* = PS bond
Chemistries used in anti-miRNA Oligos (AMOs)
6
DLDDLDDLDDLDDLDDLDDLDD
D*D*L*D*D*L*D*D*L*D*D*L*D*D*L*D*D*L*D*D
L*D*L*D*D*L*L*D*D*L*D*L*D*L*L
m*m*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*F*m*m
M
MMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMMM
M
MMMMMM MMMMMM
L*L*L*L*L*L*L*L*L
7. Luc/Luc Assay System
One perfect match miRNA binding site was cloned
Renilla
luciferase
Firefly
luciferase
miRNA
+ AMO
Translation
Light
miRNA
Renilla
Luciferase
miRNA
Binding
Site
Transfected into Cells
+
miRNA
Degradation
Renilla Luc
No Light
Cleavage
7
9. Unmodified DNA or 2’OMe oligos are rapidly degraded in
serum – need PS modification or hairpin
(Note: unmodified 2’OMe is stable in cell extracts, even though
it degrades in serum)
Stability in Serum
9
10. Interestingly, the DNA/LNA mixmers also require some PS
modification (at least on the ends)
(Note: unmodified are all stable in cell extracts)
Stability in Serum
10
11. Interestingly, the DNA/LNA mixmers also require some PS
modification (at least on the ends)
(Note: 2’F without PS rapidly degrades in cell extracts)
Stability in Serum
Degraded in
cell extracts
11
13. Insertion of “ZEN” between bases increases duplex stability
Temperature (oC)
20 30 40 50 60 70 80
%meltedduplex
0
20
40
60
80
100
Unmod DNA
Internal ZEN
5’-ATCGTTGCTA-3’
3’-TAGCAACGAT-5’
5’-ATCGTzTGCTA-3’
3’-TAGCA ACGAT-5’
vs.
+ ZEN
13
14. 2’OMe RNA is:
Natural
Safe / Nontoxic
Less expensive than LNAs or 2’F
Degraded by exonucleases in serum
Stable to endonucleases in cell extracts
The new napthyl-azo modification increases
Tm (PS decreases Tm), blocks exonuclease
action, and is compatible with RISC invasion
MzMMMMMMMMMMMMMMMMMMMMMzM
2’OMe with new napthyl-azo (“ZEN”) modifier between end bases
O P
O
O-
O
3'
N
O
N
N
5'
NO2
14
16. Small DTm can result in large DKa at 37oC
16
DNA t c a a c a t c a g t c t g a t a a g c t a 56.3 -16.4 -18.7 1.5 x1013
2′OMe U C A A C A U C A G U C U G A U A A G C U A 72.7 - -26.9 9.4 x1018
2′OMe 3PSends U*C*A*A C A U C A G U C U G A U A A G*C*U*A 72.2 -0.5 -26.4 4.3 x1018
2′OMe 5′inZEN,3′ZEN UzC A A C A U C A G U C U G A U A A G C U Az 76.3 3.6 -30.6 3.8 x1021
Ka(37°C)
(mol/L)-1Name miR-21 AMO Sequences (5′ to 3′) Tm (°C) ΔTm (°C)
ΔG o
37
(kcal/mol)
A 4.1oC increase in Tm between the 2’OMe-PSends AMO
and the ZEN-AMO results in an 880-fold increase in the
binding affinity (Ka) at 37oC
17. Importance of binding affinity for AMO potency
17
• It is generally accepted that high binding affinity improves potency for all steric
blocking antisense applications (AMO, SSO, mRNA …)
• miRNAs reside in RISC (complexed to protein) and can be stable for weeks. It is
critical to be able to invade RISC and inactivate these miRNAs
• miRNAs start as dsRNAs and get reduced to ssRNA form in RISC – thus RISC has
machinery that renders the miRNA duplex single-stranded : the AMO must
overcome these natural pathways so it does not get treated like a passenger strand
• Nuclease “slicer” function in Ago2
• Helicase “unwinding” of duplexes in Ago1, Ago2, Ago3, Ago4
• Thus the AMOs need to be nuclease resistant (cannot be cut by Ago2)
• Thus the AMOs need high enough binding affinity to overcome helicase activity
• After you reach the “threshold Tm” where helicase can no longer unwind the AMO from
the miRNA guide strand, then increases in binding affinity mostly serve to make cross-
reactivity for mismatches worse
18. miR-21 AMO length walk
18
For the miR-21 AMO with ZEN-2’OMe chemistry, the binding affinity threshold
to escape helicase unwinding must lie between the 14mer & 15mer
19. Specificity comparison of AMO chemistries
19
Mutant Type
Wildtype
MUT 1
MUT 2
MUT 3
a
Mutations are notated as blue nucleotides enclosed in red boxes
U C A A C A U C A G U C U G A U A A G C U A
U C A A C A U C A G U C A G A U A A G C U A
U C A A C C U C A G U C A G A U A A G C U A
U C A A C C U C A G U C A G A U A A C C U A
miR-21 AMO Sequences (5′ to 3′)
a
ZEN-2’OMe DNA/LNA-PS 2’OMe/LNA-PS2’OMe-PSends
“Antagomir”
20. ZEN is non-toxic, whereas PS mod and LNA mod show toxicity
2’OMe 2-Z ■Z■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■Z■
2’OMe 3PS-ends ■•■•■•■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■•■•■•■
DNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■
DNA/LNA PO ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
DNA/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■
2’OMe/LNA PO ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■ ■
2’OMe/LNA PS ■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■•■
■ = DNA bases
■ = 2’OMe bases
■ = LNA bases
Z = ZEN mod
• = PS linkages
20
0
20
40
60
80
100
120
%ViableCells
Cytotoxicity Data of "NC1" AMOs transfected
into HeLa Cells for 24hrs with INTERFERin™
50nM
100nM
2'OMe
2-Z
Reagent
Only
2'OMe
3PSends
DNA/
LNA PS
PS DNA/
LNA PO
2'OMe
LNA PS
2'OMe/
LNA PO
Stauro-
sporine
Pos Cont
22. Potency differences do not relate to transfection efficiency
22
Different AMOs were
transfected at 30 nM.
AMO transfection efficiency
was assessed using ISH
(in situ hybridization).
Cells were fixed and
hybridized with a Dig-probe
and detected with an
A647-anti-Dig antibody
23. Final design rules
MzMMMMMMMMMMMMMMMMMMMMz (N-1)-length
MzMMMMMMMMMMMMMMMMMMMMzM Full-length
Either during transfection (in serum) or exposure to cytoplasmic
nucleases, the first AMO gets degraded to the second AMO. Positioning the
ZEN at the 3’-end removes most of the Nearest Neighbor effects and avoids
any degradation. Making the final AMO be 1 base shorter than the miRNA
target can also slightly increase potency.
23
25. Insulin Regulation in Pancreatic Islets
Eran Hornstein
Weizmann Institute
• Investigated a possible role for miRNAs in the
regulation of insulin secretion
• Dicer1 conditional KO using tamoxifen inducible Cre
recombinase with rat insulin promoter
• Examine changes in insulin levels and glucose
regulation with miRNA production disrupted
• Identified SOX6 and Bhlhe22 as negative regulators of
insulin secretion, with miRNAs 22, 24, 148, and 182
regulating expression of these genes, thereby indirectly
regulating insulin secretion
25
26. Knockout of Dicer leads to reduction in miRNA levels
Studied in isolated islets; b-cells comprise ~50-60% of cell mass
26
28. Up-regulation of transcription repressors in Dicer mutants
Sox6 and Bhlhe22 are repressors of insulin transcription.
Increased levels of repressors lower insulin and raise glucose levels.
28
29. miRNA regulation of Sox6 and Bhlhe22?
• miRNA expression in pancreatic islets
was examined using microarrays
• Potential binding sites for several
highly expressed miRNAs were found
in the 3’-UTRs of Sox6 and Bhlhe22
• AMOs were synthesized to specifically
suppress these miRNAs to investigate
the role of these species in normal
cells (not Dicer mutants)
29
33. Regulation of Cystic Fibrosis (CFTR)
33
• Investigated role of miRNAs in regulation of CFTR
expression and found a major role for miR-138
• miR-138 does not directly regulate CFTR but instead
acts as a suppressor of SIN3A, which is a suppressor of
CFTR transcription
• miR-138 AMO increases SIN3A levels which lowers
CFTR levels
• miR-138 mimic lowers SIN3A levels which raises CFTR
levels (same effect is seen using anti-SIN3A siRNA)
• Not just transcription/translation effect – salvages D508
mutants!
Paul McCray
University of Iowa
35. SIN3A knockdown rescues CFTR D508 expression on cell surface
35
The D508 mutant retains Cl- channel activity, but is degraded in the EPR
and never reaches the cell surface; SIN3A knockdown not only increases
CFTR expression, it alters processing and permits the semi-functional
mutant CFTR protein to reach the cell surface.
36. miR-138 mimic restores Cl- conductance in CF airway cells
36
A new target for CF therapy?
38. Use of the ZEN modification: Splice Switching Oligos (SSOs)
Another use for this kind of antisense technology: SSOs
• Steric blocking mechanism of action
• 2’OMe RNA, LNA, PMO, PNA
• Bypass stop codon or other errors present by causing
splicing to shift and deleting the affected mutation. Many
diseases exist which could be treated by this
mechanism, including DMD, SMA, b-Thalassemia, and many
more
38
39. Duchenne Muscular Dystrophy (DMD)
Genetics – An X-linked recessive disorder
affecting approx 1 in 3500 boys
Cause - An absence of dystrophin, a protein
that helps keep muscle cells intact.
Onset - Early childhood - about 2 to 6
years.
Symptoms - Generalized weakness and
muscle wasting first affecting the muscles
of the hips, pelvic area, thighs and
shoulders. Loss of
ambulation, development of respiratory
problems (diaphragm), cardiomyopathy
and death in 20’s/30’s.
39
40. Collaboration with the Wood lab to study SSO in “mdx” mouse
C57BL/10ScSn-Dmdmdx/J mouse
• Stop codon in Exon 23
• Develops DMD phenotype
• Salvage with exon skipping SSOs = skip exon 23 and
you get an in-frame semi-functional dystrophin
protein
• PCR assay:
• Full length = 1kb product
• Skip exon 23 = 700 bp product
• Skip exons 22+23 = 550 bp product
Samir EL Andaloussi
Suzan Hammond
Graham McClorey
Matthew Wood
40
41. H2k (myoblast) cell culture
• H2k cells were grown at 33°C in 10% CO2 atmosphere using DMEM media
supplemented with 20% FBS and 0.5% chick embryo serum; grown on gelatinised
plates.
• After 24 h, cells were moved to 37°C in 5% CO2 and media is replaced with
differentiation media (DMEM with 5% horse serum). Myotubes form within 3-5
days. These are very difficult to transfect compared to the undifferentiated
myoblasts.
• Cells were transfected with LF2000 at the indicated concentrations or naked SSOs
are added at 2-4 µM concentration and cells were incubated for 48-96 h in
optiMEM.
41
42. PS linkage is important for function (not just nuclease stability)
80 40 20 10 80 40 20 10 U
PS20 EndPS
nM
Myoblasts, lipid transfection, studied at 48h
SSO-EndPS
mG*mG*mC*mC mA mA mA mC mC mU mC mG mG mC mU mU mA*mC*mC*mU
SSO-PS
mG*mG*mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC*mC*mU 42
43. Unlike AMOs, PS linkage is important for function in ZEN SSOs
Myoblasts, lipid transfection, studied at 48h
SSO-ZEN
mGzmG mC mC mA mA mA mC mC mU mC mG mG mC mU mU mA mC mCzmU
SSO-ZEN-PS
mGzmG mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC mCzmU
80 40 20 10 80 40 20 10 U
ZEN-PO ZEN-PS
nM
43
44. Pilot study: direct intramuscular injection in “mdx” mice
2’OMePS ZENPS
SSO-PS
mG*mG*mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC*mC*mU
SSO-ZEN-PS
mGzmG mC*mC*mA*mA*mA*mC*mC*mU*mC*mG*mG*mC*mU*mU*mA*mC mCzmU
• IM injection of 30 µg SSO
• Harvest muscle at day 14
• Immunohistochemical
visualization of dystrophin
protein
44
45. Functional testing in “mdx” mice
• Plan: 50 mg/kg IV weekly for 8 weeks
• Reality: 50 mg/kg IV 2x weekly for 4 weeks
1 week off
50 mg/kg IP 2x weekly for 4 weeks
Study phenotype for a week, then collect tissue
• 4 cohorts, WT untreated, “mdx” untreated, “mdx” 2’OMe-PS, “mdx” ZEN
• Study animals for functional activity
The monitoring system provides an assessment of the motor activity and behavior of
the mice, measuring both anxiety-related behavior and locomotor behavior associated
with muscle strength. IR beams of light pass through the cage. When the mouse
crosses a beam, the light is broken and this is recorded in the software, Digiscan. The
system measures 22 forms of activity including rearing, active time, static time as well
as distance travelled.
• Examine muscles for dystrophin protein and mRNA splice forms
45
46. Functional testing in “mdx” mice
• Expt was not optimal – think that
the IV phase worked but IP did not
• No splice-shifted mRNA was
detected (short half life)
• Dystrophin protein was present (long
half life)
• Repeating with a 4 week IV regimen
46
47. New ZEN (napthyl-azo) modifier inserted between the terminal
bases of a steric-blocking antisense oligo improves nuclease stability
and increases binding affinity
Particularly useful when used with 2’OMe RNA; anti-miRNA (AMO;
PO form) and splice switching (SSO; PS form) applications; more?
The designs show excellent mismatch specificity, similar to low
potency unmodified 2’OMe AMOs, yet achieve the high level of
potency normally associated with use of more toxic DNA/LNA-PS
mixmers
Both ZEN-AMOs and ZEN-SSOs are being tested in mice now
Available now “off catalog” – just call IDT Tech Support. Full product
line in catalog will be available later this year.
Summary
47
48. Integrated DNA Technologies
Kim Lennox
Scott Rose
Richard Owczarzy
Yong You
Mike Marvin
Anton Holets
Jess Alexander
Joseph Walder
Mark Behlke
Weizmann Institute
Tal Melkman-Zehavi
Sharon Kredo-Russo
Amitai Mandelbaum
Eran Hornstein
Thanks to all the scientists whose work was discussed today!
University of Iowa
Shyam Ramachandram
Michael Welch
Paul McCray
Bev Davidson
48
University of Oxford
Samir EL Andaloussi
Suzan Hammond
Graham McClorey
Matthew Wood