This document summarizes a student's research project on the role of survivin in gastric wound healing. Survivin inhibits apoptosis and cell division and may also be involved in angiogenesis and cell migration during wound healing. The student aims to prove survivin's critical role in these processes. Their methods involve cloning promoters for genes related to blood vessels and the stomach into lentiviral vectors. They successfully cloned the promoters for VE-cadherin and EGFR and will next insert these into CRISPR/Cas9 and other vectors. The overall research aims to further understanding of gastric wound healing and could translate to new treatment strategies.
Molecular Characterization of Polyglucosan Body, Cause or Consequence in the ...Ben Decker
This document describes research into polyglucosan body disease (PGBD), caused by glycogen branching enzyme (GBE) deficiency. Researchers generated two mouse models of PGBD - one with complete GBE loss and one with 20% residual activity. Both models accumulated polyglucosan (insoluble glucose polymer) in tissues like liver, heart and muscle, similar to human PGBD. The models showed physical impairment and altered glucose metabolism. The findings support that these mouse models can help study polyglucosan accumulation and determine if it is a cause or consequence of PGBD.
This study investigated the effects of lyophilized platelet-rich plasma (PRGF) on osteoblast-like MG-63 cells. The results showed that PRGF significantly increased cell proliferation, as measured by total protein content, and alkaline phosphatase (ALP) activity, a marker of osteoblast function. Specifically, cells treated with PRGF for 6 days in low-serum or serum-free medium had higher ALP activity and protein levels compared to controls. This suggests PRGF supports osteoblast proliferation and differentiation.
This document provides an overview of proteomics and protein analysis techniques. It discusses how the proteome represents all proteins expressed by a genome and how protein expression changes with health, disease, and toxicity. It also describes seven attributes of proteins, including identity, quantity, post-translational modifications, structure, interactions, spatial relationships, and function. Common techniques for protein analysis are also summarized, such as chromatography, mass spectrometry, crystallization, and sequencing. Chromatography methods separate proteins based on properties like size, charge, hydrophobicity, and specific binding interactions.
Proposal for Protein-DNA Mapping using AFM for Lab on a ChipAnthony Salvagno
This was a report written for a class I am taking in conjunction with Ken Seal, John Montoya, and Laura Pawlikowski. We discuss functionalizing an AFM tip with antibodies for protein detection. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins). This proposal details everything we imagined. We presented on this topic as well.
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
Lectut btn-202-ppt-l34. applications of site-directed mutagenesisRishabh Jain
1. Protein engineering involves using genetic manipulations to alter a gene's coding sequence and thus change a protein's properties. This allows improving properties like stability, purity, activity and modifying functions.
2. Stability of enzymes can be increased by adding disulfide bonds or replacing unstable amino acids. For example, mutating xylanase increased its stability at high temperatures.
3. Human pancreatic ribonuclease was modified through site-directed mutagenesis to form a dimer linked by disulfide bonds, creating an effective anti-cancer agent with increased solubility.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
Molecular Characterization of Polyglucosan Body, Cause or Consequence in the ...Ben Decker
This document describes research into polyglucosan body disease (PGBD), caused by glycogen branching enzyme (GBE) deficiency. Researchers generated two mouse models of PGBD - one with complete GBE loss and one with 20% residual activity. Both models accumulated polyglucosan (insoluble glucose polymer) in tissues like liver, heart and muscle, similar to human PGBD. The models showed physical impairment and altered glucose metabolism. The findings support that these mouse models can help study polyglucosan accumulation and determine if it is a cause or consequence of PGBD.
This study investigated the effects of lyophilized platelet-rich plasma (PRGF) on osteoblast-like MG-63 cells. The results showed that PRGF significantly increased cell proliferation, as measured by total protein content, and alkaline phosphatase (ALP) activity, a marker of osteoblast function. Specifically, cells treated with PRGF for 6 days in low-serum or serum-free medium had higher ALP activity and protein levels compared to controls. This suggests PRGF supports osteoblast proliferation and differentiation.
This document provides an overview of proteomics and protein analysis techniques. It discusses how the proteome represents all proteins expressed by a genome and how protein expression changes with health, disease, and toxicity. It also describes seven attributes of proteins, including identity, quantity, post-translational modifications, structure, interactions, spatial relationships, and function. Common techniques for protein analysis are also summarized, such as chromatography, mass spectrometry, crystallization, and sequencing. Chromatography methods separate proteins based on properties like size, charge, hydrophobicity, and specific binding interactions.
Proposal for Protein-DNA Mapping using AFM for Lab on a ChipAnthony Salvagno
This was a report written for a class I am taking in conjunction with Ken Seal, John Montoya, and Laura Pawlikowski. We discuss functionalizing an AFM tip with antibodies for protein detection. Conceptually, proteins bound to DNA can be flowed down a nanochannel and can be detected by an AFM tip in the channel. The detection will be based on the interaction between antibodies and their antigens (the proteins). This proposal details everything we imagined. We presented on this topic as well.
A suppressor mutation counters the effects of an original mutation by restoring the wild-type phenotype. There are two main types of suppressor mutations: intragenic mutations occur within the same gene and restore function through alternate amino acid substitutions, while intergenic mutations occur elsewhere in the genome and restore function through interacting gene products. Suppressor mutations are useful for studying protein-protein interactions and dissecting biological pathways.
Lectut btn-202-ppt-l34. applications of site-directed mutagenesisRishabh Jain
1. Protein engineering involves using genetic manipulations to alter a gene's coding sequence and thus change a protein's properties. This allows improving properties like stability, purity, activity and modifying functions.
2. Stability of enzymes can be increased by adding disulfide bonds or replacing unstable amino acids. For example, mutating xylanase increased its stability at high temperatures.
3. Human pancreatic ribonuclease was modified through site-directed mutagenesis to form a dimer linked by disulfide bonds, creating an effective anti-cancer agent with increased solubility.
This document provides an outline for a presentation on directed evolution. It discusses the process of directed evolution, which involves randomly introducing mutations at the genetic level followed by selection of variants with desired protein characteristics. The document also covers types of mutations, naturally evolutionary processes like random mutagenesis and gene recombination that directed evolution mimics, library size, selection and screening strategies, applications, and advantages of directed evolution over rational design.
protein engineering and site directed mutagenesisNawfal Aldujaily
This document discusses various techniques for protein engineering and site-directed mutagenesis. It describes altering the sequence of proteins through genetic engineering to improve properties like stability and changing amino acids near the active site to modify enzyme specificity. Directed evolution and DNA shuffling techniques are also discussed that introduce mutations and recombine protein domains to generate novel proteins with optimized functions.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This study aims to analyze the distribution of 5-hydroxymethylcytosine (5-hmC) in the hippocampus of an Alzheimer's mouse model compared to healthy mice. DNA will be isolated from the hippocampus and analyzed using a microarray containing over 20,000 promoters and 15,000 CpG islands. Antibodies specific to 5-methylcytosine and 5-hmC will isolate DNA fragments containing these modifications, which will then be amplified and compared between the transgenic and healthy mice to assess epigenetic changes associated with Alzheimer's Disease. The results are expected to show increases, decreases, or no change in 5-hmC levels in the transgenic mouse model compared to controls.
A tyrosine kinase is an enzyme that transfers a phosphate group from ATP to tyrosine residues on proteins. This phosphorylation regulates protein activity and signal transduction within cells. Tyrosine kinase inhibitors, like nilotinib, are drugs that bind to and inhibit tyrosine kinases. Nilotinib was approved to treat chronic myeloid leukemia and research found it was effective against drug-resistant forms of the disease. It works by binding the inactive form of the Abl kinase to prevent phosphorylation and cancer cell growth.
Response of egfr agents with chemotheraupatic drugs on m crcHari Prakash
Cetuximab, an EGFR inhibitor drug, has shown some effectiveness against metastatic colorectal cancer (mCRC) when used in combination with chemotherapy, according to clinical trials. Two trials found that cetuximab combined with FOLFOX or FOLFIRI reduced disease progression compared to chemotherapy alone for patients without KRAS mutations. A third trial found that the combination of cetuximab and irinotecan led to higher response rates and longer progression-free survival than cetuximab alone for patients with the rare KRAS G13D mutation. Therefore, the combination of cetuximab with chemotherapy may provide benefit for mCRC patients with certain rare KRAS mutations.
This document discusses two main theories for how enzyme systems evolved: retrograde evolution and patchwork evolution. Retrograde evolution proposes that pathways evolve backwards, with newer reactions preceding existing ones. Patchwork evolution suggests that enzymes initially had broad specificities and specialized over time through gene duplication. While some evidence supports retrograde evolution, more data favors patchwork evolution. However, the CS2 hydrolase enzyme appears to be an exception, as its structure and function indicate it did not evolve but was purposefully designed. In conclusion, the theories of enzyme evolution are still being refined as exceptions are discovered.
This document summarizes protein engineering techniques. It discusses:
1. The process of protein engineering involves diversification of genes through random mutation/recombination, selection of variants with desired properties, and amplification of selected variants.
2. Examples of techniques used for diversification include error-prone PCR and DNA shuffling. Screens and selections are used to identify variants with improved properties.
3. Multienzyme systems can be artificially synthesized through gene fusion, joining genes to create a single polypeptide with active sites from both enzymes. This allows proximity of enzymes to catalyze sequential reactions.
This document discusses site-directed mutagenesis and protein engineering. It provides an introduction to mutagenesis and defines site-directed mutagenesis. Various methods for site-directed mutagenesis are described, including using M13 bacteriophage, plasmid DNA, and PCR. Examples are given of using site-directed mutagenesis to modify lysozyme, xylanase, human pancreatic ribonuclease, and subtilisin proteins to improve properties like thermal stability and metal binding.
DESH BANDHU GANGWAR presentation on Somatic hypermutationDESH BANDHU GANGWAR
Somatic hypermutation (SHM) introduces mutations in antibody genes in B cells, increasing antibody diversity. SHM occurs at a rate 10,000 times higher than the normal mutation rate. These mutations mainly occur in hypervariable regions of antibody genes and result from nucleotide substitutions. Molecules involved in SHM include activation-induced cytidine deaminase and uracil N-glycosylase, which initiate mutations, and mismatch repair proteins, which normally eliminate mutations but instead increase the mutation rate during SHM. Future directions include using SHM to evolve proteins in mammalian cells by integrating target genes in B cells and selecting cells with desired phenotypes.
The complement system is an important part of the innate immune system that promotes clearance of foreign particles and initiation of adaptive immune responses. It involves around 30 cell-associated and fluid phase proteins that are activated in a cascade of enzyme reactions. There are three pathways of complement activation - the classical, lectin, and alternative pathways. Complement activity is tightly regulated to protect host cells, and regulation occurs through mechanisms such as component instability, differences in cell surface carbohydrates between microbes and host cells, and regulatory proteins that inhibit or destroy complement components.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
This document discusses tyrosine kinase inhibitors (TKIs), a class of targeted cancer drugs. It begins by introducing protein kinases and their role in cell signaling. There are two main categories of protein kinases - those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. Tyrosine kinases function as on/off switches in many cellular functions by adding phosphate groups to tyrosine residues on proteins. The document then discusses the different types of tyrosine kinases and how they can become mutated and cause unregulated cell growth leading to cancer. It describes targeted therapy and TKIs as targeted drugs that block specific molecules needed for tumor growth. The final sections provide examples of approved TKIs
Stem cells can differentiate into various cell types and are important for understanding diseases and developing therapies. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a molecule that regulates the expression of Sox9, a key factor in cartilage development. Methods included RT-PCR to analyze Sox9 mRNA expression, western blot to detect Sox9 proteins, ELISA to analyze protein activities, and immunofluorescence to observe GFP expression under Sox9 promoter treatment. Results showed the potential of this small molecule to induce chondrocyte differentiation as a future cellular therapy for osteoarthritis.
The document discusses the complement system and immunoglobulins. It provides an overview of:
- The components and pathways of the complement system and its role in host defense.
- The structure and classes of immunoglobulins, including IgG, IgA, IgM, IgE, and IgD.
- Deficiencies in the complement system and immunoglobulins that can cause increased susceptibility to infection.
This document discusses directed evolution, which mimics natural selection to evolve proteins or nucleic acids towards a defined goal. It does not create new organisms and focuses on specific molecular properties. The process involves randomly mutating a gene of interest, generating a library of variants, screening or selecting for desired properties, and repeating rounds of mutation and selection until the goal is achieved. Directed evolution was first used in the 1970s and has since advanced protein engineering techniques. It provides a way to customize protein reactions and improve properties like yield, substrate specificity, and stability.
1) The document discusses molecular genetic techniques for isolating and characterizing genes, including the study of mutations, DNA cloning, and recombinant DNA methods.
2) Key terms are defined for genetic analysis, such as alleles, mutants, genotypes, and phenotypes. Methods are described for identifying dominant and recessive mutations through genetic crosses and complementation analysis.
3) Techniques like conditional mutations, suppressor mutations, and synthetic lethal analysis are explained for studying essential genes and protein interactions. DNA cloning is introduced, involving restriction enzymes to cut DNA and ligases to join DNA fragments into vectors.
This document discusses how cancer cells have altered metabolism that promotes their growth and survival. Oncogenic mutations activate pathways like PI3K/Akt/mTOR that increase nutrient uptake and biosynthesis, putting cancer cells in a constant state of metabolic stress and hunger. This metabolic stress activates autophagy, which recycles cellular components and supports cancer cell metabolism and survival during starvation or chemotherapy. Autophagy degradation provides nutrients and building blocks and removes damaged mitochondria that could increase harmful reactive oxygen species. Cancer cells with oncogenic mutations like in Ras show increased basal autophagy that is essential for their survival. This suggests targeting autophagy may be an effective strategy to treat cancers dependent on it.
This study explored designing small molecule inhibitors of the NRMT1 enzyme. NRMT1 is an important methyltransferase enzyme that transfers methyl groups from SAM to its substrate RCC1. The deregulation of NRMT1 has been observed in cancers. Using molecular modeling programs Sybyl and GOLD, 14 small molecules were designed that were predicted to bind in the RCC1 binding site of NRMT1 and suppress its activity. The molecules were docked and scored using GOLD, and the top scoring molecules will be further optimized to improve their inhibitory activity against NRMT1.
Soluble protein expression optimizationBiologicsCorp
In many cases, expression of recombinant proteins often results in insoluble and/or nonfunctional proteins. Here, factors in soluble protein expression optimization and several strategies to improve the solubility of the expressed protein are reviewed.
Genetic variations in G protein-coupled receptors (GPCRs) can alter receptor function and cause diseases. Single nucleotide polymorphisms and other mutations have been linked to impaired or enhanced receptor signaling. For example, a mutation in the vasopressin V2 receptor causes nephrogenic diabetes insipidus by decreasing ligand binding and receptor expression. Similarly, mutations in chemokine receptors CCR5 and CCR2 impact HIV infection by altering receptor function or interaction with other coreceptors. Overall, GPCR polymorphisms are associated with diseases by changing ligand binding, receptor activation, trafficking, and coupling to downstream signaling pathways.
This document discusses bioassay development for drug discovery. It covers in vitro and cell-based bioassays, as well as the molecular biology techniques used to develop these assays such as cloning DNA into plasmids, propagating plasmids in bacteria, purifying plasmid DNA, sequencing DNA, and expressing proteins in bacteria and mammalian cells through techniques like transfection. Recombinant protein expression and purification using affinity tags is also summarized.
This document provides an overview of intracellular receptors, enzymes, signals, transcription factors, structural proteins, and nucleic acids. It discusses in detail the structures and functions of DNA, RNA, nuclear receptors, IP3 receptors, intracellular enzymes, transcription factors, and structural proteins like collagen, keratin, myosin, and actin. The presentation was given by S. Dinakar from the Department of Pharmacology at PSG College of Pharmacy in Coimbatore, India.
Directed enzyme evolution is a technique that mimics natural selection to engineer proteins. It involves introducing random mutations into genes and screening proteins for modified activity. The key steps are selecting a gene, creating a library of mutant genes through error-prone PCR or other mutagenesis methods, expressing the proteins, and selecting variants with improved properties. Examples where directed evolution has been applied include improving the activity of enzymes used in producing the antibiotic cephalosporin and in the cholesterol-lowering drug atorvastatin. The goal is to leverage natural selection to develop enzymes with desired industrial applications like increased stability, activity, or substrate specificity.
This study aims to analyze the distribution of 5-hydroxymethylcytosine (5-hmC) in the hippocampus of an Alzheimer's mouse model compared to healthy mice. DNA will be isolated from the hippocampus and analyzed using a microarray containing over 20,000 promoters and 15,000 CpG islands. Antibodies specific to 5-methylcytosine and 5-hmC will isolate DNA fragments containing these modifications, which will then be amplified and compared between the transgenic and healthy mice to assess epigenetic changes associated with Alzheimer's Disease. The results are expected to show increases, decreases, or no change in 5-hmC levels in the transgenic mouse model compared to controls.
A tyrosine kinase is an enzyme that transfers a phosphate group from ATP to tyrosine residues on proteins. This phosphorylation regulates protein activity and signal transduction within cells. Tyrosine kinase inhibitors, like nilotinib, are drugs that bind to and inhibit tyrosine kinases. Nilotinib was approved to treat chronic myeloid leukemia and research found it was effective against drug-resistant forms of the disease. It works by binding the inactive form of the Abl kinase to prevent phosphorylation and cancer cell growth.
Response of egfr agents with chemotheraupatic drugs on m crcHari Prakash
Cetuximab, an EGFR inhibitor drug, has shown some effectiveness against metastatic colorectal cancer (mCRC) when used in combination with chemotherapy, according to clinical trials. Two trials found that cetuximab combined with FOLFOX or FOLFIRI reduced disease progression compared to chemotherapy alone for patients without KRAS mutations. A third trial found that the combination of cetuximab and irinotecan led to higher response rates and longer progression-free survival than cetuximab alone for patients with the rare KRAS G13D mutation. Therefore, the combination of cetuximab with chemotherapy may provide benefit for mCRC patients with certain rare KRAS mutations.
This document discusses two main theories for how enzyme systems evolved: retrograde evolution and patchwork evolution. Retrograde evolution proposes that pathways evolve backwards, with newer reactions preceding existing ones. Patchwork evolution suggests that enzymes initially had broad specificities and specialized over time through gene duplication. While some evidence supports retrograde evolution, more data favors patchwork evolution. However, the CS2 hydrolase enzyme appears to be an exception, as its structure and function indicate it did not evolve but was purposefully designed. In conclusion, the theories of enzyme evolution are still being refined as exceptions are discovered.
This document summarizes protein engineering techniques. It discusses:
1. The process of protein engineering involves diversification of genes through random mutation/recombination, selection of variants with desired properties, and amplification of selected variants.
2. Examples of techniques used for diversification include error-prone PCR and DNA shuffling. Screens and selections are used to identify variants with improved properties.
3. Multienzyme systems can be artificially synthesized through gene fusion, joining genes to create a single polypeptide with active sites from both enzymes. This allows proximity of enzymes to catalyze sequential reactions.
This document discusses site-directed mutagenesis and protein engineering. It provides an introduction to mutagenesis and defines site-directed mutagenesis. Various methods for site-directed mutagenesis are described, including using M13 bacteriophage, plasmid DNA, and PCR. Examples are given of using site-directed mutagenesis to modify lysozyme, xylanase, human pancreatic ribonuclease, and subtilisin proteins to improve properties like thermal stability and metal binding.
DESH BANDHU GANGWAR presentation on Somatic hypermutationDESH BANDHU GANGWAR
Somatic hypermutation (SHM) introduces mutations in antibody genes in B cells, increasing antibody diversity. SHM occurs at a rate 10,000 times higher than the normal mutation rate. These mutations mainly occur in hypervariable regions of antibody genes and result from nucleotide substitutions. Molecules involved in SHM include activation-induced cytidine deaminase and uracil N-glycosylase, which initiate mutations, and mismatch repair proteins, which normally eliminate mutations but instead increase the mutation rate during SHM. Future directions include using SHM to evolve proteins in mammalian cells by integrating target genes in B cells and selecting cells with desired phenotypes.
The complement system is an important part of the innate immune system that promotes clearance of foreign particles and initiation of adaptive immune responses. It involves around 30 cell-associated and fluid phase proteins that are activated in a cascade of enzyme reactions. There are three pathways of complement activation - the classical, lectin, and alternative pathways. Complement activity is tightly regulated to protect host cells, and regulation occurs through mechanisms such as component instability, differences in cell surface carbohydrates between microbes and host cells, and regulatory proteins that inhibit or destroy complement components.
Protein engineering is the process of developing useful or valuable proteins. It is a young discipline, with much research taking place into the understanding of protein folding and recognition for protein design principles
This document discusses tyrosine kinase inhibitors (TKIs), a class of targeted cancer drugs. It begins by introducing protein kinases and their role in cell signaling. There are two main categories of protein kinases - those that phosphorylate tyrosine residues and those that phosphorylate serine and threonine residues. Tyrosine kinases function as on/off switches in many cellular functions by adding phosphate groups to tyrosine residues on proteins. The document then discusses the different types of tyrosine kinases and how they can become mutated and cause unregulated cell growth leading to cancer. It describes targeted therapy and TKIs as targeted drugs that block specific molecules needed for tumor growth. The final sections provide examples of approved TKIs
Stem cells can differentiate into various cell types and are important for understanding diseases and developing therapies. This study focused on differentiating adipose-derived stem cells (ASCs) into functional chondrocytes using a molecule that regulates the expression of Sox9, a key factor in cartilage development. Methods included RT-PCR to analyze Sox9 mRNA expression, western blot to detect Sox9 proteins, ELISA to analyze protein activities, and immunofluorescence to observe GFP expression under Sox9 promoter treatment. Results showed the potential of this small molecule to induce chondrocyte differentiation as a future cellular therapy for osteoarthritis.
The document discusses the complement system and immunoglobulins. It provides an overview of:
- The components and pathways of the complement system and its role in host defense.
- The structure and classes of immunoglobulins, including IgG, IgA, IgM, IgE, and IgD.
- Deficiencies in the complement system and immunoglobulins that can cause increased susceptibility to infection.
This document discusses directed evolution, which mimics natural selection to evolve proteins or nucleic acids towards a defined goal. It does not create new organisms and focuses on specific molecular properties. The process involves randomly mutating a gene of interest, generating a library of variants, screening or selecting for desired properties, and repeating rounds of mutation and selection until the goal is achieved. Directed evolution was first used in the 1970s and has since advanced protein engineering techniques. It provides a way to customize protein reactions and improve properties like yield, substrate specificity, and stability.
1) The document discusses molecular genetic techniques for isolating and characterizing genes, including the study of mutations, DNA cloning, and recombinant DNA methods.
2) Key terms are defined for genetic analysis, such as alleles, mutants, genotypes, and phenotypes. Methods are described for identifying dominant and recessive mutations through genetic crosses and complementation analysis.
3) Techniques like conditional mutations, suppressor mutations, and synthetic lethal analysis are explained for studying essential genes and protein interactions. DNA cloning is introduced, involving restriction enzymes to cut DNA and ligases to join DNA fragments into vectors.
This document discusses how cancer cells have altered metabolism that promotes their growth and survival. Oncogenic mutations activate pathways like PI3K/Akt/mTOR that increase nutrient uptake and biosynthesis, putting cancer cells in a constant state of metabolic stress and hunger. This metabolic stress activates autophagy, which recycles cellular components and supports cancer cell metabolism and survival during starvation or chemotherapy. Autophagy degradation provides nutrients and building blocks and removes damaged mitochondria that could increase harmful reactive oxygen species. Cancer cells with oncogenic mutations like in Ras show increased basal autophagy that is essential for their survival. This suggests targeting autophagy may be an effective strategy to treat cancers dependent on it.
This study explored designing small molecule inhibitors of the NRMT1 enzyme. NRMT1 is an important methyltransferase enzyme that transfers methyl groups from SAM to its substrate RCC1. The deregulation of NRMT1 has been observed in cancers. Using molecular modeling programs Sybyl and GOLD, 14 small molecules were designed that were predicted to bind in the RCC1 binding site of NRMT1 and suppress its activity. The molecules were docked and scored using GOLD, and the top scoring molecules will be further optimized to improve their inhibitory activity against NRMT1.
Soluble protein expression optimizationBiologicsCorp
In many cases, expression of recombinant proteins often results in insoluble and/or nonfunctional proteins. Here, factors in soluble protein expression optimization and several strategies to improve the solubility of the expressed protein are reviewed.
Genetic variations in G protein-coupled receptors (GPCRs) can alter receptor function and cause diseases. Single nucleotide polymorphisms and other mutations have been linked to impaired or enhanced receptor signaling. For example, a mutation in the vasopressin V2 receptor causes nephrogenic diabetes insipidus by decreasing ligand binding and receptor expression. Similarly, mutations in chemokine receptors CCR5 and CCR2 impact HIV infection by altering receptor function or interaction with other coreceptors. Overall, GPCR polymorphisms are associated with diseases by changing ligand binding, receptor activation, trafficking, and coupling to downstream signaling pathways.
This document discusses bioassay development for drug discovery. It covers in vitro and cell-based bioassays, as well as the molecular biology techniques used to develop these assays such as cloning DNA into plasmids, propagating plasmids in bacteria, purifying plasmid DNA, sequencing DNA, and expressing proteins in bacteria and mammalian cells through techniques like transfection. Recombinant protein expression and purification using affinity tags is also summarized.
This document provides an overview of intracellular receptors, enzymes, signals, transcription factors, structural proteins, and nucleic acids. It discusses in detail the structures and functions of DNA, RNA, nuclear receptors, IP3 receptors, intracellular enzymes, transcription factors, and structural proteins like collagen, keratin, myosin, and actin. The presentation was given by S. Dinakar from the Department of Pharmacology at PSG College of Pharmacy in Coimbatore, India.
protein microarray-types and approaches.pptxSachin Teotia
Protein microarrays are a high-throughput technology that allows thousands of proteins to be analyzed simultaneously. They consist of a solid support coated with thousands of different proteins in a defined array. Each protein spot represents a different protein. Protein microarrays can be used to study protein expression levels, interactions, activities, and functions on a large scale. There are different types including analytical, functional, and reverse-phase microarrays that each have their own strengths and applications.
From Ugly Duckling to Swan: Unexpected Identification from Cell-SELEXSaw Yi
An unexpected identification of an anti-Annexin A2 aptamer.
A paper presentation that I did last month. Enjoy! :)
This must have been the most easiest to understand paper that I've ever read :) Very well written piece!
This document discusses surface modification of nanoparticles for biomedical applications. It describes various methods for modifying the nanoparticle surface, including conjugating ligands for cell targeting (e.g. antibodies, peptides, aptamers), encapsulating the nanoparticle core with lipids or polymers, and attaching targeting moieties via linkers like streptavidin-biotin. Common targets for surface ligands include receptors for VEGF, folate, transferrin and others. Aptamers and peptides are also discussed as targeting options.
This document summarizes a lecture on the regulation of DNA synthesis, specifically growth factors and inhibitors. It discusses:
1. Growth factors and inhibitors that control DNA replication within the cell cycle in eukaryotes.
2. Cyclin dependent kinase complexes and MPF that drive the cell into mitosis and prevent re-replication of DNA.
3. The processes of licensing and geminin, which positively and negatively regulate replication through ORC protein complexes, Mcm proteins, and inhibition of Cdt1 by geminin to ensure one round of replication per cell cycle.
Cancer arises due to genetic aberrations that accumulate in somatic cells and alter gene expression. There are several types of genomic changes including mutations, chromosome defects, and changes to oncogenes and tumor suppressor genes. Genetic testing can identify inherited cancer risk genes and guide diagnosis and treatment, while gene therapy holds promise for directly treating cancer at the genetic level.
screening models for hepatoprotective agents slide sharebeulah43
This document summarizes several models for screening hepatoprotective drugs. It describes in vitro models including hepatic stellate cell culture, liver slice systems, and assays measuring proline hydroxylation and collagen synthesis. It also outlines several in vivo rat models, including those using CCl4, allyl alcohol, bile duct ligation, and galactosamine to induce liver fibrosis, necrosis, or damage and evaluate potential hepatoprotective effects of test compounds. Parameters measured include liver enzymes, hydroxyproline content, histology, and serum markers of collagen synthesis. These preclinical models allow evaluation of drug candidates for protection against liver injury.
The document discusses optimizing ADME and PK properties in drug development. It addresses common mistakes such as believing that intrinsic clearance cannot be optimized or that increasing plasma protein binding will always benefit PK. It emphasizes that intrinsic clearance, uptake clearance, and renal clearance all contribute to in vivo clearance. Good quality experimental data is important for accurate prediction of human PK. Formulation strategies can improve bioavailability when absorption is limited, but not if clearance is the dominant elimination pathway. The effects of plasma protein binding on free drug exposure are also explained.
This document summarizes an evaluation seminar on cell signaling and signal transduction pathways presented by Mrutyunjay B Bellad of the Department of Pharmacology at H.S.K. College of Pharmacy in Bagalkot. The seminar covered various topics related to cell signaling including introduction, types of cell signaling, signal molecules and their actions, signaling through different receptor types, second messengers, G-protein coupled receptors, and signal transduction pathways. References included standard pharmacology textbooks.
Therapeutic enzymes have a variety of uses including as oncolytics, anticoagulants, thrombolytics, and to treat metabolic deficiencies. Examples discussed include pegylated L-asparaginase for acute lymphoblastic leukemia, which exploits the metabolic differences between normal and cancer cells. Streptokinase is described as a thrombolytic that forms an activator complex with plasminogen to generate plasmin and degrade blood clots. Enzymes are also used as digestive aids, debriding agents, and to treat cystic fibrosis through administration of DNAse I to break down viscous mucus.
This presentation gives the brief idea of the various guidelines carried out to study the genetic damage to cells when there is a discover of new active molecule.
1. Beta cells in the pancreas produce the hormone insulin which regulates blood sugar levels. Impairment of beta cells leads to diabetes. Studying beta cell development in zebrafish can provide insights into treating diabetes in humans.
2. Zebrafish have similarities to humans in genes associated with disease and drug responses, making them a useful model for studying beta cell development. Their pancreas contains beta cells clustered in islets as well as single beta cells in the tail.
3. Transcription factors like Pdx1 and Ptf1a are involved in early beta cell differentiation and maturation. Understanding their roles and how to generate new beta cells could lead to strategies for treating diabetes.
Evaluation of hepatoprotective agents - Hemant KanaseHemant Kanase
1. Introduction
2. Hepatotoxicity: Mechanism
3. Therapeutic strategies available – their limitations
4. In vivo models of liver damage
- Non-invasive model
a. Chemically induced hepatotoxicity
b. Drug-induced hepatotoxicity
c. Radiation-induced hepatotoxicity
d. Metal-induced hepatotoxicity
e. Diet-induced hepatotoxicity
Models of Acute Hepatitis
Models of chronic hepatitis
Models of fibrosis
Models of cholestasis
Models of steatosis
4. Problems faced with animal studies
5. In vitro models of liver damage
6. Advantages and disadvantages of in vitro models
7. Parameters of evaluation
8. Clinical Assessment
Advances and Applications Enabled by Single Cell TechnologyQIAGEN
Over the past 5 years, single-cell genomics have become a powerful technology for studying small samples and rare cells, and for dissecting complex populations such as heterogeneous tumors. Single-cell technology is enabling many new insights into diverse research areas from oncology, immunology and microbiology to neuroscience, stem cell and developmental biology. This webinar introduces single-cell technology and summarizes the newest scientific applications in various research areas, all in the context of current literature.
Evaluation of the changes in the gene CYP3A4 expression in HepG2 cells under ...Angela Farngren
This study evaluated the effects of rifampicin and alpha-ketoglutarate on expression of the CYP3A4 gene in HepG2 liver cells. HepG2 cells were treated with either 10mM rifampicin or 4mM alpha-ketoglutarate for 7 days. RNA was extracted on days 0, 3, and 7 and analyzed via qPCR. Rifampicin treatment significantly increased CYP3A4 expression, peaking on day 3 and decreasing slightly by day 7. Alpha-ketoglutarate initially decreased CYP3A4 expression for the first 3 days but then increased expression levels. The study demonstrates that rifampicin and alpha-ket
Molecular techniques for pathology research - MDX .pdfsabyabby
This document discusses molecular techniques used in pathology research such as PCR, microarrays, next generation sequencing, immunohistochemistry, ELISA, and Western blotting. It provides details on each technique including the basic principles, applications in research, and examples of uses in studies of gene expression, cancer, bone disease, and growth retardation. The learning outcomes are to understand these techniques and their uses in basic and clinical research.
Detection of genetic diseases can be done through various techniques such as karyotyping, restriction fragment length polymorphism (RFLP), and polymerase chain reaction (PCR). Karyotyping involves analyzing chromosomes to detect abnormalities. RFLP detects variations in DNA fragments after digestion with restriction enzymes. PCR can amplify small amounts of DNA and is used for genetic testing and disease detection. Newer techniques like single strand conformation polymorphism (SSCA) and enzyme-linked immunosorbent assay (ELISA) are also used for genetic testing and disease detection.
Similar to 2015_SummerScholarsProgramPresentation- Daniel Donchev (20)
2015_SummerScholarsProgramPresentation- Daniel Donchev
1. Daniel DonchevDaniel Donchev
Wilson High School, Long Beach; University of Southern California
Michael K Jones, Ph.D.Michael K Jones, Ph.D.
Gastric Pathophysiology Molecular and Cellular
2015 Summer Scholars Research Presentation
2. Survivin
• Survivin is a member of the
inhibitor of apoptosis (IAP)
family. The survivin protein
functions to inhibit caspase
activation, thereby leading to
negative regulation of
apoptosis or programmed cell
death.
• In the event of a stomach
ulcer, survivin is up regulated
to assist with the angiogenesis
process.
• Survivin inhibits caspase 3,
helping cells move indirectly.
• Our goal is to prove survivin’s
critical role in the angiogenesis
process and cell migration.
3. Objective and Hypothesis
• Objective:Objective: To elucidate the cellular and molecular
mechanisms of gastric (stomach) wound/injury (e.g.
ulcer) healing.
• Hypothesis:Hypothesis: Survivin as an inhibitor of caspaces
during apoptosis and cell division also has a tertiary
function; it can be involved, indirectly, in
angiogenesis and cell migration, and is crucial in
wound closing.
4. Means and Methods
• Means:Means: Create gastric-specific and vascular-specific
genome editing and “gene rescue” lentiviral transfer
vectors; and, to express cDNA corresponding to genes
hypothesized to be downstream effectors of survivin.
• Methods:Methods:
- Conduct BLAST search to identify promoters of interest
in the rat genome homologous to known human
promoters.
- PCR clone the promoters for: 1) VE-Cadherin (vascular-
specific), 2) TFF2 (gastric-specific), 3) EGFR (gastric
ulcer-inducible); and, 4) Flt1 (angiogenesis-inducible) and
introduce PacI and XbaI (SpeI) restriction sites.
5. Polymerase Chain Reaction (PCR)
• Relies on three principals of molecular biology:
1.1. DenaturingDenaturing - melting double stranded DNA template
into single strands.
2.2. AnnealingAnnealing - complementary DNA strand hybridization
via DNA primers.
3.3. ExtensionExtension - DNA strand synthesis via DNA polymerase.
6. PCR Continued
• The enzyme used in PCR is DNA
polymerase.
• A special reaction buffer is also required,
called a master mix.
• Two template strands are created from the
original template after each complete cycle
of the strand synthesis reaction.
10. Results and Our Next Steps
• Results: The promoters for VE-Cadherin
VE-Cadherin and EGFR were successfully
cloned into the TA cloning vector.
• Next Step: Isolate the cloned promoters
by digest/gel extraction and ligate them
into the CRISPR/Cas9 and FUGW
vectors.
11. VALBHS Research Translates
From Drug Discovery To Patient Care
• In this program, students learned methods
that are commonly used in labs to analyze
and research many different diseases.
• The research done in the lab translates
into discovery of new treatment plans, or
even new drugs that can be used to treat
patients.
Editor's Notes
Survivin is necessary for cell division and is an anti-apoptosis protein. It’s also expressed during embryonic development and is thought not to be crucial during the adult life.
We believe that it is expressed in some tissues during adult development including the stomach. In the event of a stomach ulcer, the surrounding tissue upregulates survivin, which plays a crucial role in the healing process.
Angiogenesis is the growth of new capillary blood vessels in the body and is an important natural process used for healing and reproduction.
Our hypothesis is: Survivin as an inhibitor of caspaces during apoptosis and cell division also has a tertiary function; it can be involved, indirectly, in angiogenesis and cell migration, and is crucial in wound closing.
Survivin helps remodeling tissue grow and divide. Survivin is upregulated in cells that are migrating. Some factors are targets of caspace destruction. If survivin is upregulated, it inhibits caspaces so they aren’t destroyed. IAPs (e.g. survivin) bind to effector caspases and inhibit them from cleaving their targets (but IAPs do not “destroy” caspases). If caspases are “over inhibited,” this creates a state conducive to tumorigenesis and cancer. If caspases are “under inhibited,” apoptosis would be abnormally high and could possibly impair the processes of wound/injury healing.
Means: Create gastric-specific and vascular-specific genome editing and “gene rescue” lentiviral transfer vectors to knockout genes hypothesized to be involved in ulcer healing (i.e. Survivin) and, to express cDNA corresponding to genes hypothesized to be downstream effectors of survivin (e.g. integrin α6/β4, focal adhesion kinase) as well as mutant forms of survivin.
Methods: Conduct BLAST search to identify promoters of interest in the rat genome homologous to known human promoters. . Rat promoters were chosen because the in vivo model of study is the rat. PCR clone the promoters for: 1) VE-Cadherin (vascular-specific), 2) TFF2 (gastric-specific), 3) EGFR (gastric ulcer-inducible); and, 4) Flt1 (angiogenesis-inducible) and introduce PacI and XbaI (SpeI) restriction sites. Use these constructs to exchange (by directed restriction site ligation) the ubiquitous/constitutive hUbC promoter in the CRISPR/Cas9 genome editing vector, pLV-hUBC-Cas9-T2A-GFP; and, the lentiviral expression vector, FUGW.
Denaturing – Before DNA synthesis can begin the double stranded DNA template must be unwound and separated into single strands. In cells, this is carried out by a family of enzymes. In PCR, heat is used to melt apart, or denature, the double stranded DNA template.
Annealing – Before a target region of DNA can be amplified, you must determine short sequences of DNA upstream (at the 5’ end) and downstream (at the 3’ end) of the target loci region of interest. These areas are then used to make short pieces of DNA called primers, which are complementary to regions upstream and downstream of the target loci region. Primers serve as start and stop points for amplifying the target loci region of the DNA to be copied. The annealing temperature is very important and we were able to find the temperatures we needed by going onto the company’s website and locating it there.
Extension – Primers are needed because the DNA polymerase requires an already existing nucleotide chain to bind and add nucleotides to one at a time. Once the polymerase locates and binds to template DNA and the primer, it initiates the addition of nucleotides and synthesizes new copies of the double stranded DNA template by adding nucleotides onto the primer and extending it. Therefore, primers provide a starting point for the DNA polymerase.
DNA Polymerase must be thermally stable because PCR cycles vary between temperatures of 52 degrees Celsius and 94 degrees Celsius.
The master mix contains all of the components for PCR to occur, including the individual building blocks of DNA, a special buffer to maintain optimum pH, salts, and Magnesium ions. It’s important to keep the master mix cold before use, so that the enzyme doesn’t start to work before you add your DNA templates.
It is called the Polymerase Chain Reaction because exponential growth of the number of template molecules occurs after each cycle is complete. After 35 cycles, the DNA of interest has been amplified sufficiently to be visualized using gel electrophoresis. This allows us to determine the presence or absence of the desired PCR products.
A method that separates large molecules (including nucleic acids or proteins) on the basis of size, electric charge, and other physical properties. The large molecule we separated was DNA.
To prepare DNA for gel electrophoresis, the DNA is cut into smaller pieces when mixed with restriction enzymes.
This process is called restriction digestion, and it produces a range of DNA fragments of different lengths.
During electrophoresis, molecules are forced to move through the pores of a gel, when the electrical current is applied.
The frictional force of the gel resists the flow of the molecules, separating them by size.
Steps in the process of gel electrophoresis are:
A tray is prepared to hold the gel matrix.
A gel comb is used to create holes in the gel. The gel comb is placed in the tray.
Agarose gel powder is mixed with buffer solution (10x TBE) that carries the DNA in a stable form. The solution is heated until dissolved and poured into the tray and allowed to cool.
The gel tray is placed in an electrophoresis chamber and the chamber is filled with buffer, covering the gel. This allows the electric current from electrodes at either end of the gel to flow through the gel.
DNA samples are mixed with a “loading dye” to make the DNA sample visible. The dye also contains glycerol or sucrose to make the DNA sample heavy so that it will sink to the bottom of the well.
A safety cover is placed over the gel, electrodes are attached to a power supply and turned on.
When the dye marker has moved through the gel, the current is turned off and the gel is removed from the tray.
The DNA molecules are made visible by staining the molecules with ethidium bromide which binds to DNA and will fluoresce in UV light.
VE-Cad, PacI-AgeI Promoter Minipreps 4: The left well next to the marker worked. TA vector is at the top, and the promoter is at the bottom. It’s cut with Bam and is diagnostic. They allow us to determine what clones that we used had the insert of interest.
Bottom left:
Bottom Right: Diagnostic, Mini preps using BAM. Testing for what has our insert. BAM digest.
Top right: PCR when I was getting the promoters. They have the right flanking restriction sites that we incorporated on the PCR. Bottom left and top right are the same bands.
Bottom left: We need to purify the DNA and purify the big backbone of the vectors, and we will ligate them together, directional ligation.
Left: Third well is the correct digest, Sixth well is also a correct digest. They have the correct size.
Right: Crispr cas 9 vector is well 1. Next 4 wells are the vectors that we will ligate in our RNA guides for the crispr cas 9. Will be used in conjunction with well 1 ( in our golden gate cloning. (removes the restriction sites, and leads to the products you want) Cas9 will be expressed with four separate guide RNAs in the end.
Six well: expression vector.
Next three wells: We got the plasmids from addgene and grew them up.
The promoters for VE-Cadherin and EGFR were successfully cloned into the Results:TA cloning vector such that the proper flanking restriction sites were verified by restriction mapping.
The next step will be to isolate the cloned promoters by digest/gel extraction and ligate them into the CRISPR/Cas9 and FUGW vectors.
Site-directed mutagenesis (SDM) is an in vitro procedure that uses custom designed oligonucleotide primers to confer a desired mutation in a double-stranded DNA plasmid. There are many reasons to make specific DNA alterations (insertions, deletions and substitutions), including:
To study changes in protein activity that occur as a result of the DNA manipulation.
To select or screen for mutations (at the DNA, RNA or protein level) that have a desired property
To introduce or remove restriction endonuclease sites or tags
Used to knock out a PacI site because the vector contains two PacI sites.
The other reason for it is on the integrin alpha 6 beta 4 we want to knock out the target sites of caspace 3 to make the integrin impervious ( no longer a target of caspace3, can’t be cleaved)
Use it to mutate survivin so you can dissect the different functions of survivin with regards to the process of ulcer healing.
Do more digests with Pact I and Age I and PacI XBA I and PacI SPE I, and isolate the promoters with a low melt gel.
We will do the ligation.