- A single episomal plasmid containing Oct4, Sox2, KLF4, L-Myc driven by a hybrid CAG promoter can reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).
- The inclusion of 5 small molecules that enhance reprogramming results in the appearance of proto-colonies by 7 days and mature colonies by 21 days under low oxygen conditions.
- The iPSCs express pluripotency markers, can differentiate into neurons and cardiomyocytes, and grow robustly in a new xeno-free media. Further work will examine residual vector presence and gene expression profiles.
Canvax™ offers a wide range of high quality Human Recombinant Proteins for several research applications like ELISA, Western Blot, Antibody Production or Protein array.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
Recombinant protein expression and purification Lecturetest
The document discusses recombinant protein expression and engineering. It describes:
1) Cloning or synthesizing the gene of interest, making an expression construct, transfecting cells, purifying the recombinant protein.
2) Factors to consider like the protein's origin (prokaryotic/eukaryotic), required post-translational modifications, and available expression systems.
3) A case study expressing recombinant human alpha-1-acid glycoprotein in E. coli, including vector construction, periplasmic extraction, affinity purification, and yield.
This document discusses recombinant protein expression in different host cell systems. It begins by outlining strategies for engineering host cells to efficiently produce proteins, including optimizing transcription, translation, and protein stability. It then compares various host cell expression systems, such as bacteria, yeast, insect and mammalian cells, considering factors like post-translational modification abilities and production costs. Specific systems are covered in more detail, like using the pET and pBAD vectors to control protein expression in bacteria. The document concludes by discussing eukaryotic cell expression and challenges producing complex eukaryotic proteins in prokaryotic systems due to lack of post-translational modifications.
This study found that elevated expression of phospholipase D (PLD) suppresses DNA damage-induced apoptosis in cells overexpressing c-Src. PLD expression led to increased levels of the E3 ubiquitin ligase MDM2, which increased turnover of the tumor suppressor p53. PLD expression also suppressed increases in p53 levels induced by DNA damage. Inhibition of mTOR and MAPK pathways blocked the PLD-stimulated increases in MDM2 and suppression of p53. These findings provide evidence that PLD generates survival signals by suppressing the p53 response pathway.
- A single episomal plasmid containing Oct4, Sox2, KLF4, L-Myc driven by a hybrid CAG promoter can reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).
- The inclusion of 5 small molecules that enhance reprogramming results in the appearance of proto-colonies by 7 days and mature colonies by 21 days under low oxygen conditions.
- The iPSCs express pluripotency markers, can differentiate into neurons and cardiomyocytes, and grow robustly in a new xeno-free media. Further work will examine residual vector presence and gene expression profiles.
Canvax™ offers a wide range of high quality Human Recombinant Proteins for several research applications like ELISA, Western Blot, Antibody Production or Protein array.
Structure of a carotenoid gene cluster from Pantoea sp. strain C1B1YArunkumar K.R.
Structure of a carotenoid gene
cluster from Pantoea sp. strain C1B1Y and characterization of β-carotene hydroxylase (crtZ) gene
by functional complementation in Escherichia coli.
This study examines the degradation pathway of the thiazide-sensitive NaCl cotransporter (NCC) in yeast and mammalian cells. The authors show that NCC is a substrate of endoplasmic reticulum-associated degradation (ERAD) in yeast. Using yeast strains with mutations in ERAD components, they identify the E3 ubiquitin ligase Hrd1 and the cytoplasmic Hsp70 chaperone Ssa1/Hsp70 as important for NCC ubiquitination and degradation. Expression of NCC in mammalian kidney cells reveals similar polyubiquitination and proteasome-dependent degradation. Cytoplasmic Hsp70 preferentially associates with immature glycosylated NCC, indicating its role
Recombinant protein expression and purification Lecturetest
The document discusses recombinant protein expression and engineering. It describes:
1) Cloning or synthesizing the gene of interest, making an expression construct, transfecting cells, purifying the recombinant protein.
2) Factors to consider like the protein's origin (prokaryotic/eukaryotic), required post-translational modifications, and available expression systems.
3) A case study expressing recombinant human alpha-1-acid glycoprotein in E. coli, including vector construction, periplasmic extraction, affinity purification, and yield.
This document discusses recombinant protein expression in different host cell systems. It begins by outlining strategies for engineering host cells to efficiently produce proteins, including optimizing transcription, translation, and protein stability. It then compares various host cell expression systems, such as bacteria, yeast, insect and mammalian cells, considering factors like post-translational modification abilities and production costs. Specific systems are covered in more detail, like using the pET and pBAD vectors to control protein expression in bacteria. The document concludes by discussing eukaryotic cell expression and challenges producing complex eukaryotic proteins in prokaryotic systems due to lack of post-translational modifications.
This study found that elevated expression of phospholipase D (PLD) suppresses DNA damage-induced apoptosis in cells overexpressing c-Src. PLD expression led to increased levels of the E3 ubiquitin ligase MDM2, which increased turnover of the tumor suppressor p53. PLD expression also suppressed increases in p53 levels induced by DNA damage. Inhibition of mTOR and MAPK pathways blocked the PLD-stimulated increases in MDM2 and suppression of p53. These findings provide evidence that PLD generates survival signals by suppressing the p53 response pathway.
The document discusses cell proliferation and apoptosis. It describes how the cell cycle is regulated by cyclins, CDKs and growth factors. Growth factors such as EGF, TGF, and VEGF act through membrane receptors to stimulate cell entry into the cell cycle. Apoptosis is a tightly regulated process where cells activate enzymes to degrade DNA and proteins. Key features include DNA fragmentation, phosphatidylserine expression, and engulfment by phagocytes to prevent inflammation. Apoptosis is important for development, immune response, and removing damaged cells.
LanglaisC_2007_Systematic approach to protein experssion in cell free systemsClaudia Langlais
This document describes a study that tested the expression of 960 human full-length proteins using both in vivo and in vitro expression systems. The researchers found that E. coli cell-free expression systems and wheat germ cell-free expression were better able to express proteins that did not express in E. coli in vivo. They optimized expression in the E. coli cell-free system through sequence modifications and achieved a 93% success rate for cell-free expression overall. Wheat germ expression showed high solubility and protein yield for the proteins tested.
Recombinant proteins are manufactured using recombinant DNA technology to produce wild type and modified human and mammalian proteins in bulk. Creative Biomart produces high quality recombinant proteins through extensive quality control and lot-to-lot consistency in order to provide reliable results for researchers efficiently. They offer over 1,000 recombinant proteins, peptides, and antibodies which are rigorously tested for purity, bioactivity, stability, and safety.
Na f activates map ks and induces apoptosis in odontoblast-likeGanesh Murthi
The study examined the effects of sodium fluoride (NaF) on odontoblast-like MDPC-23 cells. The researchers found that NaF exposure induced apoptosis in a dose-dependent manner through several markers. NaF activated the mitogen-activated protein kinases (MAPKs) JNK and p38, and induced two peaks in ERK phosphorylation. Inhibition of JNK suppressed NaF-induced apoptosis, while inhibition of p38 and ERK had lesser effects, suggesting NaF-induced apoptosis depends primarily on JNK signaling.
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
OriGene Technologies Capabilities Overview Feb 2011mwatson26
Opportunity overview with OriGene Technologies. OriGene is looking to identify strategic collaboration partners for its full-length human proteins, validated monoclonal antibodies and "gene centric" tool box.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
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
Commercial production of gene products requires high levels of gene expression. Several factors can be manipulated to increase protein production, including the vector, host chromosome location, gene dosage, transcription elements like promoters and terminators, translation elements like ribosome binding sites, and codon optimization. Final localization of the protein, such as secretion extracellularly, and preventing degradation through fusion proteins or other means can also improve yields. However, expression in E. coli has limitations like the inability to perform post-translational modifications and potential endotoxin contamination.
This document discusses the production of recombinant proteins and glycoproteins from animal cells. It provides examples of several therapeutic proteins that were originally extracted from animal or human sources but are now produced recombinantly using cell culture, including insulin, interferons, erythropoietin, blood clotting factors, and tissue plasminogen activator. Recombinant production allows these proteins to be produced in larger quantities and avoids issues with variable supply or immunogenicity from animal sources. The proteins can also be engineered for improved properties when produced recombinantly compared to native forms.
This document summarizes research on the major types of cell death: apoptosis, autophagy, and necrosis. It provides a brief history of the discovery and understanding of each type of cell death. It also describes the key morphological and molecular characteristics of each type of cell death, as well as physiological and pathophysiological functions. The document then discusses experimental approaches to study cell death mechanisms using tools from QIAGEN, including gene expression analysis, epigenetic analysis, and functional studies using reporter assays, siRNA, and shRNA.
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
Cultivation strategies to enhance productivity in Pichia pastoris Muhammed Suleman
Pichia pastoris is a methylotrophic yeast used for the production of recombinant proteins. Key aspects of cultivating P. pastoris include using vectors like pPICZα which integrate the gene of interest downstream of the methanol-inducible AOX1 promoter. Developing an optimal production process involves screening clones, characterizing growth and production kinetics under controlled growth rates, and establishing a fedbatch production process to maximize protein titers. Future work may focus on single-cell analyses, in silico modeling, and improving product quality and yields.
Phosphoproteomics of collagen receptor networks reveals SHP-2Maciej Luczynski
This document describes a study that used phosphoproteomics to analyze signaling networks downstream of collagen receptors. The researchers identified 424 phosphorylated proteins over seven time points after stimulating cells with collagen. Multiple clustering analysis revealed that phosphorylation sites on proteins like SHP-2, NCK1, LYN, and PIK3C2A strongly clustered with DDR2 phosphorylation dynamics, suggesting these proteins are candidate downstream effectors of DDR2 signaling. Biochemical validation showed SHP-2 tyrosine phosphorylation depends on DDR2 kinase activity. Targeted proteomics of lung cancer DDR2 mutants showed SHP-2 is phosphorylated by some mutants. This indicates SHP-2 is a key signaling node downstream of DDR
1. The document describes a study that used site-directed mutagenesis to alter a single amino acid (phenylalanine to serine at position 67) in the furin protein sequence to examine its structure, function, and trafficking.
2. The methods included designing mutagenic primers, performing site-directed mutagenesis on the furin cDNA cloned in a plasmid, transforming E. coli with the plasmid, isolating and sequencing the plasmid DNA, and future plans to transfect mutant and wild-type furin into cells for analysis.
3. Furin is involved in development, homeostasis, and disease pathways by processing precursor proteins, and its structure and trafficking are important for understanding its functions.
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.
This study investigated whether neuronal cells show preferential growth and maturation on synthetic polymers versus decellularized nerve extracellular matrix (dECM) bioink. Several key findings were reported:
1) Neurite outgrowth of neuronal cells was greater when cultured on dECM bioink compared to matrigel or tissue culture plastic.
2) Cell viability was lower for neuronal cells grown on dECM bioink compared to matrigel or plastic, indicating dECM bioink supports longer neurites but at the expense of cell viability.
3) A blend of dECM bioink and PEG was successfully 3D printed, demonstrating a novel biomaterial containing both natural and synthetic properties that may
This document summarizes research on the structural biology of cytochrome P450 monooxygenases in the phenylpropanoid pathway of Arabidopsis thaliana. It discusses homology modeling and protein engineering methods used to study the structures of phenylpropanoid P450s. It also describes efforts to develop X-ray crystallography and NMR techniques to determine the crystal structures of these P450s to better understand their functions in metabolic pathways.
Peptides are short chains of amino acids linked by peptide bonds. They are distinguished from proteins by typically containing fewer than 50 amino acid units. Peptides are formed through condensation reactions between carboxyl and amino groups of separate amino acids, releasing a water molecule. Peptide bonds are rigid and planar, contributing to protein structure stability. Peptides serve many important biological functions and can be classified based on their production method, including through ribosomal translation, nonribosomal synthesis, and enzymatic digestion of proteins in foods. Bioactive peptides derived from food proteins can have beneficial effects like lowering blood pressure, cholesterol, and antimicrobial properties.
The document discusses cell proliferation and apoptosis. It describes how the cell cycle is regulated by cyclins, CDKs and growth factors. Growth factors such as EGF, TGF, and VEGF act through membrane receptors to stimulate cell entry into the cell cycle. Apoptosis is a tightly regulated process where cells activate enzymes to degrade DNA and proteins. Key features include DNA fragmentation, phosphatidylserine expression, and engulfment by phagocytes to prevent inflammation. Apoptosis is important for development, immune response, and removing damaged cells.
LanglaisC_2007_Systematic approach to protein experssion in cell free systemsClaudia Langlais
This document describes a study that tested the expression of 960 human full-length proteins using both in vivo and in vitro expression systems. The researchers found that E. coli cell-free expression systems and wheat germ cell-free expression were better able to express proteins that did not express in E. coli in vivo. They optimized expression in the E. coli cell-free system through sequence modifications and achieved a 93% success rate for cell-free expression overall. Wheat germ expression showed high solubility and protein yield for the proteins tested.
Recombinant proteins are manufactured using recombinant DNA technology to produce wild type and modified human and mammalian proteins in bulk. Creative Biomart produces high quality recombinant proteins through extensive quality control and lot-to-lot consistency in order to provide reliable results for researchers efficiently. They offer over 1,000 recombinant proteins, peptides, and antibodies which are rigorously tested for purity, bioactivity, stability, and safety.
Na f activates map ks and induces apoptosis in odontoblast-likeGanesh Murthi
The study examined the effects of sodium fluoride (NaF) on odontoblast-like MDPC-23 cells. The researchers found that NaF exposure induced apoptosis in a dose-dependent manner through several markers. NaF activated the mitogen-activated protein kinases (MAPKs) JNK and p38, and induced two peaks in ERK phosphorylation. Inhibition of JNK suppressed NaF-induced apoptosis, while inhibition of p38 and ERK had lesser effects, suggesting NaF-induced apoptosis depends primarily on JNK signaling.
Regulation of pten activity by its carboxyl terminal autoinhibitoryChau Chan Lao
Regulation of PTEN Activity by Its Carboxyl-terminal Autoinhibitory Domain.
Leticia Odriozola, Gobind Singh, Thuong Hoang, and Andrew M. Chan
From the Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York, 10029
THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 282, NO. 32, pp. 23306–23315, August 10, 2007
目前已知PTEN(Phosphatase and tensin homolog)是腫瘤抑制蛋白,其由403個氨基酸組成,主要分PTPase及C2 domain,C2 domain使PTEN可與細胞膜作用連結。
PTEN之C-tail(aa 350~403)被發現具有調控PTEN自身活性之功能。前人研究指出C-tail有6個可磷酸化之位置(Thr-366、Ser-370、Ser-380、Thr-382、Thr-383及Ser-385),這些位置可調控PTEN之腫瘤抑制能力、胞內之分佈及穩定性。前人產生以上位置突變之PTEN變異株,發現這些變異株具有更強的腫瘤抑制能力,但穩定性將降低,這可能是因這些變異株具有更開放結構所致。
本報告針對研究PTEN C-tail在連結細胞膜和在其本身催化活性中扮演的功能。作者先產生一系列之PTEN磷酸化位置變異株,發現S385A會促使PTEN之membrane localization in vivo及加強phosphatase活性in vitro,而且此突變會使Ser-380/Thr-382/Thr-383 cluster的磷酸化程度降低,因此知Ser-385可透過被去磷酸化以調控PTEN。而以phosphomimic residues取代Ser-380/Thr-382/Thr-383會使上述S385A所產生之PTEN催化活性反轉。之後利用免疫沉澱方法,發現C-tail之71-amino acid region會與C2 domain上之CBR3 motif作用,暗示C-tail參與連結細胞膜之調控。最後利用合成之PTEN C-tail peptide,發現其可抑制PTEN之催化活性in vitro,而在細胞表現此peptide則會抑制PTEN之membrane localization,磷酸化之Akt量亦上升。以上實驗顯示C-tail在PTEN之membrane recruitment及PTPase活性調控中扮演Autoinhibitory domain角色。
OriGene Technologies Capabilities Overview Feb 2011mwatson26
Opportunity overview with OriGene Technologies. OriGene is looking to identify strategic collaboration partners for its full-length human proteins, validated monoclonal antibodies and "gene centric" tool box.
KDM5 epigenetic modifiers as a focus for drug discoveryChristopher Wynder
A summary presentation of my scientific work.
My laboratory focused on an enzyme KDM5b (aka PLU-1, JARID1b) that was widely expressed during development and played a key role in progression of breast cancer through HER-2.
My lab focused on understanding the key biochemical activity of the enzyme through dissecting the proteomic and genomic interactors.
Our results were confirmed through the use of ES cells, adult stem cells and mouse models.
Much of this work remains unpublished, please contact me for more information and/or access to any reagents that I still have as part of this work.
crwynder@gmail.com
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
Commercial production of gene products requires high levels of gene expression. Several factors can be manipulated to increase protein production, including the vector, host chromosome location, gene dosage, transcription elements like promoters and terminators, translation elements like ribosome binding sites, and codon optimization. Final localization of the protein, such as secretion extracellularly, and preventing degradation through fusion proteins or other means can also improve yields. However, expression in E. coli has limitations like the inability to perform post-translational modifications and potential endotoxin contamination.
This document discusses the production of recombinant proteins and glycoproteins from animal cells. It provides examples of several therapeutic proteins that were originally extracted from animal or human sources but are now produced recombinantly using cell culture, including insulin, interferons, erythropoietin, blood clotting factors, and tissue plasminogen activator. Recombinant production allows these proteins to be produced in larger quantities and avoids issues with variable supply or immunogenicity from animal sources. The proteins can also be engineered for improved properties when produced recombinantly compared to native forms.
This document summarizes research on the major types of cell death: apoptosis, autophagy, and necrosis. It provides a brief history of the discovery and understanding of each type of cell death. It also describes the key morphological and molecular characteristics of each type of cell death, as well as physiological and pathophysiological functions. The document then discusses experimental approaches to study cell death mechanisms using tools from QIAGEN, including gene expression analysis, epigenetic analysis, and functional studies using reporter assays, siRNA, and shRNA.
This study examined how cellular cholesterol distribution influences the proteolytic release of the LRP-1 ectodomain in breast cancer cell lines. The researchers found that efficient cholesterol extraction and increased LRP-1 shedding occurred in MDA-MB-231 cells but not MDA-MB-435 cells. Raman microspectroscopy revealed MDA-MB-231 cells distributed cholesterol predominantly intracellularly, while MDA-MB-435 cells distributed it at the plasma membrane. Depleting cholesterol in MDA-MB-231 cells increased LRP-1 shedding by making the substrate more accessible at the cell surface, rather than increasing sheddase enzyme expression. This highlights the relationship between cellular cholesterol distribution and its impact on mod
Cultivation strategies to enhance productivity in Pichia pastoris Muhammed Suleman
Pichia pastoris is a methylotrophic yeast used for the production of recombinant proteins. Key aspects of cultivating P. pastoris include using vectors like pPICZα which integrate the gene of interest downstream of the methanol-inducible AOX1 promoter. Developing an optimal production process involves screening clones, characterizing growth and production kinetics under controlled growth rates, and establishing a fedbatch production process to maximize protein titers. Future work may focus on single-cell analyses, in silico modeling, and improving product quality and yields.
Phosphoproteomics of collagen receptor networks reveals SHP-2Maciej Luczynski
This document describes a study that used phosphoproteomics to analyze signaling networks downstream of collagen receptors. The researchers identified 424 phosphorylated proteins over seven time points after stimulating cells with collagen. Multiple clustering analysis revealed that phosphorylation sites on proteins like SHP-2, NCK1, LYN, and PIK3C2A strongly clustered with DDR2 phosphorylation dynamics, suggesting these proteins are candidate downstream effectors of DDR2 signaling. Biochemical validation showed SHP-2 tyrosine phosphorylation depends on DDR2 kinase activity. Targeted proteomics of lung cancer DDR2 mutants showed SHP-2 is phosphorylated by some mutants. This indicates SHP-2 is a key signaling node downstream of DDR
1. The document describes a study that used site-directed mutagenesis to alter a single amino acid (phenylalanine to serine at position 67) in the furin protein sequence to examine its structure, function, and trafficking.
2. The methods included designing mutagenic primers, performing site-directed mutagenesis on the furin cDNA cloned in a plasmid, transforming E. coli with the plasmid, isolating and sequencing the plasmid DNA, and future plans to transfect mutant and wild-type furin into cells for analysis.
3. Furin is involved in development, homeostasis, and disease pathways by processing precursor proteins, and its structure and trafficking are important for understanding its functions.
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.
This study investigated whether neuronal cells show preferential growth and maturation on synthetic polymers versus decellularized nerve extracellular matrix (dECM) bioink. Several key findings were reported:
1) Neurite outgrowth of neuronal cells was greater when cultured on dECM bioink compared to matrigel or tissue culture plastic.
2) Cell viability was lower for neuronal cells grown on dECM bioink compared to matrigel or plastic, indicating dECM bioink supports longer neurites but at the expense of cell viability.
3) A blend of dECM bioink and PEG was successfully 3D printed, demonstrating a novel biomaterial containing both natural and synthetic properties that may
This document summarizes research on the structural biology of cytochrome P450 monooxygenases in the phenylpropanoid pathway of Arabidopsis thaliana. It discusses homology modeling and protein engineering methods used to study the structures of phenylpropanoid P450s. It also describes efforts to develop X-ray crystallography and NMR techniques to determine the crystal structures of these P450s to better understand their functions in metabolic pathways.
Peptides are short chains of amino acids linked by peptide bonds. They are distinguished from proteins by typically containing fewer than 50 amino acid units. Peptides are formed through condensation reactions between carboxyl and amino groups of separate amino acids, releasing a water molecule. Peptide bonds are rigid and planar, contributing to protein structure stability. Peptides serve many important biological functions and can be classified based on their production method, including through ribosomal translation, nonribosomal synthesis, and enzymatic digestion of proteins in foods. Bioactive peptides derived from food proteins can have beneficial effects like lowering blood pressure, cholesterol, and antimicrobial properties.
Macromolecular structure and biological function of primary protiensArjun K Gopi
This document discusses primary proteins, their structure and functions. It begins with defining proteins as polymers of amino acids linked by peptide bonds. It then discusses the primary structure of proteins, which is the linear arrangement of amino acids. The key functions of proteins mentioned include acting as antibodies, enzymes, hormones, for transport, storage, as structural proteins, and contractile proteins. Some applications discussed are using proteins in coatings, food packaging, biotechnology, foods, microchips, and biomedical applications like hydrogels and targeted drug delivery.
The structure of a protein determines its function. For enzymes, the binding site or active site allows substrates to bind via lock-and-key or induced fit mechanisms. Myoglobin and hemoglobin both contain heme groups to bind oxygen, but differ in structure - myoglobin is spherical while hemoglobin is a tetramer. This allows hemoglobin to exhibit cooperative binding and transport oxygen more efficiently than myoglobin via conformational changes.
This document presents the case of a 29-year-old pregnant female who presented with epigastric pain, vomiting, diarrhea and later coughing up blood and nosebleeds. Investigations showed leukocytosis, low blood pH, high blood sugar, and sepsis. She had a spontaneous abortion due to sepsis and developed disseminated intravascular coagulation (DIC) and bilateral pulmonary embolism. Her condition improved with treatment but she was found to have a protein C deficiency, which can cause hypercoagulability and venous thromboembolism. The document provides details on her hospital course and explains DIC, its causes, diagnosis, and inherited and acquired hypercoagulable states.
- Amino acids are compounds that contain both an amino group and a carboxyl group. They exist in L- and D-stereoisomers and have common 1-letter and 3-letter codes.
- There are 20 standard amino acids that are classified by the properties of their variable side chains into nonpolar, polar, acidic, and basic groups.
- The ionization states of amino acids' functional groups depend on pH and can be calculated using pKa values and the Henderson-Hasselbalch equation.
- Amino acids join together via peptide bonds between amino and carboxyl groups, forming peptides and proteins of various lengths with restricted rotation around the peptide bond.
Amino acids are the building blocks of proteins and have a general structure with an acid and basic group that determine their properties. Peptides are formed from amino acids linked together by peptide bonds, which restrict their movement and give peptides directionality from the N to C terminus. Peptides can be synthesized either through purification from natural sources or chemically through solid phase synthesis to make specific sequences.
Marketing of Proteins and Peptide Pharmaceuticalsguest6c594976
The document discusses proteins and peptides as pharmaceutical drugs. It provides an overview of the protein therapeutics market, major brands that generate over $1 million in revenue, production costs, challenges in the field, and regulatory considerations for approval of protein and peptide drugs.
This document discusses protein structure and analysis techniques. It describes the four levels of protein structure: primary, secondary, tertiary, and quaternary. Common secondary structures like alpha helices and beta sheets are formed by hydrogen bonding patterns in the protein backbone. Tertiary structure refers to a protein's three-dimensional shape, while quaternary structure involves multiple polypeptide subunits. Several techniques for analyzing proteins are also outlined, including electrophoresis, chromatography, and spectroscopy.
Stability studies of proteins and peptides.SULABH910
This document discusses stability studies of proteins and peptides. It covers both chemical and physical degradation mechanisms and factors that influence degradation rates. Chemical degradation includes deamidation, racemization, hydrolysis, disulfide formation, oxidation, and cross-linking. Physical degradation involves changes in structure like denaturation, aggregation, adsorption, and precipitation. Degradation rates depend on factors like pH, temperature, moisture content, and excipients. Kinetics are often first-order and follow Arrhenius behavior, allowing prediction of long-term stability from accelerated studies. Understanding degradation mechanisms is key to developing stable protein and peptide drug formulations.
The document discusses the benefits of exercise for mental health. Regular physical activity can help reduce anxiety and depression and improve mood and cognitive function. Exercise causes chemical changes in the brain that may help protect against mental illness and improve symptoms for those who already suffer from conditions like anxiety and depression.
Our project in physics (IV-Einstein) about waves: It's nature, types, parts and measures.
I apologize for the ugly font, I used different font styles that are not available on all computers since they are downloaded from the internet.
Protein and peptide drugs can be delivered through various routes including parenteral, oral, buccal, nasal, transdermal, pulmonary, rectal, ocular, and vaginal administration. Various drug delivery systems are used to protect proteins from degradation and control release over time. These include microencapsulation, polymeric scaffolds, liposomes, magnetic targeting, and hydrogels. Recent advances provide more effective noninvasive delivery methods for these therapeutic compounds.
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The peptide bond joins amino acids in polypeptides and proteins. It has partial double bond character which makes it nearly planar. This rigidity reduces flexibility during protein folding. The peptide bond is usually in the trans configuration to reduce steric hindrance, though proline can be cis or trans due to its cyclic side chain.
This document provides an overview of peptides. It defines peptides as short chains of amino acids linked by peptide bonds. Peptides are distinguished from proteins based on their smaller size, typically containing fewer than 50 amino acid units. The document discusses peptide bond formation, characteristics of peptide bonds, classes of peptides, physical properties, examples of individual peptides, food-derived peptides with biological activity, and applications of peptides in molecular biology.
The document summarizes protein synthesis through the central dogma of genetics. It discusses how DNA is transcribed into mRNA which is then translated into proteins using tRNA and ribosomes. The main stages of translation - initiation, elongation, and termination - are also outlined. Finally, the genetic code table is shown, which demonstrates how mRNA codons correspond to specific amino acids.
Protein and-peptide-drug-delivery-systemsGaurav Kr
The document discusses protein and peptide drug delivery systems. It begins by defining proteins and peptides, noting that proteins are molecules composed of over 50 amino acids, while peptides are molecules composed of less than 50 amino acids. It then discusses how scientific advances in molecular and cell biology have led to the development of recombinant DNA and hybridoma technology to produce protein products. The document provides examples of marketed protein and peptide drugs and discusses challenges with delivering these drugs orally due to their large molecular size and susceptibility to enzymatic degradation. It explores approaches to protein and peptide delivery including non-parenteral systemic delivery methods and various considerations for developing delivery systems for these pharmaceuticals.
Protein and peptide drug delivery systemSagar Savale
Protein and Peptide drug delivery system are the Novel drug Delivery System. Proteins and peptides are the most abundant components of biological cells. They exist functioning such as
enzymes, hormones, structural element and immunoglobulin. The distinction between peptides and proteins is having a peptide contains less than 20 amino acids, having a molecular weight less, while a protein possesses 50 or more amino acids and its molecular weight lies above this value. The most of pharmaceutical proteins and peptides are absorbed IM, IV and Subcutaneous route of Absorption, but the oral route is more convenient for absorption of protein as compared to other. Various problems associated with administration of protein and peptide drugs are needed to overcome by different pharmaceutical approaches. Several approaches available for
maximizing pharmacokinetic and pharmacodynamics properties are chemical modification,
formulation vehicles, mucoadhesive polymeric system, use of enzyme inhibitors, absorption
enhancers, penetration enhancers etc.
Similar to Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery
Human, Eukaryotic And Vitro Associations Of Murine Sec...Rachel Davis
The document describes experiments to study the effects of vitamin D receptor (VDR) binding to vitamin D response elements (VDREs). Reporter plasmids containing wildtype or mutant VDREs upstream of firefly luciferase were constructed. These plasmids were transfected into HEK293T cells to assess VDR binding and activation of luciferase expression in response to vitamin D treatment.
Uncovering novel candidate genes for pyridoxine-responsive epilepsy in a cons...Golden Helix Inc
This document summarizes Hilal Al Shekaili's work on characterizing the genetic cause of pyridoxine-dependent epilepsy (PDE) in an Omani family. [1] Runs of homozygosity mapping and whole-exome sequencing identified two candidate genes involved in vitamin transport and neuropeptide processing. [2] Further studies are planned to validate the candidate genes and recruit additional families. [3] Identifying new PDE genes could improve treatment and fill knowledge gaps in pyridoxine metabolism.
Genetic Basis of Pyridoxine-Responsive Neonatal Epilepsy in Consanguineous Fa...Delaina Hawkins
Hilal Al-Shekaili is a PhD student at the University of British Columbia who conducts research in rare, autosomal recessive disorders, specifically pyridoxine-responsive epileptic encephalopathies (PREE). PREE is often characterized by recurrent seizures in the prenatal, neonatal, or postnatal period, which are typically resistant to conventional anticonvulsant treatment but are well-controlled by the administration of pyridoxine (vitamin B6). Hilal and his colleagues at UBC are undertaking a research project to identify novel genetic causes in unexplained forms of pyridoxine-dependent epilepsy (PDE), a special type of PREE with an estimated incidence of 1:20,000 to 1:750,000. In most affected infants, PDE is caused by mutations in the antiquitin gene (ALDH7A1) and subsequent inactivation of α-aminoadipic semialdehyde dehydrogenase (antiquitin, ATQ).
Currently, ALDH7A1 is the only gene for which mutations are known to underlie PDE. However, locus heterogeneity has been reported in some families and other genes seem to be involved. Nearly 5% of children with a typical clinical picture of PDE harbor no detectable mutation of ALDH7A1. Identifying causative genes in such families will likely lead to improved treatment for these patients and help unravel much of the unknown about pyridoxine metabolism in the human body.
In this webinar, Hilal will cover how he and his team used whole-genome SNP genotyping, genome-wide runs of homozygosity (RoH) mapping using SVS, and whole-exome sequencing to characterize the genetic defect underlying PREE in a consanguineous Omani Arab family with two affected children who have a PDE-like clinical picture but negative ATQ biomarkers.
Presentation On Wnt 4 and rhe role of HDAC4 & SIRT1 in bone biologyGayathri Vijayakumar
Wnt proteins are a family of conserved signaling proteins that regulate various processes including cell proliferation, fate determination, and embryonic development. They act through both canonical and non-canonical pathways. Wnt4 specifically promotes female development and suppresses male development. It also contributes to kidney and adrenal cortex development. Studies show Wnt4 enhances stem cell osteogenic differentiation by activating non-canonical pathways like P38MAPK. The NASA-Orthofix project examines the effects of pulsed electromagnetic fields on osteoblast proliferation, differentiation and mineralization using rat primary osteoblasts. Genes analyzed include osteocalcin, BAP, collagen I and GAPDH. Results show PEMF stimulation increases cell count, differentiation markers and mineralization
genes addiion\deeion\ediionthat lead to a therapeutic, prophylactic or diagnostic effect
Plasmid DNA
•Viral vectors
•Genetically engineered micro-organisms
•Human gene-editing technology
•Patient-derived cellular gene therapy products
This document provides an overview of ChEMBL, a large database of medicinal chemistry data maintained by EMBL-EBI. It describes the types of data contained in ChEMBL, including over 1.6 million compounds, 10,000 targets, and 12 million bioactivities extracted from literature. ChEMBL aims to comprehensively catalogue historical drug discovery successes and failures to identify patterns and support drug discovery. All data is freely available under an open license.
This document discusses proteomic methodologies for studying protein phosphorylation. It begins with an introduction to protein phosphorylation and its importance in regulating cellular processes. It then describes the workflow for analyzing phosphorylation through proteomics, including isolating phosphoproteins, identifying phosphorylation sites via mass spectrometry, and comparing normal and treated phosphoproteomes. Key methods discussed are 2D gel electrophoresis, phosphoprotein staining, silver staining, spot excision, in-gel digestion, and liquid chromatography-tandem mass spectrometry for identifying phosphorylation sites. The document emphasizes that proteomic analysis is needed to fully understand phosphorylation dynamics and signaling pathways.
This document discusses surface modification of nanoparticles for biomedical applications. It describes how nanoparticles can be modified on their surface with various ligands to target specific cells and tissues. These ligands include antibodies, peptides, aptamers, and other molecules like folate. Aptamers and peptides offer advantages over antibodies as targeting ligands. The document provides examples of using various ligands like VEGF, folate, and transferrin to target receptors overexpressed on cancers and other diseases. It also discusses different conjugation chemistries used to attach ligands to the nanoparticle surface, like using succinimide, biotin-streptavidin, and thiol chemistry.
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.
Genetic Dna And Bioinformatics ( Accession No. Xp EssayJessica Deakin
This document discusses natural language processing (NLP) for Sanskrit and different part-of-speech (POS) tagging methods. It introduces NLP and POS tagging, noting that POS tagging is the first step in developing NLP applications. It then discusses different tagsets and POS tagging approaches for Sanskrit like hidden Markov models and conditional random fields.
A nanomedical device should perform multiple functions, so a multifunctional nanoparticle system can be constructed from the inner core outward. The core can contain a drug, while outer layers provide targeting ligands, such as antibodies, peptides, or aptamers directed against receptors overexpressed on diseased cells. Aptamers have advantages over antibodies like uniform activity between batches. Peptides can also serve as targeting ligands if they recognize receptors. Other ligands like folate or transferrin can bind their cognate receptors, but compete with circulating levels; antibodies are more specific but expensive. A variety of conjugation strategies can be used to attach targeting ligands to nanoparticles.
Unexpected link between an antibiotic, pannexin channels and apoptosis.Salman Ul Islam
Pannexins, specifically PANX1, play an important role in apoptosis by regulating the formation and disassembly of apoptotic bodies. The study identified the antibiotic trovafloxacin as a small molecule that can inhibit PANX1 channel activity during apoptosis. Experiments showed trovafloxacin treatment and genetic deletion of PANX1 both promoted the formation of smaller apoptotic bodies. This suggests PANX1 inhibition could control the size of apoptotic structures expelled during cell death.
This document provides an overview of pharmacology of proteins and peptides. It discusses the historical perspective of peptide research beginning in the 1930s. Key developments include the elucidation of the structures of oxytocin and vasopressin by Dr. Vincent du Vigneaud in the 1950s. The document compares neuropeptides to conventional neurotransmitters and describes the biosynthesis and regulation of proteins. It also covers topics such as proteins and peptides as drugs, peptide agonists and antagonists, and techniques for identifying, isolating, and characterizing peptides. The future potential of designer proteins is also mentioned.
Current Trends in Molecular Biology and BioTechnology (ppt)Perez Eric
This document discusses current trends in molecular biology and biotechnology. It begins by defining biotechnology and explaining its importance in addressing challenges around feeding and clothing the growing global population. It then describes molecular biology as the study of biological processes at the molecular level, including DNA, RNA, protein synthesis and gene regulation. Some applications of molecular biology discussed include research, diagnosis, forensics, gene therapy and drug design. Key cellular components like DNA, RNA and proteins are also explained. Important techniques in molecular biology like PCR, DNA/RNA blotting, gene expression and cloning, microarrays, and RNA interference are summarized. The uses of embryonic and adult stem cells in research and therapy are also covered briefly.
The document summarizes several key studies from 2011 related to glomerular diseases, polycystic kidney disease, acute kidney injury, and transplantation. Notable findings include identification of bovine serum albumin as a potential antigen in membranous nephropathy, discovery of soluble urokinase receptor as a circulating factor in recurrent focal segmental glomerulosclerosis, and evidence that tolvaptan and metformin may be novel therapeutics for polycystic kidney disease by inhibiting vasopressin and mTOR pathways respectively.
This document discusses the application of proteomics in environmental studies. It begins by defining proteomics as the study of the proteome, which is the total set of proteins expressed in a cell at a given time. The document then outlines several types of proteomics including structural, functional, and expression proteomics. It provides examples of proteomics studies on various organisms like bacteria, yeast, mussels, and vertebrates that have helped understand their responses to environmental stresses. The document concludes that proteomics is a valuable tool for environmental research that can help monitor pollutants and identify protein biomarkers of toxic exposures.
The goal of this study was to analyze the effects of Csk knockouts on development of the initial segment of the epididymis. Immunofluorescence staining of control and Csk knockout mice found no differences in expression of proteins related to proliferation and differentiation. However, platelet-derived growth factor beta (PDGF-β), which enhances capillary growth, showed significantly higher expression in knockout mice at 8 weeks, suggesting increased angiogenesis over time without Csk inhibition. Further analysis is needed but this preliminary result indicates Csk knockouts may lead to time-sensitive increased vascularization in the initial epididymis segment.
Pharmacokinetics and pharmacodynamics of Biotechnological drugs-SnehalTidke
Pharmacokinetics and pharmacodynamics of biotechnological drugs along with appliations- Proteins and peptides, monoclonal antibodies, oligonucleotides, gene therapy and vaccines
Similar to Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery (20)
Ahead of the marcus evans Evolution Summit 2023, read here an interview with Erwin De beuckelaer discussing how utilising and harmonising digital measures in clinical trials will transform the pharma industry.
Ahead of the marcus evans Evolution Summit 2023 and the Evolution Europe Summit 2023, read here an interview with Alexander Fetkovsky discussing the gaps in the clinical trial supply chain.
Ahead of the marcus evans Evolution Summit 2022, read here an interview with Kristine Koontz discussing what it takes for sponsors and vendors to have a mutually beneficial relationship.
An Interactive Roundtable Discussion with Jean M. Gatewood, Vice President, Clinical Research Strategy, Fox Chase Cancer Center – Temple Health at the marcus evans Evolution Summit Fall 2019 in San Diego CA.
Presentation delivered by Lori A. Tierney, BSN, Director, Site Management Operations, Allergan, Inc. at the marcus evans Evolution Summit Fall 2019 in San Diego CA.
Presentation delivered by Sonia Sethi, Vice President, Clinical Operations & Client Engagement, Veristat and Jacqueline Mardell, Vice President, Clinical Operations, Ascendis Pharma at the marcus evans Evolution Summit Fall 2019 in San Diego CA
Ahead of the marcus evans Evolution Summit November 2019, read here an interview with Dunya Botetzayas discussing how risk-based monitoring can have a positive effect on a study site
This document discusses efforts to find a cure for a rare genetic disease called Black Bone Disease or Alkaptonuria (AKU) through several steps:
1) Understanding the disease through studies of patient symptoms, mouse models, and metabolic pathways.
2) Setting up clinical trials in the UK and Europe to test the drug nitisinone through a national research center and consortium.
3) Recruiting and retaining over 140 patients from around the world for the trials over 9 months through online outreach and regular communication.
4) Working directly with patient advocacy groups in Europe, Asia, the Middle East, and North America to support patients.
Presentation delivered by Dr Steven M. Fruchtman, Chief Medical Officer, Onconova Therapeutics at the marcus evans Evolution Summit Spring 2017 held at the Ritz-Carlton Coconut Grove, FL, May 8-10.
Presentation delivered by Dr Laura Esserman, MD, MBA, Director, UCSF Carol Franc Buck Breast Care Center, Professor of Surgery and Radiology, UCSF at the marcus evans Evolution Summit Fall 2015 held in Las Vegas, NV
This document discusses opportunities for collaboration between academia and industry to drive efficiency in research. It notes that while academia traditionally focused on pure research and industry on profit, drug discovery now requires a mixed model with contributions from both. Effective collaborations could help address challenges like rising costs and project attrition. However, differences in culture and goals can hamper partnerships. The document recommends developing focused projects and consortia, addressing intellectual property and conflicts of interest upfront, and managing collaborations strategically like an investment portfolio to maximize their potential for innovation.
Presentation by Andreas Grauer, MD, Executive Medical Director, Global Development Leader, Amgen at the marcus evans Evolution Summit Fall 2015 in Las Vegas
This document summarizes a presentation on strategies for boosting pharmaceutical innovation given by Kenneth Kaitin. It discusses the current challenging environment for drug development, including expiring patents, competitive markets, and high costs. Drug development times are long with low success rates, driving costs upwards. Oncology has become a major focus of R&D investment. The presentation proposes ways to stimulate innovation in areas of high medical need but low commercial appeal, such as through regulatory and legislative incentives.
Biomarkers to Diagnostics – The Essential Tool Box for Drug Development - Presentation delivered by Johan Luthman, Vice President, Neuroscience Clinical Development, Eisai Pharmaceuticals at the marcus evans Evolution Summit Fall 2015 in Las Vegas
1) A clinical trial was designed to evaluate the efficacy and safety of clobazam for the orphan pediatric indication of Dravet syndrome.
2) Challenges included a lack of dosing data in young children, capturing baseline seizure frequency variability, and conducting a placebo-controlled trial when clobazam was already available.
3) Pharmacokinetic modeling was used to determine appropriate dosing and sample sizes to supplement limited data, and regulatory and external input helped address challenges.
For more information contact: Slideshare@marcusevans.com.
Forming Powerful Partnerships to Drill Down into the Areas of Expertise of Each Stakeholder and Unravel Disease Mechanisms
Eric Low
Cascais, 16 March 2015
How to Get CNIC Information System with Paksim Ga.pptxdanishmna97
Pakdata Cf is a groundbreaking system designed to streamline and facilitate access to CNIC information. This innovative platform leverages advanced technology to provide users with efficient and secure access to their CNIC details.
Threats to mobile devices are more prevalent and increasing in scope and complexity. Users of mobile devices desire to take full advantage of the features
available on those devices, but many of the features provide convenience and capability but sacrifice security. This best practices guide outlines steps the users can take to better protect personal devices and information.
Ivanti’s Patch Tuesday breakdown goes beyond patching your applications and brings you the intelligence and guidance needed to prioritize where to focus your attention first. Catch early analysis on our Ivanti blog, then join industry expert Chris Goettl for the Patch Tuesday Webinar Event. There we’ll do a deep dive into each of the bulletins and give guidance on the risks associated with the newly-identified vulnerabilities.
Essentials of Automations: The Art of Triggers and Actions in FMESafe Software
In this second installment of our Essentials of Automations webinar series, we’ll explore the landscape of triggers and actions, guiding you through the nuances of authoring and adapting workspaces for seamless automations. Gain an understanding of the full spectrum of triggers and actions available in FME, empowering you to enhance your workspaces for efficient automation.
We’ll kick things off by showcasing the most commonly used event-based triggers, introducing you to various automation workflows like manual triggers, schedules, directory watchers, and more. Plus, see how these elements play out in real scenarios.
Whether you’re tweaking your current setup or building from the ground up, this session will arm you with the tools and insights needed to transform your FME usage into a powerhouse of productivity. Join us to discover effective strategies that simplify complex processes, enhancing your productivity and transforming your data management practices with FME. Let’s turn complexity into clarity and make your workspaces work wonders!
OpenID AuthZEN Interop Read Out - AuthorizationDavid Brossard
During Identiverse 2024 and EIC 2024, members of the OpenID AuthZEN WG got together and demoed their authorization endpoints conforming to the AuthZEN API
In the rapidly evolving landscape of technologies, XML continues to play a vital role in structuring, storing, and transporting data across diverse systems. The recent advancements in artificial intelligence (AI) present new methodologies for enhancing XML development workflows, introducing efficiency, automation, and intelligent capabilities. This presentation will outline the scope and perspective of utilizing AI in XML development. The potential benefits and the possible pitfalls will be highlighted, providing a balanced view of the subject.
We will explore the capabilities of AI in understanding XML markup languages and autonomously creating structured XML content. Additionally, we will examine the capacity of AI to enrich plain text with appropriate XML markup. Practical examples and methodological guidelines will be provided to elucidate how AI can be effectively prompted to interpret and generate accurate XML markup.
Further emphasis will be placed on the role of AI in developing XSLT, or schemas such as XSD and Schematron. We will address the techniques and strategies adopted to create prompts for generating code, explaining code, or refactoring the code, and the results achieved.
The discussion will extend to how AI can be used to transform XML content. In particular, the focus will be on the use of AI XPath extension functions in XSLT, Schematron, Schematron Quick Fixes, or for XML content refactoring.
The presentation aims to deliver a comprehensive overview of AI usage in XML development, providing attendees with the necessary knowledge to make informed decisions. Whether you’re at the early stages of adopting AI or considering integrating it in advanced XML development, this presentation will cover all levels of expertise.
By highlighting the potential advantages and challenges of integrating AI with XML development tools and languages, the presentation seeks to inspire thoughtful conversation around the future of XML development. We’ll not only delve into the technical aspects of AI-powered XML development but also discuss practical implications and possible future directions.
Climate Impact of Software Testing at Nordic Testing DaysKari Kakkonen
My slides at Nordic Testing Days 6.6.2024
Climate impact / sustainability of software testing discussed on the talk. ICT and testing must carry their part of global responsibility to help with the climat warming. We can minimize the carbon footprint but we can also have a carbon handprint, a positive impact on the climate. Quality characteristics can be added with sustainability, and then measured continuously. Test environments can be used less, and in smaller scale and on demand. Test techniques can be used in optimizing or minimizing number of tests. Test automation can be used to speed up testing.
Your One-Stop Shop for Python Success: Top 10 US Python Development Providersakankshawande
Simplify your search for a reliable Python development partner! This list presents the top 10 trusted US providers offering comprehensive Python development services, ensuring your project's success from conception to completion.
HCL Notes and Domino License Cost Reduction in the World of DLAUpanagenda
Webinar Recording: https://www.panagenda.com/webinars/hcl-notes-and-domino-license-cost-reduction-in-the-world-of-dlau/
The introduction of DLAU and the CCB & CCX licensing model caused quite a stir in the HCL community. As a Notes and Domino customer, you may have faced challenges with unexpected user counts and license costs. You probably have questions on how this new licensing approach works and how to benefit from it. Most importantly, you likely have budget constraints and want to save money where possible. Don’t worry, we can help with all of this!
We’ll show you how to fix common misconfigurations that cause higher-than-expected user counts, and how to identify accounts which you can deactivate to save money. There are also frequent patterns that can cause unnecessary cost, like using a person document instead of a mail-in for shared mailboxes. We’ll provide examples and solutions for those as well. And naturally we’ll explain the new licensing model.
Join HCL Ambassador Marc Thomas in this webinar with a special guest appearance from Franz Walder. It will give you the tools and know-how to stay on top of what is going on with Domino licensing. You will be able lower your cost through an optimized configuration and keep it low going forward.
These topics will be covered
- Reducing license cost by finding and fixing misconfigurations and superfluous accounts
- How do CCB and CCX licenses really work?
- Understanding the DLAU tool and how to best utilize it
- Tips for common problem areas, like team mailboxes, functional/test users, etc
- Practical examples and best practices to implement right away
CAKE: Sharing Slices of Confidential Data on BlockchainClaudio Di Ciccio
Presented at the CAiSE 2024 Forum, Intelligent Information Systems, June 6th, Limassol, Cyprus.
Synopsis: Cooperative information systems typically involve various entities in a collaborative process within a distributed environment. Blockchain technology offers a mechanism for automating such processes, even when only partial trust exists among participants. The data stored on the blockchain is replicated across all nodes in the network, ensuring accessibility to all participants. While this aspect facilitates traceability, integrity, and persistence, it poses challenges for adopting public blockchains in enterprise settings due to confidentiality issues. In this paper, we present a software tool named Control Access via Key Encryption (CAKE), designed to ensure data confidentiality in scenarios involving public blockchains. After outlining its core components and functionalities, we showcase the application of CAKE in the context of a real-world cyber-security project within the logistics domain.
Paper: https://doi.org/10.1007/978-3-031-61000-4_16
Taking AI to the Next Level in Manufacturing.pdfssuserfac0301
Read Taking AI to the Next Level in Manufacturing to gain insights on AI adoption in the manufacturing industry, such as:
1. How quickly AI is being implemented in manufacturing.
2. Which barriers stand in the way of AI adoption.
3. How data quality and governance form the backbone of AI.
4. Organizational processes and structures that may inhibit effective AI adoption.
6. Ideas and approaches to help build your organization's AI strategy.
Evolution 2013: Dr Sarah Jones, University of Wolverhampton on Exploring the Potential of Cell Penetrating Peptides for Effective Drug Delivery
1. The Latest Discovery: Exploring the Potential of
Cell Penetrating Peptides for Effective Drug
Delivery
Dr Sarah Jones
Molecular Pharmacology Research Group, University of
Wolverhampton
2. Overview
History and Background of CPPs
Hurdles and Progress
Where the field is at the moment
Targeting Protein-Protein Interactions,
from Bioportides to Sperm
3. Cell Penetrating Peptides
(CPPs)
• Inert vectors for the delivery of
bioactive cargoes into the
intracellular milieu
• Intracellular delivery of Peptides,
Proteins, Drugs,
Oligonucleotides (siRNA, PNA),
Plasmids
• Viable alternative to viral vectors
and current non-viral
intracellular delivery
4. CPP History: A Long Time Coming!
1988 - The TransActivator of
Transcription protein (TAT) derived
from the HIV virus entered cells
1994- Penetratin –Helix 3 of the
Antennapedia homeodomain
1997 - Tat48-60, a truncated
arginine-rich sequence conferred
cellular penetration.
Tat
AntHD43-58 Penetratin
6. Most CPP are polycationic sequences
Primary Sequence
Name/Source
HIV-1 Tat-derived peptides
GRKKRRQRRRPPQ
QPPRRRQRRKKRG
Tat48-60
RI-Tat
Penetratins
RQIKIWFQNRRMKWKK
RRRRRRRQIKIWFQNRRMKWKK
AntHD43-58 (Penetratin)
R6-Penetratin
Other arginine-rich peptides
RRRRRRRRR
(R(Ahx)R)4
R8 and R9 are the most common
(RXR)4
Predominantly amphipathic sequences
AGYLLGKINLKALAALAKKIL
INLKKLAKL(Aib)KKIL
LLIILRRRIRKQAHAHSK
Transportan10
Mitoparan
pVec
Hydrophobic Sequences
CSIPPEVKFNKPFVYLI
C105Y
7. Uptake mechanisms, a fixation!
Direct membrane
translocation (4oC)
Fixation artifacts (Live cell imaging)
Endocytosis (37oC, ATP)
Clathrin-mediated
Caveolin-mediated
Macropinocytosis
Dependent upon sequence, cell type, concentration and cargo
(biochemical properties and size)
8. To Endocytose or not to Endocytose? That is the question
Mishra A et al.
Translocation of HIV TAT
peptide
and analogues induced by
multiplexed membrane and
cytoskeletal interactions
PNAS (2011) 108 16883
9. Progress in CPP Technologies
PepFect Technologies
AGYLLGKINLKALAALAKKIL-NH2
TP10
PepFect3
Stearyl-AGYLLGKINLKALAALAKKIL-NH2
PepFect6
Stearyl-AGYLLGKINLKALAALAKKIL-NH2
PepFect14
Stearyl-AGYLLGKLLOOLAAAALOOLL-NH2
• Oligonucleotide therapeutics-siRNA and
splice correcting oligonucleotides
• Ecsapes endosomal entrapment
associated with the delivery of larger
cargoes
Ülo Langel Stockholm University
CPP Technologies in Clinical Development
• PsorBan (CellGate) – heptaarginine coupled to cyclsporin A (psoriasis)
• KAI-9803 (Kai Pharmaceuticals) - tat coupled to a peptide inhibitor of PKCd (reperfusion injury)
• XG-102 (Auris Medical)– tat coupled to a JNK inhibiting peptide (traumatic hearing loss)
10. What Cell Penetrating Peptides DO NOT DO!
The Lipinski Rule of Five
•Its molecular weight is less than 500.
•The compound's lipophilicity, expressed as a quantity known as logP (the logarithm of the partition
coefficient between water and 1-octanol), is less than 5.
•The number of groups in the molecule that can donate hydrogen atoms to hydrogen bonds (usually
the sum of hydroxyl and amine groups in a drug molecule) is less than 5
•The number of groups that can accept hydrogen atoms to form hydrogen bonds (estimated by the
sum of oxygen and nitrogen atoms) is less than 10.
11. Bioactive Cell Penetrating Peptides (Bioportides) Enhance the Repertoire of Druggable
Targets
Expansion of Druggable Targets
It is estimated that 8-10% of the
human genome encodes diseasemodifying proteins
Only 10% of the druggable genome
can be targeted by conventional
approaches (SMDs)
New Chemical Entities which target
Protein-Protein Interactions are
gaining momentum as an attractive
therapeutic modality
Proteomics and interactomics will
identify many other PPIs that can be
modulated by peptides
Intracellular Drug Target Space
13. Sychnologic CPP
Rhegnylogic CPP
Sequence
Target
Activity
Nosangiotide
ND
Anti-angiogenic
Sequence
Target
Activity
Pathology
Camptide
G proteins
cAMP modulation
Tat-DI1
Raf
dimerizatio
n
Inhibits proliferation of NSCLC
cell lines
Cancer
Cyt c77-101
ER
Apoptogenic
BIP
BAX
Antp-MEK1
ERK
Inhibition of ERK2 activation
Cancer
Anti-apoptogenic
(neuroprotection)
Tat48-60-P10
PCNA
Apoptogenic
Cancer
Mouse PrP1-28
Prion Proteins
Anti-prion infection
STAT-6-IP
STAT-6
Inhibitor of TH2 cytokine
production
Allergic
airways
disease
AT1AR304-318
G proteins
Blood vessel
contraction
Hph-1ctCTLA-4
TcR
signalling
Inhibition of TcR signalling.
Reduction in Th2 cytokines,
serum IgE
Allergic
airways
disease
Vasostatin-1
Heparan Sulphate
Proteoglycans
eNOS activation
(vasodilatory)
Arf (1-22)
ND
Apoptogenic
Mitoparan
VDAC (mitochondria)
Apoptogenic
Stapled p53derived peptides
p53-hDM2
Reactivation of p53
apoptosis pathway
Tat-acpTrkA666-676
Nup153-Cyt
TrkA
activation
loop
TrkA antagonism
NPC
Apoptogenic
Inflammator
y pain
Cancer
14. Bioactive Cell Penetrating Peptides - Bioportides
Signal
Transduction
Domain
Position
85
IKKKEERADLIAYLKK
2
16
86
KKKEERADLIAYLKKA
3
17
84
GIKKKEERADLIAYLKK
3
17
85
IKKKEERADLIAYLKKA
3
17
QSAR Prediction Algorithm
No. of
Amino
Acids
16
Whole
Protein
Sequence
86
KKKEERADLIAYLKKAT
2
Prediction algorithm: Hällbrink et al. Int. J. Peptide Res. Ther. 11, 249 (2005)
CPP mimic of GPCRs: Ostlund et al. Int. J. Peptide Res. Ther. 11, 237 (2005)
CPP
Probability
15. Anti-angiogenic Properties of Nosangiotide (eNOS492-507)
A Bioportide Derived from Endothelial Nitric Oxide Synthase
Nosangiotide is
located within the
a-helix domain
(shown in yellow)
that tightly binds
calmodulin. Diagram
adapted from Aoyagi et
al., EMBO J. 22, 766-775
(2003)
Sequence
RKKTFKEVANAVKISASLMG
RKKTFKEVANA
RKKTFKEVANAVK
RKKTFKEVANAVKI
RKKTFKEVANAVKISA
Early distribution
Cells were incubated for
45 mins with rhoeNOS492-507 (5mM) in
endothelial cell medium
plus growth supplement
then transferred to
DMEM w/o phenol red
for confocal visualization
CPP Index
Later nuclear and
cytoplasmic
distribution
1
3
3
3
3 Nosangiotide
Cells were incubated for
80 mins with rhoeNOS492-507 (5mM) in
endothelial cell medium
plus growth supplement
then transferred to DMEM
w/o phenol red for
confocal visualization
16. Nosangiotide (eNOS492-507) Inhibits Biological Features of FGF-induced Angiogenesis
in vitro
(a)
(b)
(c)
250
1750
90,000
80,000
70,000
mean of closed areas per field
number of migrated cells
cell number
1500
1250
1000
200
150
100
750
60,000
500
-9
-8
-7
492-507
log { [eNOS
-6
] (M) }
Proliferation
IC50 = 83.7 nM
-5
-8
-7
log { [eNOS
-6
492-507
Migration
IC50 = 38.2
nM
-5
] (M) }
-4
50
-9
-8
-7
-6
492-507
log { [eNOS
-5
-4
-3
] (M) }
Tube Formation
IC50 = 509 nM
Nosangiotide is a potent inhibitor of FGF-2 (25 ng/ml)-induced proliferation (a), migration (b) and tube
formation (c) of primary endothelial cells
17. Nosangiotide inhibits FGF-induced angiogenesis
A) Carrier control
B) FGF-2 (200ng)
C) Nosangiotide (0.5 nmole)
D) FGF-2 plus nosangiotide (0.5
nmole)
E) FGF-2 with nosangiotide (0.05
nmole)
F) FGF-2 with nosangiotide
(0.005 nmole).
Howl et al. (2012) Cell. Mol. Life Sci.
69, 2951 .
18. Identify Bioportide
Binding Partners
Future Work
Stapled
Peptides
Biotinylated CPP as
Molecular Fishing Rods
C-terminal helix
N-terminal helix
Biotinylated CPP
Primary Sequence Position
KGKKIF
KGKKIFI
GKKIFIMK
KGKKIFIM
KGKKIFIMK
EKGKKIFIMK
Swiss 3T3
Streptavidin-coated multiwell
plates
Primary Sequence
5-10
5-11
6-13
5-12
5-13
4-13
Position
VGIKKK
KMIFVGIKKK
KMIFVGIKKKEERA
KKKEERADLIAYLKKA
GIKKKEERADLIAYLKK
IKKKEERADLIAYLKKA
VGIKKKEERADLIAYLKK 83-100
GIKKKEERADLIAYLKKA
FVGIKKKEERADLIAYLKK 82-100
VGIKKKEERADLIAYLKKA
IFVGIKKKEERADLIAYLKK 81-100
FVGIKKKEERADLIAYLKKA
KMIFVGIKKKEERADLIAYLK 79-99
MIFVGIKKKEERADLIAYLKK 80-100
IFVGIKKKEERADLIAYLKKA
KMIFVGIKKKEERADLIAYLKK 79-100
MIFVGIKKKEERADLIAYLKKA
TKMIFVGIKKKEERADLIAYLKK 78-100
KMIFVGIKKKEERADLIAYLKKA79-101
GTKMIFVGIKKKEERADLIAYLKKA
TKMIFVGIKKKEERADLIAYLKKA
KMIFVGIKKKEERADLIAYLKKAT
GTKMIFVGIKKKEERADLIAYLKKA77-101
TKMIFVGIKKKEERADLIAYLKKAT
KMIFVGIKKKEERADLIAYLKKATN
83-88
79-88
79-92
86-101
84-100
85-101
84-101
83-101
82-101
81-101
80-101
77-101
78-101
79-102
78-102
79-103
Cross linking of peptide side chains
TRYPSIN
DIGEST
Metabolic stability
Enhance propensity for cellular
penetration
Dr Ashley Martin
MALDI
TOF/TOF
Proteomics Unit Cancer Studies
University of Birmingham, UK
a-aminoisobutyric
acid
20. LRRK2: Not just Parkinsons!
LRRK2 role in Inflammatory
Bowel Disease (Liu, Z. et al.,
(2011) Nature Immunology 12,10631070)
LRRK2
LRRK2 sequesters NFAT in the
cytoplasm
LRRK2 deficiency enhances
susceptibility to experimental
colitis and enhances nuclear
localization of NFAT
NFAT Luciferase reporter assay (Armesilla, A.L. et al. (1999) Mol. Cell. Biol. 19, 2032-2043)
Bioportides facilitate relocation of NFAT from the cytoplasm to the nucleus?
Bioportides abrogate PMA and Ca2+ ionophore induced relocation of NFAT?
The role of the Immune System in PD
21. Candidate Bioportides for Modulation of NFAT Translocation
LRRK21310-1331
T2397M
LRRK21124-1139
LRRK215-32
1
ARM
LRRK21116-1136
I1122V
ANK
LRR
LRRK22378-2399
LRRK21367-1386
ROC
G2385R
COR
Kinase
WD40
Leucine-Rich
Repeat domain
KLEQLILEGNKISGICSPLRLK
GNKISGICSPLRLKELKILNL
LRRK21116-1136
GNKISGVCSPLRLKELKILNL
LRRK21116-1136 (I1122V)
SPLRLKELKILNLSKN
LRRK21124-1139
LRRK21310-1331 (C-terminal helix)
Inhibits NFAT translocation
2527
Van Craenenbroeck et al., (2012) Purification and
preliminary biochemical and structural
characterisation of the leucine rich repeat domain
of LRRK2. Biochem. Biophys. Acta 1824, 450
WT inhibits NFAT translocation
Mutant enhances NFAT translocation
Enhances NFAT translocation
HIGCKAKDIIRFLQQRLKKAVP
22. LRRK22378-2399 includes the very common T2397M risk allele for Crohn’s Disease
LRRK21310-1331
T2397M
LRRK21124-1139
LRRK215-32
1
ARM
LRRK21116-1136
I1122V
ANK
LRRK2-derived bioportides
modulate NFAT translocation. HEK293 cells were transiently transfected
with a luciferase reporter vector
(pNFAT-Ta-Luc; Clontech). Luciferase
activity was measured as an indicator
of NFAT translocation to the nucleus.
Cells were stimulated with PMA (20
ng/ml) and the calcium ionophore
A23187 (1 mM) for 16 hours.
Luciferase activity was calculated as
fold induction over the value of the
reporter vector in un-stimulated cells.
LRR
LRRK22378-2399
LRRK21367-1386
ROC
COR
Kinase
G2385R
2527
WD40
23. Building Cell Selectivity into CPP Delivery
•
CPP can be readily incorporated into
multimeric complexes
•
An inherent lack of specificity can be
surmounted by the inclusion of
tissue specific targeting peptides
•
Phage Display Technology has
generated an abundance of tissue
homing peptides
25. Peptide-Based Glioma-Targeted Drug Delivery Vector gHoPe2
Coronal section of a mouse brain with U87 tumor in the right striatum. The tumour area is circled with a
dotted line. (B) H&E stained hemisphere of brain and glioma. (C) The animals received an i.v. injection of
FAM-labeled gHoPe2 3 h before tissue collection
Liver
Kidney
Intracranial Tumour Model
Intact brain
Intracranial
tumour
FAM-gHo
Eriste, E., Kurrikoff, K., Suhorutsenko, J., et al. Bioconjugate Chemistry (2013). In press
Scale 100 μm.
FAM-gHoPe2
26. Building Cell Selectivity into CPP Delivery
Activatable CPPs
Exploitation of tissue specific endopeptidases
cargo
++++++++++++++
cargo
++++++++++++++
-----------Cellular internalization
impaired
Cellular internalization
MMP2
Matthias Hallbrink – NoPe (YTA4 + MMP-2 cleavage site + inactivationg region) -tumour imaging in vivo.
27. The Challenge: Penetrating the Impenetrable
Internal Volume
Nucleus
•
•
•
Is reduced.
Mature sperm lacks a variety of
organelles: endoplasmic reticulum,
Golgi apparatus, cytosolic ribosomes.
•
Once Spermatozoa are released into the
seminiferous tubules, genomic transcription and
translation have largely been silenced.
Conventional molecular biology techniques are
thus redundant. Modulation of sperm cell
biology is restricted to cell permeable agents.
Plasma Membrane
•
•
•
•
Lipid composition of plasma membrane is highly
polarized and compartmentalized.
It appears to be incapable of endocytotic events.
Static physical barrier
Detergents are detrimental to protein function
http://www.flickr.com/photos/wellcomeimages/5814253423/
28. Understanding Sperm Biology with CPP
Sperm are transcriptionally and translationary inactive
Studies of their intracellular biology are restricted to cell permeable agents
CPP are ideal vehicles for the study of sperm intracellular biology, from motility
to capacitation and the acrosome reaction
Can be easily isolated from bulls semen with supplemented EBSS containing
0.3% BSA (“swim up method”)
sEBSS
(0.3% BSA)
semen
29. Differential Intracellular Distribution of CPP
6
5
4
3
2
1
13
21
iM
rV
1a
R
10
P
0
Ta
t
10
5Y
M
itP
TP
10
iM
it P
Rho-C105Y (5mM)
7
C
Rho-tat (5mM) + DIC
8
Rho-C105Y (5mM)
tr
at
in
Rho-penetratin (5mM)
Mitotracker (500nM)
Rho-tat (5mM) + DIC
C105Y
Fluorescence minus background (A.U.)
Rho-penetratin (5mM)
Tat
Pe
ne
Penetratin
Comparative analysis of CPP translocation
efficacies into bovine spermatozoa.
Spermatozoa were incubated with TAMRAlabelled CPP (5 mM) for 1 h at 37 oC. Data are
mean + S.E.M. from 3 experiments performed
in triplicate.
32. Why are CPP unable to deliver large proteins into sperm?
Translocation Kinetics of C105Y
Endocytosis Incompetent Spermatozoa
Swiss 3T3
Swiss 3T3
Spermatozoa
Spermatozoa
Transferrin Alexa Fluor® 488
(50 mg/ml)
Transferrin Texas Red®
(50 mg/ml)
LysoTracker® Red (75 nM))
LysoTracker® Red (75 nM))
Dextran Texas Red®
(10 mM)
Dextran Texas Red®
(10 mM)
10
8
t0.5 = 0.70 min
6
C105Y
tat
4
rV1aR102-113
2
0
0
10
20
30
40
50
60
Fluorescence minus background (A.U.)
Fluorescence minus background (A.U.)
10
12
8
6
t0.5 = 7.02 min
rV1aR102-113
2
0
0
10
20
30
40
50
60
Time (min)
Time (min)
Internalisation occurred with first order saturable
kinetics (F = Fmax x t/t0.5 + t, GraphPad Prism 5).
C105Y
tat
4
Cells were incubated at 37oC with TAMRA-labelled peptides (5mM)
for the times indicated. Normalised data (compared to tat-assigned
a value of 1) are expressed as mean fluorescence (minus
background) + s.e.m. from 3 experiments performed in triplicate.
Direct membrane translocation is the sole mechanism of CPP import into sperm
33. Bioportides as Modulators of Human Sperm [Ca2+]i Signalling
STIM1 ORAI1 Activating Region
442
334
EEELE
CC1
(248-342)
CC3
? STIM1
(485685)
KIKKK
CC2
CC2
CC2
EEEL
E
+++
CC1
CC3
(364-388) (399-432)
+
---
Occluded SOAR
CC1
KIKKK
CC3
? STIM1
(485685)
Free SOAR
Orai1
Plasma membrane
Progesterone-the
best-characterised
agonist of human
sperm [Ca2+]i
signalling.
Motility and the
acrosome reaction
Biphasic [Ca2+]i
response
Store depletion
Endoplasmic reticulum
STIM1
STIM1371-392
H-KQLLVAKEGAEKIKKKRNTLFG-NH2
Scr- STIM1371-392 H-LKNKFKGVKLAEIEKQALKGTR-NH2
34. A
140
B
120
100
80
60
Non-capacitating
MitP
nosangiotide
tat
40
20
0
0
20
40
60
80 100 120 140 160 180
120
100
80
60
Capacitating
camptide
40
Cyt c 5-13
C105Y
20
0
0
20
40
60
80 100 120 140 160 180
C
100
90
% cell viability
140
Time (min)
Time (min)
110
% motile cells (*rapid) relative to controls
% motile cells (*rapid) relative to controls
CPP Import is Compatible with Sperm Motility and Viability
A, B. Motility data were collected from human sperm cells treated with 5 mM CPP.
80
C105Y
MitP
tat
iMitP
iMP
penetratin
nosangiotide
70
60
50
40
30
Each peptide was tested on samples from 3 individual donors. Data are shown as %
rapid cells from treated samples relative to that of controls and expressed as mean +
s.e.m. *rapid cells = velocity (average path) ≥25 µms-1 and straightness ≥ 80%.
C. Isolated bovine spermatozoa were treated with CPP for 1 h at the concentrations
20
indicated. Cell viability was measured by MTS conversion and expressed as a
10
percentage of those spermatozoa treated with vehicle alone (sEBSS).
0
-6.0
-5.5
-5.0
-4.5
log { [peptide] (M) }
35. Conclusions
CPPs for site-specific delivery of bioactive
cargoes into mammalian sperm
Cargo size is critical
CPP technologies as valuable tools for the
investigation and modulation of
fundamental processes of sperm
physiology such as maturation,
capacitation, motility, hyperactivation and
fertilisation.
Pantechnia
Jones, S., Lukanowska, L., Suhorutsenko, J., Oxenham, S., Barratt, C., Publicover, S.,
Copolovici, D.M., Langel, Ü. and Howl, J. (2013) Intracellular translocation of cell
penetrating peptides into spermatozoa. Human Reproduction DOI:
10.1093/humrep/def064.
36. The Future for CPP
Therapeutics
Modifications to enhance stability are now
surmountable
Routes of administration
Mechanisms for moving forward
Marcus Evans Discovery and Evolution Summits
Mass Screening, Formulation and Analogues
37. Acknowledgements
Scientific contributors
University of Wolverhampton, UK
John Howl
Monika Lukanowska
University of Birmingham, UK
Michelle Farquhar
Ashley Martin
Steve Publicover
MRC Protein Phosphorylation Unit,
University of Dundee, UK
Dario Alessi
University of Dundee, UK
Chris Barratt
Senga Oxenham
Department of
Neurochemistry,
Stockholm University, Sweden
University of Tartu, Estonia
Ulo Langel
University of Manchester, UK
Shant Kumar