The document describes using peptidomics to monitor protease inhibition in vivo by analyzing peptides as surrogates for protease activity. Peptidomics allows the comprehensive analysis of endogenous peptides from biological samples. The study demonstrates inhibiting different proteases in rats, including DPPIV and FXA, and analyzing changes in peptide levels. Several novel protease substrates were identified, including an ITM2B peptide for DPPIV. Collagen peptide levels were also strongly affected by DPPIV inhibition, indicating its importance in collagen metabolism. Peptidomics can thus non-invasively monitor protease activity and inhibition in vivo by analyzing surrogate peptide markers.
New tools bring greater understanding to cellular metabolism research Mourad FERHAT, PhD
Presentation of Promega Solutions in the field of cellular Metabolism research. Discover new bioluminescent assays for the detection of several metabolites and metabolic process such as : Glucose Uptake, Glucose consumption, Lactate secretion and Glutamine/Glutamate metabolism.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
New tools bring greater understanding to cellular metabolism research Mourad FERHAT, PhD
Presentation of Promega Solutions in the field of cellular Metabolism research. Discover new bioluminescent assays for the detection of several metabolites and metabolic process such as : Glucose Uptake, Glucose consumption, Lactate secretion and Glutamine/Glutamate metabolism.
Measuring apoptosis in real time with a new luminescent methodMourad FERHAT, PhD
We developed a homogeneous luminogenic annexin V binding assay to detect apoptosis in real time using a multimode plate reader. The detection reagent has two different annexin V fusion proteins engineered to contain complementing domains of a binary luciferase, a substrate for luciferase and a cell impermeable fluorogenic DNA dye to detect necrotic cells. The method allow real-time monitoring of Cellular apoptosis and necrosis in microwell plates without washing steps with a highly sensitive luminescent signal. The AnnexinV luminescent method is amenable to High throughput and is a good alternative to FACS, low-throughput Annexin V-FITC based method.
Tools for improved Protein Mass Spec Sample preparation by PromegaMourad FERHAT, PhD
Proteases, glycosidases, antibody characterization tools, IdeS, IdeZ, mass spec reference protein digests, and new solutions for mass spectrometry analysis. Découvrez nos solutions pour vos analyses en spectrométrie de Masse.
Mourad FERHAT, Ph.D
Chef de Produit Analyse cellulaire
mourad.ferhat@promega.com
Promega France
Canvax™ offers a wide range of high quality Human Recombinant Proteins for several research applications like ELISA, Western Blot, Antibody Production or Protein array.
We provide You with the best biotechnology products and services that ensure high quality and innovation.
Founded in 1992 and ISO 9001:2015 certified, BioTeZ supports the research in many areas, both on the Biotechnology Campus Berlin-Buch, Germany, as well as in facilities around the world. For 25 years, we are continuously motivated for new challenges to meet Your needs.
Since 2019 Steffens-Biotec, Ebringen, ISO 13485 certified is a sister company of BioTeZ specialized in manufacturing CE in-vitro diagnostics.
We are specialized in immunochemistry and the development and production of high-quality components for ELISA, Immuno Assays & Protease Activity Kits, Immuno Affinity Chromatography and Lateral Flow Tests.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
EUGM15 - Zoltán Simon (Printnet): Drug Profile Matching - Drug Discovery by P...ChemAxon
Most drugs exert their effects via multi-target interactions, as hypothesized by polypharmacology. Here we introduce Drug Profile Matching (DPM) which is able to relate complex drug-protein interaction profiles with effect and target profiles. Structural data and registered effect profiles of all small-molecule drugs were collected and interactions to a series of non-target protein binding sites of each drug were calculated. Statistical analyses confirmed close relationships between the studied 177 effect and 77 target categories and the in silico generated interaction profiles of cca. 1,200 FDA-approved small-molecule drugs. Receiver Operating Characteristic analysis and 10-fold cross-validation was performed to assess the accuracy and robustness of the method. Based on the found relationships, the effect and target profiles of drugs can be revealed in their entirety, and hitherto uncovered effects and targets can be predicted in a systematic manner.
In order to investigate the predictive power of DPM, four effect categories (PPAR agonist, angiotensin-converting enzyme inhibitor, cyclooxygenase inhibitor and dopamine agent) were selected and predictions in the set of the FDA-approved small-molecule drugs were verified by literature analysis and experimental tests.
Moreover, a large set consisting of 600,000 druglike molecules was selected from a database of 50 million compounds and their interaction profiles were generated. Based on these profiles and chemical similarity considerations, predictions were calculated and tested experimentally to find new candidates that are chemically dissimilar to the reference drugs.
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.
Bioanalytical support plays a vital role during the lead optimization stages. The major goal of the bioanalysis is to assess the over-all ADME characteristics of the NCEs and biologics. Bioanalytical tools can play a significant role and impact the progress in drug discovery and development. Dramatic increases in investments in new modalities beyond traditional small and large molecule drugs, such as peptides, oligonucleotides, and ADC, necessitated further innovations in bioanalytical and experimental tools for the characterization of their ADME and PK properties.https://www.medicilon.com/blog/featured-stories/dmpk-bioanalysis/
Tools for improved Protein Mass Spec Sample preparation by PromegaMourad FERHAT, PhD
Proteases, glycosidases, antibody characterization tools, IdeS, IdeZ, mass spec reference protein digests, and new solutions for mass spectrometry analysis. Découvrez nos solutions pour vos analyses en spectrométrie de Masse.
Mourad FERHAT, Ph.D
Chef de Produit Analyse cellulaire
mourad.ferhat@promega.com
Promega France
Canvax™ offers a wide range of high quality Human Recombinant Proteins for several research applications like ELISA, Western Blot, Antibody Production or Protein array.
We provide You with the best biotechnology products and services that ensure high quality and innovation.
Founded in 1992 and ISO 9001:2015 certified, BioTeZ supports the research in many areas, both on the Biotechnology Campus Berlin-Buch, Germany, as well as in facilities around the world. For 25 years, we are continuously motivated for new challenges to meet Your needs.
Since 2019 Steffens-Biotec, Ebringen, ISO 13485 certified is a sister company of BioTeZ specialized in manufacturing CE in-vitro diagnostics.
We are specialized in immunochemistry and the development and production of high-quality components for ELISA, Immuno Assays & Protease Activity Kits, Immuno Affinity Chromatography and Lateral Flow Tests.
His Tag Protein Production and PurificationExpedeon
The study of protein regulation, structure, and function relies heavily on the expression and purification of recombinant proteins. Many recombinant proteins are expressed as fusion proteins, meaning that they contain an affinity / epitope tag. A tag is a short sequence of DNA that codes for a specific amino acid, which is frequently inserted into a target gene at the point of coding for expression at either the N or C terminal of the protein required.
EUGM15 - Zoltán Simon (Printnet): Drug Profile Matching - Drug Discovery by P...ChemAxon
Most drugs exert their effects via multi-target interactions, as hypothesized by polypharmacology. Here we introduce Drug Profile Matching (DPM) which is able to relate complex drug-protein interaction profiles with effect and target profiles. Structural data and registered effect profiles of all small-molecule drugs were collected and interactions to a series of non-target protein binding sites of each drug were calculated. Statistical analyses confirmed close relationships between the studied 177 effect and 77 target categories and the in silico generated interaction profiles of cca. 1,200 FDA-approved small-molecule drugs. Receiver Operating Characteristic analysis and 10-fold cross-validation was performed to assess the accuracy and robustness of the method. Based on the found relationships, the effect and target profiles of drugs can be revealed in their entirety, and hitherto uncovered effects and targets can be predicted in a systematic manner.
In order to investigate the predictive power of DPM, four effect categories (PPAR agonist, angiotensin-converting enzyme inhibitor, cyclooxygenase inhibitor and dopamine agent) were selected and predictions in the set of the FDA-approved small-molecule drugs were verified by literature analysis and experimental tests.
Moreover, a large set consisting of 600,000 druglike molecules was selected from a database of 50 million compounds and their interaction profiles were generated. Based on these profiles and chemical similarity considerations, predictions were calculated and tested experimentally to find new candidates that are chemically dissimilar to the reference drugs.
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.
Bioanalytical support plays a vital role during the lead optimization stages. The major goal of the bioanalysis is to assess the over-all ADME characteristics of the NCEs and biologics. Bioanalytical tools can play a significant role and impact the progress in drug discovery and development. Dramatic increases in investments in new modalities beyond traditional small and large molecule drugs, such as peptides, oligonucleotides, and ADC, necessitated further innovations in bioanalytical and experimental tools for the characterization of their ADME and PK properties.https://www.medicilon.com/blog/featured-stories/dmpk-bioanalysis/
Comprehensive Investigation of the Utilization of SFC/ESI Positive Mode MS fo...Waters Corporation
Bioanalysis and drug metabolism studies are critical parts of the drug development process. The aim of these studies is to identify and quantify drugs and their associated metabolites in biofluids such as plasma and urine. Typically reversed-phase chromatography coupled with mass spectrometry is often the analytical technique of choice utilized in the analyses due to the specificity and sensitivity of the technique. However, due to the complexity of the biofluid samples accurate and precise measurements can become challenging due to poor chromatographic peak shape, insufficient chromatographic resolution from matrix components and incompatible sample compositions.
Recent advancements in the field of super critical fluid chromatography (SFC) have lead to the development of sub 2 micron chromatographic separations coupled mass spectrometry operating under positive ESI mode. In the work presented here the applicability of SFC for both chiral and achiral bioanalysis is shown. The orthogonal separation selectivity compared with reversed-phase separations and tolerance for high organic sample compositions will be discussed. This work further presents a critical evaluation of the influence of mobile phase and make up flow modifiers on the sensitivity and selectivity of probe pharmaceuticals analyzed under SFC/ESI positive mode MS conditions.
Paul Rainville of Waters Corporation gave this Oral presentation at Pittcon 2015.
Antibodies Newsflash - September 2014 - BBI SolutionsBBISolutions
In this month’s newsflash you can see the new range of veterinary antibodies available from BBI Solutions along with new analysis data for ST2 antibodies and CRP antibodies.
Next generation biotherapeutics production system Trichoderma reeseiChristopher Landowski
The filamentous fungus Trichoderma reesei is an important production organism used by industrial enzyme companies world-wide. It is a low cost production system that secretes its native enzymes at levels exceeding 100 g/L of culture medium. Several T. reesei produced enzymes have obtained the generally recognized as safe status by the U.S. Food and Drug Administration. T. reesei has tremendous prospects to be a cost efficient and high yield system for producing therapeutic proteins. We have adapted the fungus to become more suitable for biotherapeutic production by reducing secreted protease activity and altering glycosylation pathways needed for adding mammalian glycoforms.
Expression strains for monoclonal antibodies, Fab antibody fragments, interferon alpha-2b, insulin-like growth factor 1, and fibroblast growth factor 21 were constructed, cultivated in bioreactors, and expression levels were measured from the culture medium. After deleting 13 of the most critical protease genes, the general secreted protease activity was reduced over 30-fold. Monoclonal antibodies could be produced up to 7.6 g/L, Fab antibody fragments up to 8.2 g/L, interferon alpha-2b at 7.9 g/L, and insulin-like growth factor fusion protein at 8 g/L. With protease inhibitor treatment interferon alpha-2b could be produced at over 10 g/L, insulin-like growth factor fusion protein at 19 g/L, and full length fibroblast growth factor 21 at 200 mg/L in addition to a shorter form at 3.5 g/L. Human glycoforms such as G0 and FG0 were produced on monoclonal antibodies.
Expression levels and product quality improved dramatically after multiple protease deletions and optimization of culture conditions. While the production levels achieved are already relatively high, the strains could be developed further to reach the 100 g/L potential of the organism. This study demonstrates the excellent prospects of T. reesei as a host for therapeutic protein production.
Preparation of protein-loaded PLGA-PVP blend nanoparticles by nanoprecipitati...Nanomedicine Journal (NMJ)
Abstract
Objective(s):
Despite of wide range applications of polymeric nanoparticles in protein delivery, there are some problems for the field of protein entrapment, initial burst and controlled release profile.
Materials and Methods:
In this study, we investigated the influence of some changes in PLGA nanoparticles formulation to improve the initial and controlled release profile. Selected parameters were: pluronic F127, polysorbate 80 as surfactant, pH of inner aqueous phase, L/G ratio of PLGA polymer, volume of inner aqueous phase and addition of polyvinylpyrrolidone as an excipient. FITC-HSA was used as a model hydrophilic drug. The nanoparticles were prepared by nanoprecipitation.
Results:
Initial release of FITC-HSA from PLGA-tween 80 nanoparticles (opt-4, 61%) was faster than control (PLGA-pluronic) after 2.30 h of incubation. Results showed that decrease in pH of inner aqueous phase to pI of protein can decrease IBR but the release profile of protein is the same as control. Release profile with three phases including a) initial burst b) plateau and c) final release phase was observed when we changed volume of inner aqueous phase and L/G ratio in formulation. Co-entrapment of HSA with PVP and pluronic reduced the IBR and controlled release profile in opt-19. Encapsulation efficiency was more than 97% and nanoparticles size and zeta potentials were mono-modal and -18.99 mV, respectively.
Conclusion:
In this research, we optimized a process for preparation of PLGA-PVP-pluronic nanoparticles of diameter less than 300 nm using nanoprecipitation method. This formulation showed a decreased initial burst and long lasting controlled release profile for FITC-HSA as a model drug for proteins.
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
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.
In his public lecture, Christian Timmerer provides insights into the fascinating history of video streaming, starting from its humble beginnings before YouTube to the groundbreaking technologies that now dominate platforms like Netflix and ORF ON. Timmerer also presents provocative contributions of his own that have significantly influenced the industry. He concludes by looking at future challenges and invites the audience to join in a discussion.
20 Comprehensive Checklist of Designing and Developing a WebsitePixlogix Infotech
Dive into the world of Website Designing and Developing with Pixlogix! Looking to create a stunning online presence? Look no further! Our comprehensive checklist covers everything you need to know to craft a website that stands out. From user-friendly design to seamless functionality, we've got you covered. Don't miss out on this invaluable resource! Check out our checklist now at Pixlogix and start your journey towards a captivating online presence today.
zkStudyClub - Reef: Fast Succinct Non-Interactive Zero-Knowledge Regex ProofsAlex Pruden
This paper presents Reef, a system for generating publicly verifiable succinct non-interactive zero-knowledge proofs that a committed document matches or does not match a regular expression. We describe applications such as proving the strength of passwords, the provenance of email despite redactions, the validity of oblivious DNS queries, and the existence of mutations in DNA. Reef supports the Perl Compatible Regular Expression syntax, including wildcards, alternation, ranges, capture groups, Kleene star, negations, and lookarounds. Reef introduces a new type of automata, Skipping Alternating Finite Automata (SAFA), that skips irrelevant parts of a document when producing proofs without undermining soundness, and instantiates SAFA with a lookup argument. Our experimental evaluation confirms that Reef can generate proofs for documents with 32M characters; the proofs are small and cheap to verify (under a second).
Paper: https://eprint.iacr.org/2023/1886
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!
GraphSummit Singapore | The Future of Agility: Supercharging Digital Transfor...Neo4j
Leonard Jayamohan, Partner & Generative AI Lead, Deloitte
This keynote will reveal how Deloitte leverages Neo4j’s graph power for groundbreaking digital twin solutions, achieving a staggering 100x performance boost. Discover the essential role knowledge graphs play in successful generative AI implementations. Plus, get an exclusive look at an innovative Neo4j + Generative AI solution Deloitte is developing in-house.
Generative AI Deep Dive: Advancing from Proof of Concept to ProductionAggregage
Join Maher Hanafi, VP of Engineering at Betterworks, in this new session where he'll share a practical framework to transform Gen AI prototypes into impactful products! He'll delve into the complexities of data collection and management, model selection and optimization, and ensuring security, scalability, and responsible use.
Alt. GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using ...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
Maruthi Prithivirajan, Head of ASEAN & IN Solution Architecture, Neo4j
Get an inside look at the latest Neo4j innovations that enable relationship-driven intelligence at scale. Learn more about the newest cloud integrations and product enhancements that make Neo4j an essential choice for developers building apps with interconnected data and generative AI.
Unlock the Future of Search with MongoDB Atlas_ Vector Search Unleashed.pdfMalak Abu Hammad
Discover how MongoDB Atlas and vector search technology can revolutionize your application's search capabilities. This comprehensive presentation covers:
* What is Vector Search?
* Importance and benefits of vector search
* Practical use cases across various industries
* Step-by-step implementation guide
* Live demos with code snippets
* Enhancing LLM capabilities with vector search
* Best practices and optimization strategies
Perfect for developers, AI enthusiasts, and tech leaders. Learn how to leverage MongoDB Atlas to deliver highly relevant, context-aware search results, transforming your data retrieval process. Stay ahead in tech innovation and maximize the potential of your applications.
#MongoDB #VectorSearch #AI #SemanticSearch #TechInnovation #DataScience #LLM #MachineLearning #SearchTechnology
GDG Cloud Southlake #33: Boule & Rebala: Effective AppSec in SDLC using Deplo...James Anderson
Effective Application Security in Software Delivery lifecycle using Deployment Firewall and DBOM
The modern software delivery process (or the CI/CD process) includes many tools, distributed teams, open-source code, and cloud platforms. Constant focus on speed to release software to market, along with the traditional slow and manual security checks has caused gaps in continuous security as an important piece in the software supply chain. Today organizations feel more susceptible to external and internal cyber threats due to the vast attack surface in their applications supply chain and the lack of end-to-end governance and risk management.
The software team must secure its software delivery process to avoid vulnerability and security breaches. This needs to be achieved with existing tool chains and without extensive rework of the delivery processes. This talk will present strategies and techniques for providing visibility into the true risk of the existing vulnerabilities, preventing the introduction of security issues in the software, resolving vulnerabilities in production environments quickly, and capturing the deployment bill of materials (DBOM).
Speakers:
Bob Boule
Robert Boule is a technology enthusiast with PASSION for technology and making things work along with a knack for helping others understand how things work. He comes with around 20 years of solution engineering experience in application security, software continuous delivery, and SaaS platforms. He is known for his dynamic presentations in CI/CD and application security integrated in software delivery lifecycle.
Gopinath Rebala
Gopinath Rebala is the CTO of OpsMx, where he has overall responsibility for the machine learning and data processing architectures for Secure Software Delivery. Gopi also has a strong connection with our customers, leading design and architecture for strategic implementations. Gopi is a frequent speaker and well-known leader in continuous delivery and integrating security into software delivery.
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.
Pushing the limits of ePRTC: 100ns holdover for 100 daysAdtran
At WSTS 2024, Alon Stern explored the topic of parametric holdover and explained how recent research findings can be implemented in real-world PNT networks to achieve 100 nanoseconds of accuracy for up to 100 days.
GraphSummit Singapore | The Art of the Possible with Graph - Q2 2024Neo4j
Neha Bajwa, Vice President of Product Marketing, Neo4j
Join us as we explore breakthrough innovations enabled by interconnected data and AI. Discover firsthand how organizations use relationships in data to uncover contextual insights and solve our most pressing challenges – from optimizing supply chains, detecting fraud, and improving customer experiences to accelerating drug discoveries.
2. Peptides are Products of Protease Activity
Peptidomics is defined as the systematic, comprehensive, qualitative and
quantitative multiplex analysis of endogenous peptides in a biological
sample at a defined time point and location. In contrast to Proteomics
peptides possess a lower molecular weight and a higher degree of
proteolytic processing.
Expert Rev Mol Diagn. 2007 Sep;7(5):605-13
3. Peptidomics - Process
Characteristics [Plasma]
• Sensitivity : 50 pM
• Recovery: 40-110%
• CV : 30-35 %
• Throughput: 32 samples/week
• Volume: 500 -1000 µl
• ~ 10,000 signals/display
• ~ 1500 signals identified
• Quality controlled process Samples are prepared, separated by
chromatography and measured by mass
spectrometry. Mass spectra are visualized and
analyzed. Peptides of interest are identified by
MS/MS.
Comb Chem High Throughput Screen. 2005 Dec;8(8):725-33
4. Protease Inhibition
Protease 1 Protease 2
[Substrate] [Product] [Product 2]
Accumulation of substrates Reduction of products
Protease 3
Alternative product
The figure demonstrates principle effects of protease inhibition.
Since peptides are generated by proteases they are excellent
surrogate markers for protease activity.
5. Example: Peptides Mirror Protease Activity
COAGULATION FIBRINOLYSIS
Thrombin Plasmin
Fibrinogen 20-866 Protein
20-35 582-602 603-620 Peptides
Peptides are suitable to encode conditions diametrically opposed to
each other (Coagulation versus Fibrinolysis), which are not accessible
by classical Proteomics methods (e.g. after Tryptic Digestion)
6. In Vivo FXa Inhibition
Proof-of-concept study to demonstrate the usefulness of
Peptidomics for analysis of proteases and inhibitors in vivo
Anti-Factor Xa-Activity
Thromborel S
<0.01- 0.02 U/ml
Fondaparinux
>1.10 U/ml
Peptides. 2008 Dec;29(12):2188-95.
7. In Vivo FXa Inhibition - Results
Abbreviation Precursor SwissProt. Sequence Comment
Protein Acc.No.
1 FIBA_227-239 Fibrinogen alpha P06399 AIK..MSPVPDLVPGSFK..SQL
2 FIBA_520-538 Fibrinogen alpha P06399 SYK..MADEAASEAHQEGDTRTTK..RGR
3 PROC_199-212 Protein C P31394 KRD..IDPEDEELELGPR..IVN Activation Peptide
4 FIBA_20-36 Fibrinogen alpha P06399 VWT..ADTGTTSEFIEAGGDIR..GPR Fibrinopeptide A
5 FIBA_24-36 Fibrinogen alpha P06399 TAD..TGTTSEFIEAGGDIR..GPR
6 FIBA_26-36 Fibrinogen alpha P06399 DTG..TTSEFIEAGGDIR..GPR
7 FIBB_19-32 Fibrinogen beta P14480 TQA..ATTDSDKVDLSIAR..GHR Fibrinopeptide B
8 FIBG_89-110 Fibrinogen gamma P02680 LIK..AIQVYYNPDQPPKPGMIEGATQ..KSK
9 THRB_25-41 Prothrombin P18292 VHS..QHVFLAPQQALSLLQRVR..RAN c-terminal truncated propeptide
10 THRB_25-42 Prothrombin P18292 VHS..QHVFLAPQQALSLLQRVRR..ANS c-terminal truncated propeptide
Peptides were identified, which derived from proteins related
to coagulation or fibrinolysis and it could be shown how
Fondaparinux (FXa inhibitor) influences these physiological
processes.
9. for vildagliptin were shown in studies using obese Zucker fa/fa
rats [2,4].
Experimental Setup : In-Vivo Inhibition DPPIV 3.2. Differential peptide display
To profile peptides in each individual sample by generating
8 week old Wistar-Rats (315-320g)
peptide displays, platelet free EDTA plasma was used. Using
plasma in such a study is an important prerequisite since serum
or residual platelets exhibit enzymatic activities, which are
Vehicle vs. DPP-IV inhibitors leading to an ex vivo generation of peptides, which hampers the
sensitive detection of endogenously generated peptides [23].
From the 124 generated peptide displays, 7 samples were
identified as technical outliers on the basis of visual inspection
of the peptide displays and principal component analysis of the
data matrix. They were excluded from further analysis. A mean
DPPIV - Inhibitors
(Vildagliptin/Ab192: 0-3 mg/kg)
0 1
time [h]
DPPIV activity DPPIV activity
Peptide Displays Peptide Displays
Fig. 1 – DPP4 activity of rat plasma samples. Following
injection of the two inhibitors, a dose-dependent decrease
in DPP4 activity was measured as described in Section 2.
All of the tested doses led to a significant decrease in
Biochem Pharmacol. 2009 Jan 15;77(2):228-37
activity – with vildagliptin more potent already in the
lower range. Significance was tested using the Man–
10. Differential Peptide Display
molecular mass
hydrophobicity
The figure shows the differences before and after treatment with DPPIV
inhibitors. The treatment leads to an accumulation ( ) or depletion ( )
of substrates, which could be correlated to clinical data (e.g. side effects)
11. Product and Substrate
PxBVN Controls
Group I
(Saline)
Δ m/z = 200
Group B
(Vehicle)
Group C
(Protease inhibitor)
Each lane represents the same region from 27 peptide displays. The blue square
indicates a peptide (and a PTM) which is decreased in samples derived from animals
treated with a DPPIV inhibitor. With a mass difference of 200 Da a second signal
(red square) shows the opposite regulation. The identification revealed that both
peptides are products and substrates of DPPIV. The delta of 200 Da corresponds
to a truncation of two amino acids
12. Sequencing Results
All identified peptides represent novel entities, which were not described before:
Table 1 – Identified DPP4 substrates and products in rat plasma.
Index m/z Correlation coeffi- p-value Protein precursor, amino acid range Sequence, SwissProt. accession number (www.expasy.com)
cient
AB 192 vildagliptin AB 192 vildagliptin
1 2234 À0.303 À0.609 0.209 0.002 Collagen alpha 1 (I) chain (216–238) GPP_GKNGDDGEAGKPGRPGERGPPGP_QGA (P02454)
2 2814 À0.233 À0.494 0.259 0.021 Collagen alpha 1 (I) chain (295–325) PRG_LPGERGRPGPPGSAGARGNDGAVGAAGPPGP_TGP (P02454)
3 2805 0.554 0.598 0.003 0.000 Collagen alpha 1 (I) chain (802–832) PAG_FAGPPGADGQPGAKGEPGDTGVKGDAGPPGP_AGP (P02454)
4 3493 À0.251 À0.672 0.026 0.000 Collagen alpha 1 (I) chain (804–843) GFA_GPPGADGQPGAKGEPGDTGVKGDAGPPGPAGPAGPPGPIG_NVG (P02454)
5 2192 À0.328 À0.516 0.097 0.006 Collagen alpha 1 (I) chain (809–832) PGA_DGQPGAKGEPGDTGVKGDAGPPGP_AGP (P02454)
6 3267 À0.305 À0.703 0.259 0.004 Collagen alpha 1 (I) chain (996–1030) GLA_GPPGESGREGSPGAEGSPGRDGAPGAKGDRGETGP_AGP (P02454)
7 3210 À0.150 À0.603 0.383 0.009 Collagen alpha 1 (I) chain (997–1030) LAG_PPGESGREGSPGAEGSPGRDGAPGAKGDRGETGP_AGP (P02454)
8 3333 0.582 0.621 0.002 0.001 Collagen alpha 1 (III) chain (519–554) PRG_VAGEPGRDGTPGGPGIRGMPGSPGGPGNDGKPGPPG_SQG (P13941)
9 3132 À0.468 À0.629 0.026 0.000 Collagen alpha 1 (III) chain (521–554) GVA_GEPGRDGTPGGPGIRGMPGSPGGPGNDGKPGPPG_SQG (P13941)
10 1938 À0.182 À0.409 0.209 0.009 Collagen alpha 1 (III) chain (665–686) GEA_GAPGVPGGKGDSGAPGERGPPG_TAG (P13941)
11 2579 À0.371 À0.685 0.073 0.001 Collagen alpha 1 (III) chain (812–839) GFP_GAPGQNGEPGAKGERGAPGEKGEGGPPG_AAG (P13941)
12 4922 À0.216 À0.585 0.620 0.001 Collagen alpha 1 (III) chain (1001–1053) GLP_GQPGTAGEPGRDGNPGSDGQPGRDGSPGGKGDRGENGSP-
GAPGAPGHPGPPGP_VGP (P13941)
13 4228 À0.375 À0.692 0.017 0.001 Collagen alpha 2 (I) chain (194–238) LDG_LKGQPGAQGVKGEPGAPGENGTPGQAGARGLPGERGRVGAPGPAG_ARG (P02466)
14 2638 À0.284 À0.538 0.259 0.008 Collagen alpha 2 (I) chain (460–488) GLP_GSPGNVGPAGKEGPVGLPGIDGRPGPIGP_AGP (P02466)
15 4086 À0.078 À0.598 0.383 0.004 Collagen alpha 2 (I) chain (649–692) GLP_GERGAAGIPGGKGEKGETGLRGEIGNPGRDGARGAPGAIGAPGP_AGA (P02466)
16 2855 À0.335 À0.432 0.017 0.002 Collagen alpha 2 (I) chain (931–958) GEA_GRDGNPGSDGPPGRDGQPGHKGERGYPG_NIG (P02466)
17 5490 0.734 0.062 0.002 0.612 Vitronectin (Somatomedin B) (20–67) ALA_DQESCKGRCTEGFNVDKKCQCDELCSYYQSCCTDYTAECKPQVTRGDV_FTM
(P04004.1)
18 2675 0.665 0.573 0.001 0.000 Integral membrane protein 2B (244–266) QKR_EASNCFTIRHFENKFAVETLICS (Q5XIE8)
19 2475 À0.421 À0.575 0.029 0.014 Integral membrane protein 2B (246–266) REA_SNCFTIRHFENKFAVETLICS (Q5XIE8)
The table shows the mass-to-charge ratios (m/z), the experimental group (AB192 or vildagliptin) with the corresponding correlation coefficient (signal intensity and inhibitor dosage) and p-value
obtained by the Mann–Whitney U-test comparing groups of control and highest applied inhibitor dosage for each differentially regulated peptide. This is followed by the precursor name and sequence
range, the amino acid sequence and the SwissProt accession number in parenthesis. The sequenced peptide is depicted in bold. Preceding and succeeding amino acids are depicted before and after the
horizontal lines.
Biochem Pharmacol. 2009 Jan 15;77(2):228-37
13. Substrate Accumulation in Various Species
molecular mass
2200 2480
Species Year
(Data acquisition)
- Pig 2001
+ new plasma
preparation
- Human 2004
+ new pre-analytical
procedures
- Ape 2007
+ enhanced
sensitivity
-
+ Rat 2008
The peptide (red square) possesses the same amino acid sequence in various organisms (highly
conserved). It can be regarded as a pan-marker for DPPIV inhibition. It is detectable at the same
position in a peptide display in all samples.
- = control += DPPIV inhibitor (Val-Pyr, Vildagliptin, AB192, Sitagliptin)
14. Outcome
• DPP4 inhibition influenced massively collagen metabolism
• ITM2B peptide was detected in plasma following in vivo DPP4 inhibition
• Somatomedin B shows solely an accumulation under AB192
• DPP4 catalyzed cleavage kinetics of the ITM2B peptide were
determined in vitro:
k(cat) and K(m) 5.2s-¹ and 14µM
specificity constant k(cat)/K(m) of 0.36 x 106 s-1M-1
ITM2B = Integral membrane protein 2B (Accession number: Q5XIE82)
15. Conclusion
• Peptidomics is capable to monitor protease action in vivo
• ITM2B peptide was identified as a novel DPP4 substrate.
• Somatomedin B was differentially regulated between both inhibitors
and may indicating a potential side effect
• Collagen accumulation may cause long-term problems
16. Frequently Asked Questions
What is the extent of a project ?
A discovery project approx. includes the analysis of up to 100 samples, the identification (sequence) of up to
40 peptides, the biological interpretation of peptides in the project context and compiling of interim reports
and a final project summary report within a time frame of 3-5 months.
What is the amount/volume of a sample necessary for analysis ?
The amount/volume depends on the biological matrix and the desired analytical sensitivity.
The necessary volumes vary between 400 µl (e.g. CSF) - 4 ml (e.g. FCS free cell culture supernatant). For the
analysis of human blood plasma we recommend a volume of 0.5 - 1 ml. The amounts of tissues can vary
between 1 mg (e.g. tumor tissue) - 100 mg (muscle tissue)
What are the costs of sample analysis (per sample) ?
The costs are calculated individually for each project and depend on the number of analyzed samples. The
cost are approx. 650-1500 Euro per sample.
What is the required number of samples per project ?
This number is determined by the quality of differences. To achieve relevant results we recommend a
minimum of 20 samples per arm.
What are the costs for sequencing ?
This depends on the candidates and their abundance and can be between 500 - 4000 Euro/candidate, since
low abundant candidates require an extra preparation of up to 20 mL plasma.
What ways of candidate validation are possible ?
An analytical as well as a biological validation is possible. The exact approach is highly dependent on the
project background.