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lizseyi · 1 year

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Mass Spectrometry Analytics For Biologics – Selvita

Biological drugs, also known as biologics, are medications derived from living organisms. Unlike traditional small molecule drugs, which are chemically synthesized, biologics are produced using biotechnology methods, such as recombinant DNA technology or protein engineering. They are used to treat various medical conditions, including autoimmune diseases, cancer, and rare genetic disorders. Biologics are often administered through injection or infusion, and they have become an increasingly important part of modern medicine.

In general, therapeutic proteins exhibit high molecular complexity, making them sensitive to various environmental factors, such as temperature, light, and pH. Even minor changes can cause the biologic to become unstable, leading to decreased efficacy or harmful side effects. Therefore, proper analytical characterization and usage of state-of-the-art techniques allow for mitigating potential risks.

Mass spectrometry (MS) has become an essential tool for characterizing proteins in modern biotechnology and pharmaceutical research. This technology is based on the principles of ionization and mass-to-charge (m/z) separation. It allows researchers to identify and quantify the individual components of a protein sample with a high degree of accuracy and precision. Undoubtedly, MS is a valuable tool for confirming the primary structure of a protein and identifying any post-translational modifications that may affect its function.

Primary structure determination

Primary structure confirmation using mass spectrometry allows for identifying and confirming a protein’s amino acid sequence. It involves breaking down the protein into its constituent peptides and analyzing these peptides to determine their mass-to-charge ratio (m/z). The first step is to cleave the protein into smaller peptides using a protease enzyme, such as trypsin or others. The resulting peptides are then purified and separated using liquid chromatography. The complex peptide mixture can be separated by a nano-LC system where the separation of the analytes takes place into capillary columns to enhance sensitivity. Next, the peptides are ionized using an electrospray ionization source and introduced into the mass spectrometer. The difficulty of obtaining complete coverage of the protein sequence using MS alone is probably the main limitation of this technique, especially for larger proteins. Additionally, some amino acids may be challenging to detect using MS, such as those that are modified or have unusual chemical properties. Therefore utilization of MS-based analysis requires thorough experience.

Modifications of proteins

One of the key applications of mass spectrometry in protein characterization is the identification of post-translational modifications (PTMs). PTMs are chemical modifications that occur after the translation of a protein and can significantly alter its biological activity and function. Almost all proteins, whether produced as recombinant proteins or isolated from natural sources, will carry, to some degree, modified amino acids. Modification may also occur during the sample handling process. Therefore, they need to be monitored to ensure the consistency of production batches. Modifications, like deamidation, oxidation, pyroglutamate formation, etc., may affect protein functionality. Hence, identifying protein modifications and their position is essential in protein characterization.

Among others formation of disulfide bridges significantly influences protein structure and function. Incorrectly paired disulfide bonds result in changing protein properties. For that reason, their mapping is a significant step for confirming proper tertiary structure. Mass spectrometry can be used to identify the specific sites of PTMs, such as phosphorylation or glycosylation, and to determine their abundance. The glycosylation-related heterogeneity of proteins arises from the differences in localization and occupancy of the glycosylation sites and the diversity of the glycan structures expressed on a specific site. To address both levels, glycopeptides analysis and site occupancy evaluation should be utilized.

Aggregation and oligomerization

Oligomers, aggregates, and fragments are biologically active protein proteoforms and common product-related impurities in biopharmaceuticals that impact efficacy, safety, and stability. Identification of low-level impurities may involve labor-intensive chromatographic fraction collection and follow-up experiments. In addition, a significant part of the total protein aggregates can be caused by non-covalent molecular interactions, which are dissociable in denaturing buffers and cannot be characterized by conventional mass spectrometry. Using ammonium acetate solution for SEC allows for preserving non-covalent protein interactions and registers them by MS with high sensitivity and accuracy.

Besides the assessment of biomolecule size variants, native MS allows the characterization of charge heterogeneity through the combination of ion exchange chromatography and native MS. Characterization of variants such as deamidation, which are traditionally unattainable by an intact mass due to their minimal molecular weight differences, can be measured unambiguously by mass and retention time.

Conformational dynamics analysis

Hydrogen–deuterium exchange mass spectrometry (HDx-MS) allows insight into the behavior of the protein in the solution and the correlation of HDx with the structure and dynamics of the molecule. The technique enables the analysis of proteins in terms of interactions with ligands/drugs, other proteins, and lipids or the study of the effect of mutations and post-translational modifications under the same experimental conditions in solution. The main application of HDx-MS is the comparative analysis of different protein conformers. HDx-MS is a complementary method to 3D static structures, allowing for a “dynamic” image of a protein that can explain many biological processes.

In conclusion, mass spectrometry is a powerful tool for the characterization of proteins in modern biotechnology and pharmaceutical research. It allows for identifying post-translational modifications, protein-protein interactions, and quantifying protein abundance, among other applications. With ongoing advances in mass spectrometry technology and sample preparation techniques, mass spectrometry will continue to play a critical role in protein characterization and the development of new therapeutics.

For more information visit our website: https://selvita.com

#small molecule

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samsontjacob · 2 years

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What is the role of epigenetic changes in cancer cells?

DNA methylation and histone modifications alter the epigenetic landscape of cells, influencing cellular function. They play a critical role in tumorigenesis and have been found to interact with genetic alterations to promote cancer development.

The epigenetic landscape undergoes extensive distortion in cancer (4). This includes global DNA methylation patterns, altered expression profiles of chromatin-modifying enzymes, and changes in non-coding RNAs.

DNA methylation is a modification in which a small molecule called a methyl group is added to a DNA strand. This can affect how the genes in your DNA work or make proteins.

Many of the genes in your body have been methylated at certain places. This methylation changes how they can make proteins and can increase your risk of getting cancer.

Another important role of methylation is that it can turn off some genes. For example, if the BRCA1 gene is methylated at a specific place, it does not make the protein needed to protect your body from breast and other types of cancer.

A large-scale study has found that aberrant DNA methylation is present in more than 15% of tumors, including primary and secondary ones (Taby and Issa, 2010). This indicates that DNA methylation could be essential in determining whether or not cancer cells will develop.

In eukaryotes, an octamer of histones H2A, H2B, H3, and H4 are wrapped by 147 bp of DNA to form the nucleosome - a core particle determining chromatin structure and gene expression. These histones are subject to various post-translational modifications, including acetylation (ac) and methylation (me) at their N termini, phosphorylation, ubiquitylation, glycosylation, sumoylation, ADP-ribosylation and carbonylation.

Histone modifications are essential determinants of epigenetic status, and cancer cells exhibit aberrations in the levels of some histone modifications. These include decreased acetylation levels at H4-lysine 16 (H4K16ac) and H4-lysine 20 trimethylation (H4K20me3) and increased methylation at H3K9me2/3 in some cancers.

These changes are believed to result from perturbation of the various nodes of epigenetic regulation, which determine chromatin stability and cellular identity. They may also reflect chromatin heterochromatinization, which limits DNA exposure and provides a protective response against genotoxic stress.

Several classes of noncoding RNAs play a crucial role in regulating gene expression and cellular functions. They include microRNAs, long noncoding (lncRNAs), and circular RNAs.

In addition to their importance in the normal functioning of cells, ncRNAs have also been found to be deregulated during cancer onset and progression. Many of these deregulations can be used as diagnostic and prognostic biomarkers in patients with cancer.

However, the precise ncRNAs that regulate gene expression in cancer initiation and progression are still unknown. Despite this, several potential ncRNAs have been linked to cancer pathogenesis.

One such ncRNA is H19, a transcription inhibitor inhibiting cell proliferation and evading cell death. This lncRNA is also known to be reduced in lung cancer. It has also been shown to act on Ras and HMGA2 proteins in lung tumors. In addition, H19 expression levels have been associated with a decreased risk of bladder cancer.

MicroRNAs play a critical role in the cancer cell, as they target genes involved in drug resistance and influence gene expression associated with proliferation, cell cycle, and apoptosis. A single miRNA may affect several genes, and the effect is tissue-specific.

One such example is the c-Myc and E2F1 interacting network that controls the expression of many miRNAs in hepatocellular carcinoma (HCC). The p53 tumor suppressor system promotes cell growth in the cancer cell by inhibiting the c-Myc-regulated microRNAs.

In addition to targeting the genes, miRNAs can influence their DNA methylation and histone modification levels. These epigenetic changes alter chromatin structure, which in turn drives the development of cancer cells.

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rnewspost · 2 years

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Study identifies a potential therapeutic target for triple-negative breast cancer in Black patients

Protein diversity due to alternative mRNA splicing or post-translational modifications (PTMs) plays a vital role in various cellular functions. The mitotic kinases polo-like kinase 1 (PLK1) and Aurora B (AURKB) phosphorylate survivin, an inhibitor of apoptosis (IAP) family member, thereby regulating cell proliferation. PLK1, AURKB, and survivin are overexpressed in triple-negative breast cancer…

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biomedres · 2 years

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Epigenetic concerns in therapy for Chronic Obstructive Pulmonary Disease

Epigenetic concerns in therapy for Chronic Obstructive Pulmonary Disease in Biomedical Journal of Scientific & Technical Research

https://biomedres.us/fulltexts/BJSTR.MS.ID.005971.php

COPD (Chronic Obstructive Pulmonary Disease) is a prevalent chronic adult disease that, by 2020, will be the world’s 3rd leading cause of death. COPD is characterized by Mucociliary dysfunction, lung inflammation, airway fibrosis, and alveolar destruction [1]. There are many factors at hand, such as chronic inflammation, the theory of elastase/anti-elastase, apoptosis, the balance of oxidantantioxidant, and infective repair that cause COPD pathogenesis [2,3]. Cigarette smoke contains over 4700 organic compounds and 1014 free radicals/oxidants, making it the primary source of COPD and inflammation [4]. It stimulates multiple redox-sensitive transcription factors such as NF-kB (nuclear factor kappa-B), most important to increased pro-inflammatory cytokine and chemokine assembly in COPD [5,6]. Post-translational changes in histone and other DNA proteins are known as epigenetic alterations [7]. Without altering the primary gene sequence, DNA methylation and histone methylation can influence gene expression such as post-translational histone protein modifications are acetylation, phosphorylation, and ubiquitination (H2A, H2B, H3, and H4) [8].

For more articles in Journals on Biomedical Sciences click here bjstr

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#open access clinical and medical journal #family medicine #nano medicine #journals on biomedical engineering #biomedical open access journals

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botanyone · 2 years

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Doctoral Research Associate

Doctoral Research Associate https://ift.tt/Ua7rjOT The research group of Prof. Iris Finkemeier, Institute of Plant Biology and Biotechnology in the Faculty of Biology at the University of Münster, Germany, is seeking to fill the positions of 2 Doctoral Research Associates (Wissenschaftliche Mitarbeiter/innen salary level TV-L E 13, 65%) at the earliest possible date. We are offering fixed-term part-time positions (65%) for 3 years. The Finkemeier group investigates the role of post-translational protein modifications (i.e. acetylation, phosphorylation and cysteine redox) in the acclimation of plants towards changing environmental conditions. We are particular interested in the biochemical characterization of the writers and erasers of these modifications and to uncover their regulation. For more details see WWU Münster. This message expires at 9:00am on Sunday February 26th, 2023 The post Doctoral Research Associate appeared first on Botany One. via Botany One https://botany.one/ January 29, 2023 at 07:52PM

#Botany #Plant Science #Botany One

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leedsomics · 5 months

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Parallel phosphoproteomics and metabolomics map the global metabolic tyrosine phosphoproteome

Tyrosine phosphorylation of metabolic enzymes is an evolutionarily conserved post-translational modification that facilitates rapid and reversible modulation of enzyme activity, localization or function. Despite the high abundance of tyrosine phosphorylation events detected on metabolic enzymes in high-throughput mass spectrometry-based studies, functional characterization of tyrosine phosphorylation sites has been limited to a subset of enzymes. Since tyrosine phosphorylation is dysregulated across human diseases, including cancer, understanding the consequences of metabolic enzyme tyrosine phosphorylation events is critical for informing disease biology and therapeutic interventions. To globally identify metabolic enzyme tyrosine phosphorylation events and simultaneously assign functional significance to these sites, we performed parallel phosphoproteomics and polar metabolomics in non-tumorigenic mammary epithelial cells (MCF10A) stimulated with epidermal growth factor (EGF) in the absence or presence of the epidermal growth factor receptor (EGFR) inhibitor erlotinib. We performed an integrated analysis of the phosphoproteomic and metabolomic datasets to identify tyrosine phosphorylation sites on metabolic enzymes with functional consequences. We identified two previously characterized (PKM, PGAM1) and two novel (GSTP1, GLUD1) tyrosine phosphorylation sites on metabolic enzymes with purported functions based on metabolomic analyses. We validated these hits using a doxycycline-inducible CRISPR interference (CRISPRi) system in MCF10A cells, in which target metabolic enzymes were depleted with simultaneous re-expression of wild-type, phosphomutant or phosphomimetic isoforms. Together, these data provide a framework for identification, prioritization and characterization of tyrosine phosphorylation sites on metabolic enzymes with functional significance. http://dlvr.it/T6vbZl

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michellem123 · 7 months

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Acetyltransferases & Deacetylases

Acetylation is one of the major protein modifications in cells, where the acetyl group from acetyl coenzyme A (acetyl-CoA) is transferred to a polypeptide chain. Protein acetylation can occur at N-terminal as a co-translational covalent modification by N-terminal acetyltransferases (NATs), or on lysine as a reversible post-translational modification (PTM). Like phosphorylation, acetylation also affects protein function by altering the properties of proteins such as hydrophobicity, solubility, protein conformation and affinity with other macromolecules.

Thousands of acetylation have been identified in human proteome, among which chromatin proteins and metabolic enzymes are highly represented such as histones, P53, and STAT3. Consequently, dysregulation of acetylation can lead to metabolic disorders, cancers, neurodegeneration, and cardiovascular diseases.

CD BioSciences offers a complete portfolio of solutions to study protein acetylation including acetylation profiling, acetyltransferase/deacetylase identification, substrate identification, and inhibitor/activator screening.

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ignited-minds-era · 2 years

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What is Western Blotting? A Brief Overview

Western blotting is a common laboratory technique used to detect specific proteins in a sample. It's a key part of many research studies, as well as diagnostics for medical conditions. In this post, we'll give a brief overview of what western blotting is, how it works, and some of its applications. We'll also touch on some of the challenges associated with this technique.

What is Western Blotting?

In brief, Western blotting is a technique used to detect specific proteins in a sample. This technique is commonly used in biomedical research to study the proteins involved in various cellular processes.

In more detail, Western blotting involves separating proteins by size using gel electrophoresis. The proteins are then transferred to a membrane, where they are exposed to antibodies that recognize and bind to specific proteins. The bound proteins are then detected using chemiluminescence or radioactivity.

Western blotting is a powerful tool that can be used to study the function of specific proteins in cells. It can also be used to diagnose disease, as certain diseases are associated with changes in the levels of specific proteins.

The History of Western Blotting

Western Blotting (WB) is a technique used to detect and analyze proteins. It is an essential tool in molecular and cell biology, as it allows researchers to study the structure and function of proteins in different cellular contexts.

The history of Western blotting dates back to the early 1970s, when scientists first began using antibodies to detect proteins. The technique was initially developed for use in immunology, but soon found applications in other fields such as cancer research.

Over the years, WB has undergone many refinements and today it is one of the most commonly used techniques in molecular biology.

If you would like to learn more about Western blotting, please see our other blog articles or contact us for more information.

The Western Blotting Process

The Western blotting process is a common laboratory technique used to detect specific proteins in a sample. The procedure involves separating proteins by size using gel electrophoresis and then transferring them to a membrane. The proteins are then incubated with antibodies that recognize and bind to the target protein. Finally, the membrane is incubated with a detection reagent that visualizes the bound antibodies, allowing for detection of the target protein.

Western Blotting Applications

Western blotting, also known as protein immunoblotting, is a widely used analytical technique used to detect and quantify specific proteins in a sample. Western blotting has many applications, including determining the identity of a protein, determining the abundance of a protein, and detecting post-translational modifications.

One common application of western blotting is to confirm the identity of a protein. To do this, antibodies that specifically bind to the protein of interest are used. If the protein is present in the sample, it will bind to the antibody and can be detected using an appropriate detection method.

Another common application of western blotting is to determine the abundance of a specific protein in a sample. By quantifying the amount of protein that binds to an antibody, one can get an estimate of how much protein is present in the sample.

Finally, western blotting can also be used to detect post-translational modifications (PTMs). PTMs are changes that occur to a protein after it has been synthesized and can affect its function. Common PTMs include phosphorylation, acetylation, and ubiquitination. By detecting PTMs with specific antibodies, one can learn about how a protein has been modified and what effect that modification may have on its function.

Conclusion

Western blotting is a powerful technique that can be used to detect the presence of specific proteins in a sample. While it is often used in research settings, it also has applications in diagnostics and drug development. If you are working with proteins, learning how to perform a Western blot can be a valuable skill.

#western blotting #western blotting market #blotting

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agadignostic1 · 2 years

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Things to Consider While Developing Assays

The development of a biological assay must take a number of fundamental elements into account, regardless of the application or the individual molecule to be analysed. It is crucial to give the assay significant consideration, as well as the entire process that needs to be designed from sample preparation through analysis of the assay's data quality output. The main assay development considerations covered in this article are generally applicable.

No matter the test being created (e.g., antibody-based assays like ELISA and quantitative PCR, electrochemiluminescence, measurement of enzyme activity assays, cell-based assays or total protein/protein concentration, or customised assay like single-molecule activation assay), all are legitimate. Assays come in a wide variety. However, there are some crucial components of test construction that apply to all assays. Here is a simple checklist to help you get ready to build a new assay.

What chemical and objective are being assessed?

The first step is to be crystal clear about the molecule to be tested and the exact attribute that should be assessed. Although it may seem obvious, this issue is very important and forms the basis for all subsequent assay development efforts. For instance, the researcher may want to determine whether they want to assess the overall amount of a specific protein in a cell lysate, only the phosphorylated form, or both. Similar to this, the study can mandate that measurements be made of particular isoforms or splice variants of the relevant protein. The assay needs to be created to yield precise data on the molecule being studied.

What’s the source of the molecule?

It is crucial to take into account the molecule's source before doing the analysis. Do you want to measure the molecule in a body fluid like serum or urine? Is the molecule to be evaluated in a biopsy sample from a human or an organ taken from an experimental animal? It's possible that the source will be in vitro-cultured cells, in which case it's crucial to assess whether the cells are limited primary cells or an easily scaled-up immortalised cell line.

The availability and number of samples will depend on the molecule's source. As mentioned in the sections that follow, it will also establish the molecule's concentration and could significantly affect its stability. Therefore, it is likely that the origin of the target molecule will have a substantial impact on the final test workflow while doing tests like silver assay, gold assay etc.

Molecular stability

Understanding the stability of the chemical for which the assay is to be built is also crucial. Does it require additional care to be taken during sample collection and preparation for the test in order to produce valid assay results, or is it moderately or severely unstable? Even molecules that are stable when they are isolated may become unstable when they are exposed to the complex biological environment of the samples that will be tested, where they may be vulnerable to oxidation, proteolysis, or the loss of post-translational modifications.

Semi-quantitative v/s quantitative

It's critical to decide up front whether a semi-quantitative measurement of the molecule, such as a Western blot, would suffice for the project's needs or whether a rigorously quantitative assay is necessary in order to create an assay that is appropriate for the task at hand.

The quantity of samples to be analysed

How many samples will need to be analysed is another crucial factor to take into account. A labour-intensive, multi-step manual assay approach may be appropriate if just a small number of samples will be analysed. Conversely, it will be crucial to simplify, streamline, and automate the assay procedure as much as lab resources permit, for example, with a format like microarray, if hundreds or tens of thousands of tests are to be conducted, maybe as part of a chemical profiling exercise. But arrays have their own unique set of issues, namely intra-assay spatial fluctuation.

Our approach to effective assay development could benefit your company whether you're wanting to increase your competitive edge or release a new diagnostic product. AgPlus Diagnostics tried-and-true method for assay development could improve your offering and your company if you're seeking a competitive advantage or want to introduce a new diagnostic product to the market.

#assay development #gold assay

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medchemexpressinhibitor-blog · 2 years

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Deciphering the mechanism of p53-mediated ferroptosis

Background of p53

It is well known that p53 is a tumor suppressor gene and essential for regulating DNA repair and cell division. Since its discovery in 1979, p53 gene has been a hot topic in molecular biology and oncology. According to Dolgin, E. et al.’s literature published in Nature, TP53 becomes the most popular human genome in the list of most studied genes in PubMed database[1]. The TP53 gene provides instructions for making a p53 protein.

According to integrated cancer genomic and epidemiological data analyses, TP53 is the most commonly mutated gene (35%) among mutated driver genes in human cancers[2](Figure 1), it is also the most studied gene in the human genome based on PubMed database. TP53 is located on the short arm of human chromosome 17 and encodes the p53 tumor suppressor protein.

The p53 protein is often referred to as the “Guardian of the Genome”. The main biological function of the p53 protein is the protection of the DNA integrity of the cell.

Figure 1: Left, List of most studied genes in the PubMed database as of 2017

[3]

Right, Proportion of epidemiologically weighted gene mutations in important genomes in the cancer patient population

[2]

In response to intrinsic and extrinsic stress signals, the p53 protein is activated by a variety of post-translational modifications including phosphorylation, acetylation, methylation, ubiquitination, or SUMOylating, etc. These modifications at key sites allow p53 protein to become stabilized, oligomerize as a tetramer, interact with cofactors, bind to the p53RE, execute the transcription of the target genes in a tightly controlled and context dependent manner [Figure 2]. In recent years it was found that p53 plays roles in the regulation of ferroptosis. In this article, the connection between p53 and ferroptosis will be explored.

Figure 2. Central role of p53 protein as a tumor suppressor

[4]

Ferroptosis is a novel form of regulated cell death. The morphological features of ferroptosis include shrunk mitochondria with condensed mitochondrial membrane densities, reduction or vanishing of mitochondria crista, and rupture of outer mitochondrial membrane. The p53 (especially acetylation-defective mutant p53, p533KR) positively regulates ferroptosis by inhibiting expression of SLC7A11 (a specific light-chain subunit of the cysteine/glutamate antiporter).

SLC7A11 is a subunit of System Xc-, which is responsible for maintaining redox homeostasis by importing cystine. After transported into cells, cystine is quickly reduced to cysteine, a critical precursor for glutathione and subsequent reduced glutathione (GSH). GSH biosynthesis is critical to functional activity of membrane lipid repair enzyme GPX4. Inhibiting System Xc- activity by inhibition of SLC7A11 expression leads to decreased uptake of cystine, eventually resulting in impaired antioxidant capability of cells and initiation of ferroptosis [Figure 3]. “Ferroptosis as a p53-mediated activity during tumor suppression”, Jiang et al. reported that p53 inhibits expression of SLC7A11, reduces cystine uptake and induces ferroptosis of cancer cells.

Figure 3: The mechanism of p53 mediated ferroptosis

[6]

.The SLC7A11 gene is a target of p53-mediated transcriptional repression

Jiang, L et al. has demonstrated in the "Ferroptosis as a p53-mediated Activity during Tumour Suppression" published in Nature in 2015 demonstrates that p53 supposes SLC7A11 (a key component of the cystine/glutamate antitransporter) to inhibit cystine uptake and sensitize cells to ferroptosis[7].

By microarray analysis of Tetracycline -controlled (tet-on) p53 induced and non-induced cells, SLC7A11 was identified as a novel p53 target gene. Western blot showed that p53 activation significantly reduced SLC7A11 protein levels [Figure 4a]. While in U2OS cells under p53-knockdown conditions treated Nutlin-3 (a p53-MDM2 inhibitor), downregulation of SLC7A11 was abrogated. These data suggest that SLC7A11 gene is a target of p53-mediated transcriptional repression.

The acetylation defective mutant p533KR cells that fails to induce cell cycle arrest, senescence and apoptosis maintain the ability to regulate SLC7A11 expression and induce ferroptosis. Tet-on p533KR inducible H1299 cells were resistant to erastin-mediated ferroptosis in the absence of p533KR induction, while significant cell death was observed upon p533KR induction together with Erastin treatment. But SLC7A11overexpression abrogated p533KR induced ferroptosis [Figure 4c]. In xenograft tumor models implanted with p53-null H1299 cells, tumor size is markedly reduced upon p533KR expression induced by tetracycline, while this tumor suppression effects of p533KR were abrogated by SLC7A11 overexpression[7] [Figure 4d-e].

Figure 4. SLC7A11 regulates p53-mediated ferroptosis

[7]

a: Western blot of Doxycycline-treated tet-on p53 stable line cells; b: Western blot analysis of Nutlin-treated p53-knockdown U2OS cells; c-d: Ferroptosis in Tet-on p53

3KR

cells transfected with a control or SLC7A11-overexpressing plasmid and xenograft tumor weight of cells.

In addition, it was found that high levels of reactive oxygen species (ROS) can trigger p53-mediated ferroptosis. As shown in Figure 5a, no significant cell death was observed in p533KR induction alone or in ROS activator treatment; whereas the p533KR and ROS group induced substantial cell death, which was rescued by overexpression of SLC7A11 (Figure 5b)[7].

Figure 5. Effects of high levels of ROS on p53-mediated ferroptosis

[7]

a-b: Cell death of Tet-on p53

3KR

cells treated with tetracycline and ROS as control or transfected with SLC7A11-overexpressing plasmid;ALOX12 is essential for the p53-mediated ferroptosis pathway

In a follow-up study, Chu B. et al. reported that ALOX12-mediated, ACSL4-independent ferroptosis pathway is critical for p53-dependent tumor suppression[8]. The ALOX12 gene is located on human chromosome 17p13.1, a labile site for monoallelic deletions in human cancers.

ALOX12 was found to be essential for p53-mediated ferroptosis under ROS stress. ALOX12 depletion had no apparent effect on p53 levels or the expression of its transcriptional targets such as SLC7A11, Mdm2, and p21, but it rescued p53-mediated ferroptosis (Figure 6a-b)[8].

Next, it was found that p53-mediated ferroptosis under ROS stress was regulated independently of GPX4. As shown in Figure 6c, high levels of endogenous lipid peroxidation could be detected in GPX4-knockout cells, whereas lipid peroxidation levels were significantly reduced after ectopic expression of GPX4. At the same time, RSL-3 (GPX4 inhibitor) could abrogate the reducing effect of GPX4 on lipid peroxidation, while the activation of p53 had no effect on it[8].

Then, the effect of ALOX12 on p53-mediated tumor growth inhibition was investigated. Tetracycline-induced p533KR expression significantly reduced tumor cell growth. Moreover, ALOX12 knockdown ablated the tumor suppressive effect of p533KR [Figure 6c-d]. These data suggested that ALOX12 is critical for the tumor cell growth inhibitory activity of p53[8].

Figure 6. The role of ALOX12 in p53-mediated ferroptosis under ROS stress

[8]

a: Western blot analysis of U2OS cells; b: U2OS cell death after treatment with different drugs; c: Changes in cellular lipid peroxidation levels; d: Xenograft tumors in H1299 Tet-on p533KR and ALOX12 knockout mice.iPLA2β is a key regulator of the p53-mediated ferroptosis pathway

In June 2022, Chen Du et al. reporteda mechanism in an article "iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4" in Nature CommunicationsIn this proposed mechanism iPLA2β is a key regulator of p53 activation-induced ferroptosis under high ROS stress conditions and p53 induces ferroptosis in a GPX4-independent manner[9][Figure 7].

Figure 7. Model for the role of ALOX12 and iPLA2β in regulating p53-mediated ferroptosis

[9]

The p53 levels were not affected by ACSL4 and GPX4, in the ACSL4/GPX4 double knockout (ACSL4

-/-

/GPX4

-/-

) human osteosarcoma cell line U2OS. At the same time, p53-mediated transcriptional activation of p21 or repression of SLC7A11 remained unchanged (Fig. 8a). However, when ACSL4

-/-

/GPX4

-/-

cells were exposed to TBH and Nutlin, ferroptosis cell death apparently occurred, which was specifically blocked by ferroptosis inhibitors (Fig. 8b).

Figure 8. p53 mediates ferroptosis in a GPX4-independent manner under ROS stimulation

[9]

a: Effect of Nutlin on p53 protein in U2OS cells; b-d: The effects of cell death influenced by TBH, Nutlin, Ferr-1, Lipro-1, 3-MA, Necrostatin-1, Z-VAD-FMK on WT,ACSL4

-/-

, GPX4

-/-

and U2OS cellsConclusion:The tumor suppressor gene

TP53

is the most studied gene in cancer research. Ferroptosis is a novel form of regulated cell death that has sparked a research frenzy since its discovery. The discovery of connection between p53 and ferroptosis paves a new way for the development of p53-related drugs for cancer treatment.

Related products

Nutlin-3

A potent p53-MDM2 inhibitor with Ki of 90 nM.

Idasanutlin

A potent and selective MDM2 antagonist that inhibits p53-MDM2 binding with IC50 of 6 nM.

Pifithrin-α hydrobromide

A p53 inhibitor that blocks its transcriptional activity and prevents apoptosis; an agonist of aryl hydrocarbon receptor (AhR).

Erastin

A ferroptosis inducer that binds and inhibits voltage-dependent anion channels (VDAC2/VDAC3).

RSL3

An inhibitor of glutathione peroxidase 4 (GPX4) (ferroptosis agonist) that reduces GPX4 expression. .

Ferrostatin-1

A selective ferroptosis inhibitor; antioxidant.

References

[1]. Liz J Hernández Borrero 1, Wafik S El-Deiry, et al. Tumor suppressor p53: Biology, signaling pathways, and therapeutic targeting. Biochim Biophys Acta Rev Cancer. 2021 Aug;1876(1):188556.

[2]. Gaurav Mendiratta, et al. Cancer gene mutation frequencies for the U.S. population. Nat Commun. 2021 Oct 13;12(1):5961

[3]. Elie Dolgin, et al. The most popular genes in the human genome. Nature. 2017 Nov 23;551(7681):427-431.

[4]. Sandra L Harris, et al. The p53 pathway: positive and negative feedback loops. Oncogene. 2005 Apr 18;24(17):2899-908.

[5]. Ou M, et al. Role and mechanism of ferroptosis in neurological diseases. Mol Metab. 2022 Jul;61:101502.

[6]. Y Xie, R Kang, D Tang et al. Ferroptosis: process and function. Cell Death Differ. 2016 Mar;23(3):369-79.

[7]. Le Jiang, Wei Gu, et al. Ferroptosis as a p53-mediated activity during tumour suppression. Nature. 2015 Apr 2;520(7545):57-62.

[8]. Bo Chu, Wei Gu, et al. ALOX12 is required for p53-mediated tumour suppression through a distinct ferroptosis pathway. Nat Cell Biol. 2019 May;21(5):579-591.

[9]. Delin Chen, Wei Gu, et al. iPLA2β-mediated lipid detoxification controls p53-driven ferroptosis independent of GPX4. Nat Commun. 2021 Jun 15;12(1):3644.

#p53-mediated

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colonyfact3 · 4 years

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Bicyclic Peptides

Dbal Kinds, Steroids On Keto

Content

Everything About Peptides.

What Are Sarms? (Component.

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Iaaf Values Board Choice: Timeline

Procollagen Peptide Type 3 (p3np Or Piiinp).

At Paula's Option, we take a multi-ingredient 'mixed drink strategy' to skincare. direct-peptides online Buy best quality peptides Direct Belgium means the skincare products you purchase ought to also consist of other reliable ingredients such as antioxidants and skin-repairing ingredients. The increasing recognition on the scientific, technical and also financial worth of peptides is starting to energise investment on peptide research study and peptide production facilities. One example is the Peptide Synthesis Facility of the Faculty of Sciences of the University of Porto, which Gomes coordinates.

Androgen is an all-natural product of the body that allows us to build skeletal muscular tissue mass.

Efficacy tests of several SARMs in people remain in beginning and have typically shown moderate increments in fat-free mass.

GSK, the research medicine, comes from a new class of medications called non-steroidal, selective androgen receptor modulators, which can mimic several of the advantageous effects of testosterone whilst preventing several of the undesirable negative effects.

The first generation SARMs do not go through aromatization or 5-alpha decrease; it is unidentified whether this may posture long term risks.

The term 'SARMs' represent Selective Androgen Receptor Modulators.

The efficacy as well as the safety and security of SARMs as function advertising treatment is simply beginning to be assessed.

This cycle of deprotection as well as combining is repeated till the unabridged peptide is created. Due to the fact that amino acids have several responsive groups, peptide synthesis have to be thoroughly done to avoid side responses that can lower the size and also cause branching of the peptide chain. To promote peptide formation with marginal side reactions, chemical groups have actually been established that bind to the amino acid responsive teams as well as block, or secure, the useful group from nonspecific response. Furthermore, artificial peptides are made use of to examine enzyme-substrate communications within crucial enzyme classes such as kinases as well as proteases, which play a critical function in cell signaling. Any individual from young adults to pensioners can take advantage of applying products with peptides, much like they can from various other healthy and balanced skin components, like niacinamide, vitamin C, anti-oxidants and omega fats and so on . For younger people, it will certainly assist fend off the look of aging and for those who are a little older, they can enhance the skin look and feel. Copper peptide is one of one of the most raved-about skin care ingredients.

Everything About Peptides.

He reported that the peptide group (Carbon Monoxide-- NH) has an inflexible planar structure. This structure is because of interactions between electrons of the dual bond of the carbonyl team and also those of the C-- N bond such that the latter gets partial (regarding 40%) double-bond buildings. In proteogenomic evaluations it is vital to recognize the loci triggering peptides in order to boost genomic note as well as the useful characterization of protein products in their biological context. With next-generation sequencing of DNA and RNA for each and every example examined by proteomic mass spectrometry combination as well as visualisation in a common coordinate system, i.e. the genome, is vital for systems biology. Developments in modern technology in mass spectrometry currently allow nearly total quantification of the example proteome. In addition, moving in the direction of even more individual multi-omics researches comparative visualisation of proteomic data on a genome has actually been doing not have. To promote this kind of combination not just the genomic areas of customized peptides yet especially the genomic loci of related to these adjustments is needed.

Will rad140 show up on a drug test?

RAD140 and the majority of the identified in vitro metabolites were detected in post‐administration urine samples. For controlling the misuse of RAD140 in horses, RAD140 and its metabolite in sulfate form gave the longest detection time in hydrolysed urine and could be detected for up to 6 days post‐administration.

The hype started due to the reality that the foundation of your skin are formed by copper. So logic would dictate that you require copper peptide in your skincare products to repair your skin. It holds true that copper peptide is a skin-restoring component, but there are lots of other peptides with the exact same otherwise more remarkable residential properties. While it's clear that peptides are special, as an isolated active ingredient they are not nearly enough.

What Are Sarms? (Part.

This molecular weight distinction makes heavy peptides valuable devices for measurable peptide evaluation or protein structure and also dynamics decision by mass spectrometry or nuclear magnetic vibration spectroscopy, respectively. Phosphorylated tyrosine, serine or threonine can be positioned anywhere on a provided peptide. Although numerous phosphorylated amino acids can be added, they can negatively influence peptide synthesis and also filtration. Although peptide synthesis methods have actually been maximized and can be mass-produced, the process to create peptides is by no means excellent. Events such as insufficient deprotection or response with complimentary shielding teams can trigger abbreviated or removal sequences, isomers or other side items. These events can take place at any kind of step during peptide synthesis, and consequently the longer the peptide sequence, the better possibility that something will adversely influence the synthesis of the target peptide. Thus, peptide yield is vice versa associated with peptide length.

Is Using SARMS for Running a Good Idea? Your Questions, Answered - - VENTS Magazine

Is Using SARMS for Running a Good Idea? Your Questions, Answered -.

Posted: Sat, 09 May 2020 07:00:00 GMT [source]

There are 20 amino acids, each with different residential properties, and combining these creates functionally different peptides. The series of amino acids in a peptide is called the main framework. Proteins as well as peptides are both chains of amino acids, however peptides have much shorter chain lengths.

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The chemical uniqueness of amino acids, and also the ease with which they can be paired with each other, give exciting opportunities for designing-in functionality as well as structure. This research study manipulated the distinctions in amino acid side chains to control exactly how peptides engage with each other and after that self-assemble into bigger frameworks. As appeal pioneers captured on, they started including them in items. Using circulation chemistry, it is simple to pre-heat and also pre-activate the amino acid.

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This lowers the expense and amount of waste generated by the synthesis. Assimilation of inline real-time analysis, such as UV/Vis spectroscopy and also our Variable Bed Circulation Reactor into the circulation after the reactor authorization immediate recognition of any non-standard coupling events. This allows the user to identify and also optimise hard combining events during tiny range syntheses. The peptide synthesis can after that be scaled up using the optimised sequence. Important to an understanding of protein structure is an expertise of the framework of the peptide bond. Linus Pauling, in the 1930s, used X-ray diffraction to examine the nature of the peptide bond formed between two amino acids.

Iaaf Principles Board Choice: Timeline

Below, we supply a mapping device, PoGo, to promptly as well as effectively identify genomic loci of peptides as well as post-translational alterations as well as couple these mappings with associated quantitative values over numerous examples. This ultra-hydrating formula incorporates the latest age-defying innovation to proactively fight the look of expression lines and also decreased quantity. The 30% Peptide Complicated includes professional drone-targeted Copper Peptides which particularly target the cells in charge of collagen and also elastin synthesis, promoting manufacturing to aid soften and smooth the look of prematurely ageing skin.

What exactly are SARMs?

Selective Androgen Receptor Modulators (SARMs) are a class of therapeutic compounds that have similar anabolic properties to anabolic steroids, but with reduced androgenic (producing male characteristics) properties. As an example, the androgen receptor is activated by binding androgens, such as testosterone.

It likewise assists to secure cells from oxidative tension as well as supports the body immune system. Based upon these searchings for, it was especially essential for us to combine these 3 components in the formula of this particular item.

Procollagen Peptide Kind 3 (p3np Or Piiinp).

This center, yet unparalleled in Portugal, was open to provide peptide synthesis solutions in 2016, many thanks to Portuguese/European Union co-funding to acquire a cutting edge multiple synthesizer the only one in the country. Imaginative Peptides is specialized in the process advancement as well as the manufacturing of bioactive peptides. It is committed to providing custom-made peptide synthesis, process advancement, GMP production in addition to directory items for clients. Nonetheless, different types of peptides can profit the skin in a different way. Some can aid to ravel as well as reduce wrinkles, whilst others can fix obstacle function as well as increase firmness and also hydration in the skin. Having these numerous types combined in one product can permit you to target several different signs of ageing at once.

After succeeding cycles of amino acid deprotection and combining, all continuing to be securing groups should be removed from the incipient peptide. When appropriately carried out, cleavage lead to the elimination of the N-terminal protecting team of the last amino acid added, the C-terminal securing group from the initial amino acid as well as any kind of side-chain safeguarding teams. As with award-winning direct-peptides honored as top-rated and best Buy best quality peptides Direct Norway , scavengers are also included throughout this action to react with complimentary shielding groups.

This strategy greatly raises the rate of each combining cycle. Complete combining and deprotection cycles of just 15 minutes are conveniently achieved. This much more effective coupling also significantly decreases the requirement for excesses of reagent.

Collagen, elastin as well as keratin are the most common healthy proteins that compose our skin. Including peptides topically can help boost manufacturing of these healthy proteins as well as reinforce the skin's repair barrier function. In contrast to standard amino acids, isotopically classified "hefty" amino acids are synthesized by substituting12C and14N atoms with13C and/or15N atoms, specifically. Hefty amino acids are non-radioactive and also have known molecular weights that are much heavier than common amino acids.

Due to the significance of cleavage in proper peptide synthesis, this step ought to be optimized to avoid acid-catalyzed side responses. The N-terminal protecting group on the C-terminal amino acid of the peptide to be manufactured is initial deprotected. The brand-new N-terminus of the expanding peptide is then deprotected as well as coupled to the following amino acid.

What's really amazing regarding having this component in your skin care products is that it works to assist skin revitalise itself and makes it extra resistant to future damages. Study shows that the right peptide skincare item can play a crucial role in assisting to recover injuries as well as boost skin disease like dermatitis and dermatitis. You see words peptides pop up in all kinds of skincare items, however what exactly are they? In these peptides, the I3 theme is extremely hydrophobic, and also the amino acid K is hydrophilic. In this way, the scientists had the ability to methodically compare the second frameworks developed and evaluate the 'polar zipper' device.

With Female's Finest Collagen Peptides and its Plus variation, we have actually managed to smartly combine efficiency as well as quality. Our collagen powder is highly absorbable as well as includes essential amino acids which provide the body and also as a result advertise all-natural beauty from within. As we age, the production of hyaluronic acid decreases, the skin loses wetness as well as the joints become a lot more immobile. Scientific studies have actually shown that vitamin C supports the production of collagen in bones, cartilage material, skin and also teeth.

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