Analysis Of Target Proteins Research Articles

Label-Free Quantitative Proteomics Analysis of COVID-19 Vaccines by Nano LC-HRMS.

A nanoliter liquid chromatography-high resolution mass spectrometry-based method was developed for quantitative proteomics analysis of COVID-19 vaccines. It can be used for simultaneous qualitative and quantitative analysis of target proteins and host cell proteins (HCPs) in vaccine samples. This approach can directly provide protein information at the molecular level. Based on this, the proteomes of 15 batches of COVID-19 inactivated vaccine samples from two companies and 12 batches of COVID-19 recombinant protein vaccine samples from one company were successfully analyzed, which provided a significant amount of valuable information. Samples produced in different batches or by different companies can be systematically contrasted in this way, offering powerful supplements for existing quality standards. This strategy paves the way for profiling proteomics in complex samples and provides a novel perspective on the quality evaluation of bio-macromolecular drugs.

Comparative case study on NAMs: towards enhancing specific target organ toxicity analysis

Traditional risk assessment methodologies in toxicology have relied upon animal testing, despite concerns regarding interspecies consistency, reproducibility, costs, and ethics. New Approach Methodologies (NAMs), including cell culture and multi-level omics analyses, hold promise by providing mechanistic information rather than assessing organ pathology. However, NAMs face limitations, like lacking a whole organism and restricted toxicokinetic interactions. This is an inherent challenge when it comes to the use of omics data from in vitro studies for the prediction of organ toxicity in vivo. One solution in this context are comparative in vitro–in vivo studies as they allow for a more detailed assessment of the transferability of the respective NAM data. Hence, hepatotoxic and nephrotoxic pesticide active substances were tested in human cell lines and the results subsequently related to the biology underlying established effects in vivo. To this end, substances were tested in HepaRG and RPTEC/tERT1 cells at non-cytotoxic concentrations and analyzed for effects on the transcriptome and parts of the proteome using quantitative real-time PCR arrays and multiplexed microsphere-based sandwich immunoassays, respectively. Transcriptomics data were analyzed using three bioinformatics tools. Where possible, in vitro endpoints were connected to in vivo observations. Targeted protein analysis revealed various affected pathways, with generally fewer effects present in RPTEC/tERT1. The strongest transcriptional impact was observed for Chlorotoluron in HepaRG cells (increased CYP1A1 and CYP1A2 expression). A comprehensive comparison of early cellular responses with data from in vivo studies revealed that transcriptomics outperformed targeted protein analysis, correctly predicting up to 50% of in vivo effects.

Identification of overlapping molecular mechanisms in tuberculosis and sarcoidosis: A bioinformatics approach

BackgroundTuberculosis (TB) and sarcoidosis are chronic granulomatous diseases sharing similar symptoms, immune responses, and radiological characteristics. Transcriptome analysis offers insights into gene expression, regulation, and cellular processes, facilitating the understanding of shared molecular mechanisms. MethodsMicroarray datasets from the NCBI Gene Expression Omnibus (NCBI-GEO) were analysed to identify differentially expressed genes (DEGs) in TB and sarcoidosis compared to controls. DEGs were identified using the GEO2R tool, and subsequent functional enrichment analysis was conducted using EnrichR. Protein-protein interaction (PPI) networks, as well as gene-miRNA and transcription factor-DEG interaction networks, were constructed. In addition, pathway analysis and molecular docking of target proteins were conducted to further elucidate the biological mechanisms involved in both diseases. ResultsFifteen genes, including ANKRD22, BATF2, DHRS9, EPSTI1, ETV7, FCGR1A, FCGR1B, GBP1, GBP5, SERPING1, NELL2, CCR7, PASK, LRRN3, and SLC16A10, were commonly altered in TB and sarcoidosis as compared to controls. Gene network analysis revealed 48.89% co-expression and 26.10% physical interaction between these overlapping genes. PPI networks showed a total of 15 nodes and 28 edges present between the connected proteins (PPI enrichment p-value:<1.0e−16). MiRNAs and transcription factors that exhibited the highest interaction with DEGs included hsa-miR-26a-5p, hsa-miR-16-5p, hsa-miR-335-5p, and EPAS1, HIF1A, KLF2, respectively. Pathway analysis indicated enrichment of IFN gamma signaling in both diseases. Molecular docking revealed weighted scores of −884.4, −851.9, and − 637.1 between three key proteins (PASK-GBP1, PASK-GBP5, and GBP1-GBP5). ConclusionThe shared dysregulated genes in TB and sarcoidosis demonstrate notable co-expression and physical interaction, constituting a PPI network enriched in the IFN-gamma signaling pathway.

Qianyang yuyin granule ameliorates mitochondrial dysfunction of hypertensive myocardial remodeling

Ethnopharmacological relevanceClinical studies have found that Qianyang Yuyin granule (QYYYG), a kind of oral Chinese patent medicine, had definite clinical effect for hypertensive myocardial remodeling. However, the potential mechanism is not entirely clear. Aim of the studyThe purpose of this research was to explore the underlying mechanism QYYYG on the treatment of hypertensive myocardial remodeling. Materials and methodsAnalysis the transcriptome data from the NCBI public platform GEO database and our study to explore the key pathological change of myocardial tissues in hypertensive mice and the main pathway of QYYYG in treating hypertensive myocardial remodeling. Network pharmacological analysis was used to predict the potential target of QYYYG. The molecular docking and molecular dynamics simulation was used for molecular binding analysis of specific compounds and target proteins. In the experiment in vivo, the effect of QYYYG on hypertensive myocardial remodeling and myocardial mitochondrial dysfunction in hypertensive mice caused by Ang Ⅱ was estimated. In the experiment in vitro, the Ang Ⅱ-induced myocardial remodeling model in H9c2 cells was constructed, and the effect of QYYYG on ameliorating myocardial remodeling and mitochondrial dysfunction was evaluated. ResultsTranscriptome analysis suggested that mitochondrial dysfunction was a key pathological change of myocardial tissues in hypertensive mice, and QYYYG could improve hypertensive myocardial remodeling through enhancing mitochondrial biogenesis to repair myocardial mitochondrial dysfunction. Network pharmacological analysis predicted that SIRT1 was an important potential target of QYYYG in treating hypertensive myocardial remodeling, and basically all the active components, especially quercetin, had a great binding affinity with SIRT1. Experiments in vivo proved that QYYYG had great efficacy hypertensive myocardial remodeling in Ang Ⅱ-treated mice. It was found that QYYYG improved the quality and quantity of mitochondria, and increased SIRT1 levels in myocardial tissue of Ang Ⅱ-treated mice. In Ang Ⅱ-treated H9c2 cells, with intervention of QYYYG, myocardial remodeling and myocardial mitochondrial dysfunction was ameliorated. In addition, QYYYG up-regulated SIRT1 expression and enhanced mitochondrial biogenesis in Ang Ⅱ-treated H9c2 cells. ConclusionThis study suggested that mitochondrial dysfunction was an important pathological change of myocardial tissues in hypertensive mice. QYYYG might ameliorate the mitochondrial dysfunction of hypertensive myocardial remodeling through up-regulating SIRT1 expression to enhance the mitochondrial biogenesis.

Role of Hemigraphis alternata in wound healing: metabolomic profiling and molecular insights into mechanisms

Hemigraphis alternata (H. alternata), commonly known as Red Flame Ivy, is widely recognized for its wound healing capabilities. However, the pharmacologically active plant components and their mechanisms of action in wound healing are yet to be determined. This study presents the mass spectrometry-based global metabolite profiling of aqueous and ethanolic extract of H. alternata leaves. The analysis identified 2285 metabolites from 24,203 spectra obtained in both positive and negative polarities. The identified metabolites were classified under ketones, carboxylic acids, primary aliphatic amines, steroids and steroid derivatives. We performed network pharmacology analysis to explore metabolite–protein interactions and identified 124 human proteins as targets for H. alternata metabolites. Among these, several of them were implicated in wound healing including prothrombin (F2), alpha-2A adrenergic receptor (ADRA2A) and fibroblast growth factor receptor 1 (FGFR1). Gene ontology analysis of target proteins enriched cellular functions related to glucose metabolic process, platelet activation, membrane organization and response to wounding. Additionally, pathway enrichment analysis revealed potential molecular network involved in wound healing. Moreover, in-silico docking analysis showed strong binding energy between H. alternata metabolites with identified protein targets (F2 and PTPN11). Furthermore, the key metabolites involved in wound healing were further validated by multiple reaction monitoring-based targeted analysis.

Molecular docking, ADMET profiling of gallic acid and its derivatives (N-alkyl gallamide) as apoptosis agent of breast cancer MCF-7 Cells

Background In 2020, breast cancer has become the most common cancer in the world and in Indonesia. Searching for anticancer drugs using computational methods is considered more effective and selective than other methods. Gallic acid and its derivatives (esters and amides) are compounds that have biological activities such as anticancer effects. The purpose of this study was to analyse the molecular modelling and ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) profile of gallic acid derivative compounds (N-alkyl gallamides) as anticancer agents. Methods Target proteins were selected by analysis of protein-protein and drug-protein interactions. Molecular modelling was done by molecular docking and molecular dynamic simulation. Predictive analysis of the ADMET profile of gallic acid and its derivatives (N-alkyl gallamide) was conducted using Marvin Sketch, Swissadme, protox II, and pkCSM pharmacokinetics. The selected target proteins were JUN, AKT1, CASP3, and CASP7. Results Compounds N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide were the three best gallic acid derivatives based on molecular modelling analysis of target proteins associated with breast cancer. The ADMET profile of the N-alkyl gallamide compound is predictable and shows a good profile as a candidate for anticancer drugs. Conclusion N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide have potential as anti-breast cancer agents.

A Network Pharmacology and Molecular Dynamics Simulation-Based Study of Qing Run Hua Jie Decoction in Interstitial Pneumonia Treatment.

This study is dedicated to revealing the potential mechanism of Qin Run Hua Jie (QRHJ) decoction in Interstitial pneumonia (IP) treatment. The TCMSP database predicted the chemical components and targets of QRHJ decoction, and the IP-related genes were from the Genecards database. Cytoscape software was used to establish the interaction network. R package clusterProfiler was utilized for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The molecular docking analysis of target proteins and the corresponding active pharmaceutical ingredients in the core position of the interaction network was conducted. Then, molecular dynamics (MD) simulations of a potential active substance and its key targets were performed. The binding efficiency of EGFR and luteolin, HIF1A and diosgenin was detected by cellular thermal shift assay (CETSA), and protein expression was measured by Western blot. CCK-8 was used to detect cell activity. A total of 153 active ingredients, 127 targets and 362 IP-related genes were obtained. KEGG enrichment analysis identified IP-related signaling pathways including HIF-1 signaling pathway and TNF signaling pathway. The two key components luteolin and diosgenin stably bound to the key targets EGFR and HIF1A. Cell experiments further showed that EGFR and luteolin, HIF1A and diosgenin bound to exert anti-fibrotic effects. As an active ingredient of QRHJ decoction, luteolin and diosgenin may exert therapeutic effect on IP through binding to the key target EGFR and HIF1A. This work initially revealed the key molecular mechanism of QRHJ decoction in IP treatment and offered theoretical evidence.

3D evaluation of the extracellular matrix of hypoxic pancreatic islets using light sheet fluorescence microscopy

ABSTRACT Pancreatic islet transplantation is a promising treatment for type 1 diabetes, but the survival and function of transplanted islets are hindered by the loss of extracellular matrix (ECM) during islet isolation and by low oxygenation upon implantation. This study aimed to evaluate the impact of hypoxia on ECM using a cutting-edge imaging approach based on tissue clearing and 3D microscopy. Human and rat islets were cultured under normoxic (O2 21%) or hypoxic (O2 1%) conditions. Immunofluorescence staining targeting insulin, glucagon, CA9 (a hypoxia marker), ECM proteins (collagen 4, fibronectin, laminin), and E-cadherin (intercellular adhesion protein) was performed on fixed whole islets. The cleared islets were imaged using Light Sheet Fluorescence Microscopy (LSFM) and digitally analyzed. The volumetric analysis of target proteins did not show significant differences in abundance between the experimental groups. However, 3D projections revealed distinct morphological features that differentiated normoxic and hypoxic islets. Under normoxic conditions, ECM could be found throughout the islets. Hypoxic islets exhibited areas of scattered nuclei and central clusters of ECM proteins, indicating central necrosis. E-cadherin was absent in these areas. Our results, demonstrating a diminution of islets’ functional mass in hypoxia, align with the functional decline observed in transplanted islets experiencing low oxygenation after grafting. This study provides a methodology combining tissue clearing, multiplex immunofluorescence, Light Sheet Fluorescence Microscopy, and digital image analysis to investigate pancreatic islet morphology. This 3D approach allowed us to highlight ECM organizational changes during hypoxia from a morphological perspective.

Molecular pathways underlying lung-brain axis signaling in asthma: Relevance for psychopathology and neuroinflammation

Accumulating evidence indicates that asthma has systemic effects and affects brain function. Although airway inflammation is proposed to initiate afferent communications with the brain, the signaling pathways have not been established. We sought to identify the cellular and molecular pathways involved in afferent lung-brain communication during airway inflammation in asthma. In 23 adults with mild asthma, segmental bronchial provocation with allergen (SBP-Ag) was used to provoke airway inflammation and retrieve bronchoalveolar lavage fluid for targeted protein analysis and RNA sequencing to determine gene expression profiles. Neural responses to emotional cues in nodes of the salience network were assessed with functional magnetic resonance imaging at baseline and 48 hours after SBP-Ag. Cell deconvolution and gene coexpression network analysis identified 11 cell-associated gene modules that changed in response to SBP-Ag. SBP-Ag increased bronchoalveolar lavage eosinophils and expression of an eosinophil-associated module enriched for genes related to TH17-type inflammation (eg, IL17A), as well as cell proliferation in lung and brain (eg, NOTCH1, VEGFA, and LIF). Increased expression of genes in this module, as well as several TH17-type inflammation-related proteins, was associated with an increase from baseline in salience network reactivity. Our results identify a specific inflammatory pathway linking asthma-related airway inflammation and emotion-related neural function. Systemically, TH17-type inflammation has been implicated in both depression and neuroinflammation, with impacts on long-term brain health. Thus, our data emphasize that inflammation in the lung in asthma may have profound effects outside of the lung that may be targetable with novel therapeutic approaches.

Molecular docking, ADMET profiling of gallic acid and its derivatives (N-alkyl gallamide) as apoptosisagentof breast cancer MCF-7 Cells.

In 2020, breast cancer has become the most common cancer in the world and in Indonesia. Searching for anticancer drugs using computational methods is considered more effective and selective than other methods. Gallic acid and its derivatives (esters and amides) are compounds that have biological activities such as anticancer effects. The purpose of this study was to analyse the molecular modelling and ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) profile of gallic acid derivative compounds (N-alkyl gallamides) as anticancer agents. Target proteins were selected by analysis of protein-protein and drug-protein interactions. Molecular modelling was done by molecular docking and molecular dynamic simulation. Predictive analysis of the ADMET profile of gallic acid and its derivatives (N-alkyl gallamide) was conducted using Marvin Sketch, Swissadme, protox II, and pkCSM pharmacokinetics. The selected target proteins were JUN, AKT1, CASP3, and CASP7. Compounds N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide were the three best gallic acid derivatives based on molecular modelling analysis of target proteins associated with breast cancer. The ADMET profile of the N-alkyl gallamide compound is predictable and shows a good profile as a candidate for anticancer drugs. N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide have potential as anti-breast cancer agents.

Abstract P3036: Circular RNA Decoys Targeting MiR-146a As A Promising Therapeutic Strategy To Improve Vascular Regeneration

Heart failure and cardiac remodeling following myocardial infarction (MI) depend on capillary density and neovascularization. MicroRNAs (miRNAs), which are small non-coding RNAs that modulate gene expression, play a major role in the pathogenesis of these conditions. Inhibition of these pathways through miRNA sponges, such as antagomiRs or circular decoys (circRNAs), might represent a therapeutic approach for MI. Here, we evaluate the role of miR-146a and present a promising treatment strategy using synthetically designed circular decoys. miRNA-146a expression was analyzed in human vascular cells and mice tissue following either myocardial infarction or hind limb ischemia. CircRNAs were synthetically designed and evaluated. Methods to characterize the effect of inhibition of miR-146a included the quantification of the target gene and protein expression and analysis of cellular functions.miR-146a was significantly upregulated in vitro in senescent cells ( P &lt;0.05) and in vivo in the ischemic myocardium following ligation of the LAD ( P &lt;0.001) in C57BL/6 mice. A circular RNA decoy targeting miR-146a was successfully designed and transfected into vascular cells in vitro . The following analysis showed a significant target upregulation for NOX4, KLF4, and TRAF6 ( P &lt;0.001) after circRNA transfection, also compared to antagomiR transfected cells. Functional analysis revealed improved migration and proliferation capacity in vascular endothelial cells ( P &lt;0.05). First results indicate that inhibition of the upregulation of endothelial miR-146a in mice results in significantly enhanced angiogenesis and revascularization ( P &lt;0.05) accompanied by a significantly reduced myocardial infarct size ( P &lt;0.01) and preserved cardiac function post-MI ( P &lt;0.01). In summary, we could identify miR-146a as a critical regulator of angiogenesis and vascular regeneration, and present a first strategy for angiogenic therapy of ischemic diseases. However, in vitro analysis showed a higher efficiency of circRNAs in the context of inhibiting miR-146a functions in vascular cells. CircRNAs are more stable and more efficient when transfected, compared to antagomiRs, and might be a better alternative and a realistic approach for future clinical studies.

The role of NLRP3 in regulating gingival epithelial cell responses evoked by Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has myriads of virulence factors among which leukotoxin provides A. actinomycetemcomitans with the advantage to thrive in the surrounding hostile environment and evade host immune defences. The NLRP3 inflammasome has been associated with periodontal disease development. However, our understanding of the involvement of caspase-1, caspase-4, and NLRP3 in the release of IL-1β and other inflammatory mediators from gingival epithelial cells during a A. actinomycetemcomitans infection is limited. The aim of this study was to investigate how the inflammasome-associated proteins caspase-1, caspase-4 and NLRP3 regulate the immune response of gingival epithelial cells during a A. actinomycetemcomitans infection. Human gingival epithelial cells (Ca9-22) deficient in NLRP3, caspase-1 or caspase-4 were created using CRISPR/Cas9. Gingival epithelial cells were stimulated with the A. actinomycetemcomitans low-leukotoxic strain NCTC9710 or the highly leukotoxic JP2 strain HK 165 for 6, 12 and 24 h. The results showed that the JP2 strain HK1651 induced higher IL-1β and IL-1RA release and mediated more epithelial cell death compared to the NCTC9710 strain. These findings were found to be capsase-1, caspase-4 and NLRP3-dependant. A targeted protein analysis of inflammation-related proteins showed that the expression of 37 proteins were identified as being significantly altered after HK1651 infection compared to unstimulated Cas9 and NLRP3-deficient cells. Of the 37 proteins, 23 of these inflammation-related proteins released by NLRP3-deficient cells differed significantly compared to Cas9 cells after infection. This suggests that NLRP3 has a broad effect on the inflammatory response in gingival epithelial cells.

Abstract 3331: Highly sensitive blood-based multi-cancer screening device with tiered specificity based on diagnostic workflow

Abstract Background. Multi-cancer blood-based tests may yield clinical benefit by improving compliance to guideline recommended screening with a more patient-friendly modality, and also by detection of early (stage I/II) tumors in cancer types that lack screening tests, yet early intervention can save lives. A single test with clinically meaningful performance which addresses both opportunities has yet to be developed. Methods. We evaluated a blood-based multi-modal device based on cfDNA epigenomic and targeted protein analysis that enables high performance for early-stage cancer detection in colon, lung, bladder, gastric, liver, ovarian, and pancreas cancers. Blood samples were obtained from multiple case-control cohorts of individuals with colorectal (N &amp;gt; 2,000), lung (N &amp;gt; 300), and other solid tumor cancers (bladder, gastric, liver, ovarian, pancreas (N &amp;gt; 300)) as well as individuals without cancer (N &amp;gt; 3,000). The assay is based on Guardant Shield™ blood-based CRC screening test and the bioinformatic pipeline is augmented with a multi-cancer screening caller for the detection of additional cancers. Sensitivity for CRC and lung cancer detection is calculated at 90% target specificity thresholds, due to availability of guideline recommended screening tests, colonoscopy and LDCT scan to adjudicate blood-based test results. Specificity for all other cancers were targeted at an overall specificity of 98%. The specificity thresholds for CRC, lung, and multi-cancer are selected to yield assay performance tailored for the cancer type and clinical diagnostic pathway. Results. In this study cohort, this integrated, single device, multi-cancer test yielded CRC sensitivity of 91% (stage I/II: 93%) and lung cancer sensitivity of 85% (stage I/II: 75%) at 90% specificity. The overall sensitivity in bladder, gastric, liver, ovarian, and pancreas cancers was 75% (stage I/II: 66%) at 98% overall specificity. Overall prevalence-adjusted sensitivity of this device in the above 7 cancers were 79% overall sensitivity (stage I/II: 78%). This blood-based test could detect 25% of the expected cancer diagnoses in 2022 according to SEER estimates. Conclusions. This highly-sensitive, integrated, blood-based cancer screening device yields performance on par with currently available screening tests for cancers with screening guidelines (CRC and lung) and clinically meaningful early-stage detection in cancer types without screening guidelines where early intervention can bring clinical benefit, highlighting the ability of this technology to yield clinically meaningful results for the detection of early stage cancer. The performance of this device is under investigation in prospective screening trials. Citation Format: Yupeng He, Anton Valouev, Liyang Xiong, William W. Young Greenwald, Victoria M. Raymond, Sven Duenwald, AmirAli Talasaz. Highly sensitive blood-based multi-cancer screening device with tiered specificity based on diagnostic workflow [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3331.

Measurement of protein in vivo turnover rate with metabolic labeling using LC-MS.

Understanding the protein dynamics of a drug target is important for pharmaceutical research because it provides insight into drug design, target engagement, pharmacodynamics and drug efficacy. Nonradioactive isotope labeling has been the method of choice for protein turnover measurement thanks to the advancement of high-resolution mass spectrometry. While the changes in proteome in cell cultures can be monitored precisely, as the culture media can be completely replaced with 2 H-, 15 N- or 13 C-labeled essential amino acids, quantifying rates of protein synthesis in vivo is more challenging. The amount of isotope tracer that can be administered into the body is relatively small compared with the existing protein, thus requiring more sensitive detection, and the precursor-product labeling relationship is more complicated to interpret. The purpose of this review is to provide an overview of the principles of in vivo protein turnover studies using deuterium water (2 H2 O) with an emphasis on targeted protein analysis by hybrid LC-MS assay platforms. The pursuit of these opportunities will facilitate drug discovery and research in preclinical and clinical stages.

Highly Accurate and Robust Absolute Quantification of Target Proteins in Formalin-Fixed Paraffin-Embedded (FFPE) Tissues by LC-MS.

Accurate, absolute liquid chromatography-mass spectrometry (LC-MS)-based quantification of target proteins in formalin-fixed paraffin-embedded (FFPE) tissues would greatly expand sample availability for pharmaceutical/clinical investigations but remains challenging owing to the following issues: (i) efficient/quantitative recovery of target signature peptides from FFPE tissues is essential but an optimal procedure for targeted, absolute quantification is lacking; (ii) most FFPE samples are long-term-stored; severe immunohistochemistry (IHC) signal losses of target proteins during storage were widely reported, while the effect of storage on LC-MS-based methods was unknown; and (iii) the proper strategy to prepare calibration/quality-control samples to ensure accurate targeted protein analysis in FFPE tissues remained elusive. Using targeted quantification of monoclonal antibody (mAb), antigen, and 40 tissue markers in FFPE tissues as a model system, we extensively investigate those issues and develope an LC-MS-based strategy enabling accurate and precise targeted protein quantification in FFPE samples. First, we demonstrated a surfactant cocktail-based procedure (f-SEPOD), providing high/reproducible recovery of target signature peptides from FFPE tissues. Second, a heat-accelerated degradation study within a roughly estimated 5 year storage period recapitulated the loss of protein IHC signals while LC-MS signals of all targets remained constant. This indicates that the storage of FFPE tissues mainly causes decreased immunoreactivity but unlikely chemical degradation of proteins, which strongly suggests that the storage of FFPE tissues does not cause significant quantitative bias for LC-MS-based methods. Third, while a conventional spike-and-extract approach for calibration caused substantial negative biases, a novel approach, using FFPE-treated calibration standards, enabled accurate and precise quantification. With the pipeline, we conducted the first-ever pharmacokinetics measurement of mAb and its target in FFPE tissues, where time courses by FFPE vs fresh tissues showed excellent correlation.

Molecular docking, ADMET profiling of gallic acid and its derivatives (N-alkyl gallamide) as an anti-breast cancer agent

Background In 2020, breast cancer has become the most common cancer in the world and in Indonesia. Searching for anticancer drugs using computational methods is considered more effective and selective than other methods. Gallic acid and its derivatives (esters and amides) are compounds that have biological activities such as anticancer effects. The purpose of this study was to analyse the molecular modelling and ADMET (Adsorption, Distribution, Metabolism, Excretion and Toxicity) profile of gallic acid derivative compounds (N-alkyl gallamides) as anticancer agents. Methods Target proteins were selected by analysis of protein-protein and drug-protein interactions. Molecular modelling was done by molecular docking. Predictive analysis of the ADMET profile of gallic acid and its derivatives (N-alkyl gallamide) was conducted using Marvin Sketch, Swissadme, protox II, and pkCSM pharmacokinetics. The selected target proteins were JUN, AKT1, CASP3, and CASP7. Results Compounds N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide were the three best gallic acid derivatives based on molecular modelling analysis of target proteins associated with breast cancer. The ADMET profile of the N-alkyl gallamide compound is predictable and shows a good profile as a candidate for anticancer drugs. Conclusion N-octyl gallamide, N-ters-butyl gallamide, and N-isoamil gallamide have potential as anti-breast cancer agents.

Predicting and Validating the Mechanism of Qingyi II Granules in the Treatment of Acute Pancreatitis by Network Pharmacology.

Network pharmacology, reverse molecular docking, and rat acute pancreatitis (AP) models were used to analyze the mechanism of protection by Qingyi II granules. The chemical components of 7 Chinese herbal medicines in Qingyi II granules were searched through the TCMSP (traditional Chinese medicine systems pharmacology database and analysis platform) database. The active ingredients were screened out in the OB (oral bioavailability) and DL (drug likeness) filters as a condition for inclusion. Then, the prediction analysis of potential targets was performed through databases. A GO (gene ontology) enrichment analysis of target proteins related to AP and KEGG (Kyoto Encyclopedia of Genes and Genomes) pathway annotation was performed using the DAVID (The Database for Annotation, Visualization, and Integrated Discovery) database. Finally, the "Herbal-Compound-Target" network was constructed using Cytoscape software. The active component structure and target name were uploaded to the Systems Dock database for reverse molecular docking. With octreotide as a positive control, Qingyi II decoction and Qingyi II granules were administered to AP rats at low, medium, and high doses. The pathological changes in the pancreas were observed using HE staining. The levels of Bcl-2, AMS, BAX, IL-2, and CASP3 in plasma were determined by an ELISA kit. Real-time PCR detected the expression of AKT1 and PIK3CA mRNA in the pancreas. The database predicted 94 active components of Qingyi II granules, 76 potential targets, and 64 signaling pathways. Twenty pathways were directly or indirectly associated with acute pancreatitis, including the TNF signaling pathway and the PI3K-AKT signaling pathway. In the reverse molecular docking experiment, the matching scores of the active components and the target were mainly between 6.0 and 7.0, with strong binding activity. Compared to the normal group, the plasma concentrations of BAX, IL-2, Bcl-2, AMS, and CASP3 in the model group were significantly increased (P < 0.05). Compared with the model group, the low-dose group of Qingyi II granules only significantly reduced IL-2 levels and had no effect on other indicators. The other groups could significantly reduce the levels of AMS, BAX, and CASP3 (P < 0.05). Compared with the model group, the octreotide group and Qingyi II granules high-dose group significantly increased the Bcl-2 level (P < 0.05), and there was no statistical difference in other drug-administered groups. Compared with the normal group, the expression of AKT1 and PIK3CA in the pancreas of the model group was significantly higher. Compared to the model group, the expression of PIK3CA was low in all drug-administered groups. In addition to the low-dose group, the other drug-administered groups significantly reduced the expression of AKT1. Qingyi can reduce the levels of AMS, BAX, IL-2, and CASP3 and increase the levels of Bcl-2. This mechanism may be related to the PI3K- AKT signaling pathway.

Supramolecular self-assembled AIE molecules are used in the search for target proteins in norcantharidin

Norcantharidin (NCTD), a demethylated derivative of cantharidin, is an anticancer active component in traditional Chinese medicine. At present, the main methods for finding its target proteins are pharmacological methods and biophysical screening, which cannot achieve the purpose of efficient and accurate screening. Here we established a new analytical method for specific fishing and assisted imaging for norcantharidin target proteins. For the AIE supramolecule probe, the benzophenone azide (BPA) fluorescent nanoparticles with strong AIE properties were encapsulated in biocompatible DSPE-PEG that covalently coupled with NCTD (named BPA@NCTD NPs). The target proteins of NCTD can be captured by BPA@NCTD NPs, and then be detected to investigate the potential signaling pathways. The screened differential proteins were analysed through the protein and signaling pathway database, and multiple signaling pathways were obtained and verified. The mechanism of norcantharidin in inhibiting the migration and invasion of A549 cells through the P53 signaling pathway was confirmed by Western blot experiments. Our research showed that AIE supramolecule probe BPA@NCTD NPs has the dual functions of specific screening of A549 cells target proteins and biological imaging, which not only offers a good anti-fluorescence quenching ability for the dynamic imaging process of NCTD, but also provides a novel and efficient specific method for efficient analysis of target proteins and signal pathways.

Proteomics-Based Identification of Dysregulated Proteins in Breast Cancer.

Immunohistochemistry (IHC) is still widely used as a morphology-based assay for in situ analysis of target proteins as specific tumor antigens. However, as a very heterogeneous collection of neoplastic diseases, breast cancer (BC) requires an accurate identification and characterization of larger panels of candidate biomarkers, beyond ER, PR, and HER2 proteins, for diagnosis and personalized treatment, without the limited availability of antibodies that are required to identify specific proteins. Top-down, middle-down, and bottom-up mass spectrometry (MS)-based proteomics approaches complement traditional histopathological tissue analysis to examine expression, modification, and interaction of hundreds to thousands of proteins simultaneously. In this review, we discuss the proteomics-based identification of dysregulated proteins in BC that are essential for the following issues: discovery and validation of new biomarkers by analysis of solid and liquid/non-invasive biopsies, cell lines, organoids and xenograft models; identification of panels of biomarkers for early detection and accurate discrimination between cancer, benign and normal tissues; identification of subtype-specific and stage-specific protein expression profiles in BC grading and measurement of disease progression; characterization of new subtypes of BC; characterization and quantitation of post-translational modifications (PTMs) and aberrant protein–protein interactions (PPI) involved in tumor development; characterization of the global remodeling of BC tissue homeostasis, diagnosis and prognostic information; and deciphering of molecular functions, biological processes and mechanisms through which the dysregulated proteins cause tumor initiation, invasion, and treatment resistance.

A Highly Sensitive Fluorescence and Screen-Printed Electrodes-Electrochemiluminescence Immunosensor for Ricin Detection Based on CdSe/ZnS QDs with Dual Signal.

A sensitive dual-readout immunosensor for fluorescence and electrochemiluminescence (ECL) detection of ricin was established, which was combined with a streptavidin–biotin signal amplification system. CdSe/ZnS quantum dots with fine fluorescence and ECL properties were used as the dual-signal function probes of the sandwich immunocomplex. Under the optimum experimental conditions, the dual signal intensity increased significantly with the rise in ricin concentration. The fluorescence intensity of the senor exhibited a good liner relationship toward the ricin concentrations with 0.1~100 ng/mL and the limit of detection (LOD) was 81.7 pg/mL; taking ECL as the detection signal, the sensor showed a linear relationship with the ricin concentrations ranging from 0.01 ng/mL to 100 ng/mL and the LOD was 5.5 pg/mL. The constructed sensor with high sensitivity had been successfully applied to the detection of ricin in complex matrices with satisfactory recoveries. The proposed immunosensor model can be extended to the analysis and detection of others target proteins.