Label-free target identification for natural products based on proteomics / 药学学报

Target identification and verification of natural products is an important and challenging work in the field of chemical biology. It is also an important job for researchers to apply chemical proteomics technology to biomedicine in order to identify target proteins of natural products. Target identification is critical to understanding its mechanisms and developing natural products as molecular probes and potential therapeutic drugs. Traditional approaches of small molecule target identification based on affinity have been shown to be successful, such as click-chemical probes, radioisotope labeling or photosensitized small-molecule probes. Nevertheless, these technologies require purified candidate target proteins, and modified small molecules with probes or linkers, such as adding agarose beads, biotin labels, fluorescent labeling or photo-affinity labeling. Many structure-activity relationship studies should be performed to ensure that the addition of small molecule labels undisturbed the original biological activity of the small molecules. Unfortunately, all these modifications are likely to alter their biological activity or binding specificity. To overcome the bottleneck of "target recognition", researchers have developed a series of new techniques for unmodified drug target identification. In this article, we reviewed the target identification techniques of natural product without structural modification in order to provide reference for the development of natural products.

Label-free target discovery technology of small molecule drug and its application in traditional Chinese medicines / 药学学报

The discovery of drug targets plays a crucial role in drug research. Accurate information of small molecule drug-protein interaction can be provided by label-free target discovery technology without any structural modification at the small molecule. So, the label-free drug target discovery technology had become the powerful tool to discover the targets of drugs. Due to the “multi-component and multi-target” characteristics of traditional Chinese medicines (TCMs), the research on its targets and mechanism had been restricted. Based on potential of the label-free target discovery technology in the research of TCMs, this paper summarized the label-free target discovery technology and its application in TCMs research. It will provide a reference for the discovery of targets of TCMs and a new view for promoting the modernization of TCMs.

Identification techniques of small molecule drug target proteins without chemical modification and its applications: a review / 生物工程学报

Identification of the target proteins of small molecule drugs is crucial for understanding the mechanisms of drug actions and its side effects. Conventional methods require chemical modification, which might alter the activities of the drugs. Various label-free techniques have been developed to identify drug target proteins without chemical modifications. This includes drug affinity responsive target stability (DARTS), stability of proteins from rates of oxidation (SPROX), cellular thermal shift assay (CETSA), thermal proteome profiling (TPP) and many others. Here we review the principles and applications of these label-free techniques, their advantages and limitations, as well as the most recent advances.

Regeneration and differentiation of decellularized adipose matrix in periosteal microenvironment / 中华医学美学美容杂志

Objective:To observe the lipogenic or osteogenic differentiation of decellularized adipose matrix (DAM) in the periosteal microenvironment.Methods:From June 2020 to December 2020, adipose tissue obtained by human liposuction was prepared as DAM at the National Institute of Plastic Surgery. Six male SD rats of 4 to 6 weeks were selected and implanted into the subperiosteum of the rat parietal bone according to the same initial volume. After 12 weeks, adipogenesis and osteogenic differentiation of DAM were observed by gross specimens, histological staining, and immunofluorescence staining. Label-free quantitative protein mass spectrometry was used for detection of osteogenic-associated proteins in DAM.Results:Vascularization around the DAM was evident. Adipogenesis and angiogenesis were observed in DAM by H&E and Masson staining, while OCN immunofluorescence staining confirmed osteogenic differentiation of DAM. The osteogenic differentiation related proteins were screened by mass spectrometry.Conclusions:In the microenvironment of periosteum, DAM mainly differentiates towards adipose tissue, but a few of them havs the potential to differentiate towards osteogenesis, which might be related to some of the osteogenesis-related proteins contained in DAM.

Label-free quantitative proteomic analysis of aqueous humor in patients with high myopia / 中华实验眼科杂志

Objective:To characterize proteomic profile in aqueous humor of patients with high myopia using quantitative proteomic analysis.Methods:Sixty-eight age-related cataract patients were divided into high myopic cataract group and simple cataract group according to that they had high myopia or not, with 34 patients (34 eyes) in each group.Aqueous humor samples (100 μl/patient) were collected from each patient using a 1 ml tuberculin syringe during cataract surgery at Tianjin Medical University Eye Hospital from January 2019 to August 2019.Sixteen samples from each group were selected for protein quantification and comparison by BCA method.The differentially expressed proteins between the two groups were analyzed using label-free liquid chromatography tandem mass spectrometry.The function and signal transduction pathways of differentially expressed proteins were further analyzed by Gene Ontology enrichment and Kyoto Encyclopedia of Genes and Genomes.Eighteen aqueous humor samples from each group were selected to verify the results of mass spectrometry by enzyme-linked immunosorbent assay (ELISA). This study protocol adhered to the Declaration of Helsinki.The use of human samples was approved by an Ethics Committee of Tianjin Medical University Eye Hospital (No.2020KY[L]-40). Written informed consent was obtained from each patient prior to surgery.Results:The mean protein mass concentration of aqueous humor sample in the high myopic cataract group was (1 134.91±104.78) ng/L, which was significantly higher than that in the simple cataract group (706.71±85.43) ng/L, showing statistically significant difference ( t=11.977, P<0.01). A total of 463 proteins were identified and 86 proteins were found to be differentially expressed, including 49 up-regulated proteins and 37 down-regulated proteins in the two groups.These differentially expressed proteins were mainly protein-binding activity modulator, extracellular matrix protein, carrier protein, intercellular signal molecule, protein modifying enzyme and so on, accounting for 32.70%, 14.50%, 9.10%, 9.10% and 7.30%, respectively.Bioinformatics analysis showed that 86 differentially expressed proteins were mainly related to biological processes such as complement activation and regulation, acute inflammatory response, and extracellular matrix tissue remodeling.Among them, 21 differentially expressed proteins were enriched in the complement and coagulation cascades pathways, 15 in the extracellular matrix-receptor interaction pathway, and 8 in the PI3K-Akt signaling pathway.ELISA results showed that the expression trends of three randomly selected differentially expressed proteins of the two groups were consistent with the results of label-free quantitative proteomic analysis. Conclusions:There are significant changes in proteomic profiles of aqueous humor between the high myopia cataract patients and simple cataract patients.High myopia is closely associated with inflammation and immune interactions, and remodeling of extracellular matrix.

TAB1——Potential Biomarker of Paclitaxel Resistance in Ovarian Cancer Based on Label-Free Quantitative Proteomics / 中国实验方剂学杂志

Objective:To explore the potential targets and related mechanism involved in the paclitaxel resistance to ovarian cancer. Method:Ovarian cancer A2780 cells and A2780 paclitaxel-resistant cells （A2780/T） were treated by 2， 4， 8， 16， 32， 64， 128， 256 μmol·L^-1 paclitaxel （PTX） for 24 h or 48 h respectively <italic>in vitro</italic>. The proliferation rate of A2780 cells and A2780/T cells treated with paclitaxel was determined by methyl thiazolyl tetrazolium （MTT） colorimetric method assay. A2780 and A2780/T cells were analyzed by LC-MS/MS Label-Free quantitative proteomics to identify and screen differentially expressed proteins in the two groups of cells. Gene ontology （GO） annotation and Kyoto encyclopedia of genes and genomes （KEGG） pathway analysis were used to determine the potential biomarkers of paclitaxel resistance in ovarian cancer. Conventionally cultured A2780 cells were used as a control group， and A2780/T cells were treated with 0， 1， 4 μmol·L^-1 PTX. Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot methods were used to detect and verify the mRNA and protein expression levels of potential target transforming growth factor-<italic>β</italic>-activated kinase 1 binding protein 1 （TAB1） and its downstream related molecules transforming growth factor-<italic>β</italic>-activated kinase （TAK1） and p38. Result:After PTX treatment for 24 h and 48 h， the cell viability of A2780 and A2780/T cells decreased. The inhibitory rate of PTX on A2780 cells was significantly higher than that of A2780/T cells. In A2780 cells， the IC₅₀ of PTX treatment for 48 h was 0.002 μmol·L^-1， while in A2780/T cells， the IC₅₀of PTX was greater than the maximum concentration of 128 μmol·L^-1， indicating that A2780/T cells were resistant to PTX compared with A2780 cells. 441 differentially expressed proteins and 421 special differentially expressed proteins between A2780/T and A2780 cells were screened by label-free quantitative proteomic analysis. GO function enrichment analysis showed that the binding proteins accounted for the majority （80%） among the differentially expressed proteins. According to the results of KEGG pathway analysis and expression site analysis， TAB1 might be a potential biomarker in paclitaxel-resistant ovarian cancer. Compared with A2780 cells， mRNA and protein expression levels of TAB1 in A2780/T cells were significantly reduced （<italic>P</italic><0.01）. mRNA expression of TAK1 and p38 that interacted with TAB1 were also significantly reduced （<italic>P</italic><0.05， <italic>P</italic><0.01）， while there was no significant change in protein expression. Conclusion:TAB1 may be a potential biomarker of paclitaxel resistance to ovarian cancer ， and its mechanism may be related to the TAB1/TAK1/p38 MAPK pathway.

Label-free quantitative peptidomics-based analysis on glycopeptides in deerhorn gelatin and deer-hide gelatin / 中国中药杂志

Nano-LC-MS/MS was used to analyze trypsin digested deer-horn gelatin( DCG) and deer-hide gelatin( DHG) samples.The glycopeptides in DCG and DHG were quantified by Label-free quantitative( LFQ) peptidomics,on the basis of which the glycopeptides with significant difference in DCG and DHG were determined. As a result,5 736 peptides were identified from DCG samples,including 213 galactosyl-hydroxylysine containing peptides( Gal-Hyl-peptides) and 102 glucosyl-galactosyl-hydroxylysine containing peptides( Glc-Gal-Hyl-peptides),while 6 836 peptides were identified from DHG samples,among which there were 250 Gal-Hyl-peptides and 98 Glc-Gal-Hyl-peptides. With over 3-fold peak area difference and highly significant intergroup difference( P < 0. 01) as the screening criteria,444 differential peptides were determined in DCG and DHG,including 16 Gal-Hyl-peptides and 5 Glc-Gal-Hyl-peptides. Then XIC peak shapes,standard deviation of peak area,and fold change were applied for further screening and 5 glycopeptides with significant differences in DCG and DHG were confirmed,which could serve as potential biomarkers for distinguishing DCG and DHG. The present study provided ideas and strategies for the in-depth investigation on the discrimination of DCG and DHG and is of good theoretical significance and application value for the further research on chemical constituents and quality control of gelatin derived Chinese medicinals.

Optimization of a cucurbit6uril-based real-time label-free method for analyzing the activity of ornithine decarboxylase / 生物工程学报

Ornithine decarboxylase (ODC) is a key enzyme in the biosynthetic pathway of polyamines and catalyzes the decarboxylation of ornithine to produce putrescine. Inhibition of ODC activity is a potential approach for the prevention and treatment of many diseases including cancer, as the expression levels and the activities of ODC in many abnormal cells and tumor cells are generally higher than those of normal cells. The discovery and evaluation of ODC inhibitors rely on the monitoring of the reaction processes catalyzed by ODC. There are several commonly used methods for analyzing the activity of ODC, such as measuring the yield of putrescine by high performance liquid chromatography, or quantifying the yield of isotope labelled carbon dioxide. However, the cumbersome operation and cost of these assays, as well as the difficulty to achieve high-throughput and real-time detection, hampered their applications. In this work, we optimized a real-time label-free method for analyzing the activity of ODC based on the macromolecule cucurbit[6]uril (CB6) and a fluorescent dye, DSMI (trans-4-[4-(dimethylamino) styryl]-1-methylpyridinium iodide). Finally, the optimized method was used to determine the activities of different ODC inhibitors with different inhibition mechanisms.

Label-free quantitative proteomic study of RUNX3 regulating Herceptin resistance in gastric cancer cells / 药学学报

Resistance of tumor cells is a complex biological process involving multiple mechanisms and factors, in which anti-apoptosis is the most important cause of drug resistance. Previous studies have shown that the DNA binding activity of Runt related transcription factor 3 (RUNX3) increased prominently in Herceptin resistant gastric cancer cells (NCI N87R) while the relevance of which to drug resistance has not yet been confirmed. In this study, we employed CRISPR/Cas9 to establish RUNX3 knock-out cell line (△RUNX3/NCI N87R) to investigate the functions of RUNX3 in Herceptin resistance of NCI N87R cells and its potential mechanisms. We investigated proteomics profiling of △RUNX3/NCI N87R cells based on label free quantitative proteomics. Differentially expressed proteins were screened out according to fold change and significance level between △RUNX3/NCI N87R and NCI N87R cells. Pathway enrichment analysis was done using GeneAnalytics database, and gene ontology analysis was conducted by DAVID Bioinformatics Resources database. Protein-protein interaction networks were constructed based on STRING database. The results showed that △RUNX3/NCI N87R cells increased the sensitivity to Herceptin. Proteomic data demonstrated that the expression of 577 genes changed significantly in △RUNX3/NCI N87R cells, among which 191 genes were up-regulated while 386 ones down-regulated comparing with NCI N87R cells. Pathway analysis showed that autophagy, cell cycle, apoptosis, mitochondrial fatty acid β oxidation, neurogenic locus notch homolog protein 1 (NOTCH1), mammalian target of rapamycin (mTOR), Hedgehog and DNA damage response pathways exhibited notable changes based on pathway enrichment ratio and significance level (P < 0.05). These results indicated that RUNX3 knock-out altered multiple signaling pathways of NCI N87R cells. Western blotting manifested that the expression of autophagy regulatory molecules autophagy-related protein (ATG) 13, 7 and BECN1 increased remarkably while cell cycle molecules serine/threonine-protein kinase Chk2 (CHEK2) and apoptosis regulator Bcl-2 (BCL2) decreased prominently in △RUNX3/NCI N87R cells. The p-AKT expression decreased significantly in △RUNX3/NCI N87R cells compared with NCI N87R cells (P < 0.01) and was suppressed by Herceptin. These results indicated that RUNX3 knock-out altered cell cycle, increased inhibition to p-AKT by Herceptin, promoted autophagy and induced cell apoptosis of NCI N87R cells. These results suggested that RUNX3 may be a potential therapeutic target for reversing or reducing Herceptin resistance in gastric cancer cells.

Effect of heavy metal lead on anticoagulant activity of leech based on label-free quantitative proteomics / 中国中药杂志

Whitmania pigra is the most widely distributed species of leeches in the market. In this study, the effect of heavy metal lead pollution on the anticoagulant activity of Wh. pigra was studied and the potential mechanism was explored. Pb(NO_3)_2 was used to contaminate the breeding soil which was then used to rear Wh. pigra for 50 days(lead-contaminated group, LC group), and meanwhile the blank control group(CG group) was set. Proteins were extracted from the obtained leech samples, and the differentially expressed proteins between LC and CG groups were analyzed by label-free proteomics technology. In this study, a total of 152 differentially expressed proteins were screened out, of which 93 proteins were up-regulated and 59 proteins were down-regulated in LC group. Bioinformatics analysis showed that the biological processes enriched with the differentially expressed proteins were mainly vesicle-mediated transport and transport positive regulation; the enriched cell components were mainly endocytosis vesicles and apical plasma membrane; the enriched molecular functions mainly included carbohydrate binding. The differentially expressed proteins were enriched in 76 KEGG pathways, which mainly involved metabolic pathways, biosynthesis of secondary metabolites, and bacterial invasion of epithelial cells. In this study, two differentially expressed proteins with Antistasin domain were presumed, which provides reference for further exploring the regulatory mechanism and signal transduction underlying the effect of lead pollution on the anticoagulant activity of leech.

Effects of Fe₃O₄ on the denitrification performance of Pseudomonas stutzeri / 生物工程学报

Biological denitrification is the most widely used technology for nitrate removal in wastewater treatment. Conventional denitrification requires long hydraulic retention time, and the nitrate removal efficiency in winter is low due to the low temperature. Therefore, it is expected to develop new approaches to enhance the denitrification process. In this paper, the effect of adding different concentrations of Fe₃O₄ nanoparticles on the denitrification catalyzed by Pseudomonas stutzeri was investigated. The maximum specific degradation rate of nitrate nitrogen improved from 18.0 h⁻¹ to 23.7 h⁻¹ when the concentration of Fe₃O₄ increased from 0 mg/L to 4 000 mg/L. Total proteins and intracellular iron content also increased along with increasing the concentration of Fe₃O₄. RT-qPCR and label-free proteomics analyses showed that the relative expression level of denitrifying genes napA, narJ, nirB, norR, nosZ of P. stutzeri increased by 55.7%, 24.9%, 24.5%, 36.5%, 120% upon addition of Fe₃O₄, and that of denitrifying reductase Nap, Nar, Nir, Nor, Nos increased by 85.0%, 147%, 16.5%, 47.1%, 95.9%, respectively. No significant difference was observed on the relative expression level of denitrifying genes and denitrifying reductases between the bacteria suspended and the bacteria adhered to Fe₃O₄. Interestingly, the relative expression level of electron transfer proteins of bacteria adhered to Fe₃O₄ was higher than that of the bacteria suspended. The results indicated that Fe₃O₄ promoted cell growth and metabolism through direct contact with bacteria, thereby improving the denitrification. These findings may provide theoretical support for the development of enhanced denitrification.

Serum biomarkers and bioinformatics of Alzheimer's disease based on LC-MS/MS proteomics / 药学学报

To identify potential serum proteins that might serve as biomarkers for Alzheimer's disease (AD), we performed comparative proteomic profiling of sera from AD and healthy control subjects using label-free LC-MS/MS. Our study identified 387 proteins, 61 of which showed significant changes in the serum of AD patients compared to healthy controls. Gene ontology (GO) enrichment analysis showed that some GO terms related to the pathogenesis of AD were significantly enriched in differentially expressed proteins, including cholesterol and lipid metabolism, inflammation, coagulation and hemostasis processes, and immune responses. Therefore, based on the above results and the consistency of protein content changes in the 8 comparison groups, 18 proteins were selected as candidate biomarkers. Protein-protein interaction results suggest that these 18 proteins can directly or indirectly interact with APP. Therefore, changes in the levels or functions of these proteins may affect Aβ metabolism and participate in the occurrence of AD, and have the potential to become AD blood biomarkers.

A label-free and real-time method for evaluating cytotoxicity of CD19 CAR-T cells / 医学研究生学报

ObjectiveUsing Chromium-51 release assay, lactate dehydrogenase release assayand other methods to detect the cytotoxicity of CD19 CAR-T cells is cumbersome, with low repeatability and poor stability. This study aims to establish a label-free and real-time method for detectingspecific cytotoxicity of CD19 CAR-T cells.MethodsIn order to establish target cell models for cytotoxic assay of CD19 CAR-T cells by using Real Time Cellular Analysis (RTCA) system,the adherent human breast cancer cells were infected with lentiviral vectors encoding CD19. CD19 expression on the transduced cells was detected by flow cytometry. The cellsexpressing CD19 stably werethen sorted by fluorescence activated cell sorting (FACS).With such cells as target cells, CD19 CAR-T cells and BCMA CAR-T cells as effector cells, RTCAsystem was used to evaluate the cytotoxicity of CAR-T cells against target cells.ResultsMDA-MB-231 and SKBR3cells with stable expression CD19were obtained in this study.The results of flow cytometry showed that positive expression rate of CD19 in MDA-MB-231/CD19 cells and SKBR3/CD19 monoclonal cells were 99.03% and 98.91%,respectively.RTCA results showed that with MDA-MB-231 and MDA-MB-231/CD19 cells as target cells,CD19 CAR-T cells showed significant cytotoxicity to MDA-MB-231/CD19 cellsat the effector-target ratio of 5∶1, 1∶1 and 1∶5,but not to MDA-MB-231 cells. With SKBR3 and SKBR3/CD19 cells as target cells, CD19 CAR-T cells showed significant cytotoxicity to SKBR3/CD19 cellsat the effector-target ratio of 5∶1and 1∶1. When the effector-target ratio was 1∶5, there was no obvious cytotoxicity.The data of MDA-MB-231/CD19 or SKBR3/CD19 as target cells and CD19 CAR-T as effector cells were analyzed separately, showing that when the number of target cells was the same, the cytotoxicity detected by RTCA increased as the number of CD19 CAR-T cells increased.The cytotoxic assays of CD19 CAR-T cells showed specificity and dose-response relationship of CD19 CAR-T cytotoxicity against the target cells.ConclusionThis study established a method for evaluating cytotoxicity of CD19 CAR-T cells that is real-time, label-free, simple and convenient.

Application of methods on target identification and validation of label-free natural products / 药学学报

Natural products areone source of new drugs, and their target identification is pivotal forelucidating the mechanism of action. Most methods of target discovery and validation utilize labeling natural products with probes. This is time-consuming and laborious, and often results in activity decrease or change of the natural products. Recent methods withoutchemicalmodificationhave become the main force in the target identification of natural products, including direct or indirect methods. Direct methods are mainly based on the principle of affinity and stability of protein, and indirect methods infer the drug target from the change in physiological responses or biochemical signatures. This review summarizes recent methods for target identification and validation of label-freenatural products, providing new ideas and strategies for future research in natural products.

Analysis of different proteins in primary open angle glaucoma based on aqueous proteome / 中华实验眼科杂志

Objective@#To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma (POAG), and to explore the biological markers and potential therapeutic targets associated with disease occurrence.@*Methods@#A retrospective case-control study was adopted.Ten patients with age-related cataract and 10 patients with POAG combined with cataract.were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017.In the process of surgery, 100 μl of aqueous humor were collected with 1 ml syringe and No.1 needle through the surgical access.Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry.The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P<0.05 and difference multiple>2.The biological big data was annotated by the function of GO and KEGG.The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No.2013KY[L]-10). Patients and their guardians all signed the informed consent.@*Results@#Ninty-seven differential proteins were detected in this proteomic analysis, including 48 up-regulated proteins and 49 down-regulated proteins.GO analysis significantly differs in protein function involved in many aspects, some different proteins including lipopolysaccharide-binding protein (LBP), cluster of Differentiation 163(CD163), C-reactive protein (CRP), annexin A1 (ANXA1) were involved in inflammation; redox-related proteins were glutathione S-transferase P (GSTP1), thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein (COMP), desmocollin-2 (DSC2) and laminin subunit beta-2 (LAMB2); procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1), solid protein (tenascin, TNC) are associated with fibrosis; some different proteins related to nerve growth were reelin (RELN), semaphorin-3F(SEMA3F), semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase (PKM), carb oxypeptidase N subunit 2 (CPN2) and so on.KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups.@*Conclusions@#The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased, which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways.GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG.

Analysis of different proteins in primary open angle glaucoma based on aqueous proteome / 中华实验眼科杂志

Objective To analyze the changes of protein expression in aqueous humor of patients with primary open angle glaucoma ( POAG ) , and to explore the biological markers and potential therapeutic targets associated with disease occurrence. Methods A retrospective case-control study was adopted. Ten patients with age-related cataract and 10 patients with POAG combined with cataract. were collected at the Tianjin Medical University Eye Hospital from October 2016 to December 2017. In the process of surgery,100 μl of aqueous humor were collected with 1 ml syringe and No. 1 needle through the surgical access. Those proteins extracted from aqueous humor were analyzed by quantitative proteomic mass spectrometry. The differential significance test was performed by Maxquant significances A. The differential proteins of the two groups were screened and determined with the conditions of P<0. 05 and difference multiple>2. The biological big data was annotated by the function of GO and KEGG. The study was approved by the Ethics Committee of the Tianjin Medical University Eye Hospital (No. 2013KY[L]-10). Patients and their guardians all signed the informed consent. Results Ninty-seven differential proteins were detected in this proteomic analysis,including 48 up-regulated proteins and 49 down-regulated proteins. GO analysis significantly differs in protein function involved in many aspects,some different proteins including lipopolysaccharide-binding protein (LBP),cluster of Differentiation 163(CD163),C-reactive protein (CRP),annexin A1 (ANXA1) were involved in inflammation;redox-related proteins were glutathione S-transferase P (GSTP1),thioredoxin (TXN), some different proteins related with cell adhesion movement were cartilage oligomeric matrix protein ( COMP ) , desmocollin-2 ( DSC2 ) and laminin subunit beta-2 ( LAMB2 );procollagen-lysine, 2-oxoglutarate 5-dioxygenase 1 (PLOD1),solid protein (tenascin,TNC) are associated with fibrosis;some different proteins related to nerve growth were reelin (RELN),semaphorin-3F(SEMA3F),semaphorin-4B(SEMA4B). Metabolism-related proteins included pyruvate kinase ( PKM ) , carb oxypeptidase N subunit 2 ( CPN2 ) and so on. KEGG analysis indicated that the expression of NrF2/ERK signaling pathway and TGF-β/NF-κB signaling pathway were different between the two groups. Conclusions The expression of GSTP1 and TXN in the aqueous humor of POAG is significantly decreased,which may regulate cell adhesion and activity and expression of extracellular matrix by regulating NrF2/ERK1/2 and TGF-β/NF-κB signaling pathways. GSTP1 and TXN may serve as a potential biomarker and therapeutic target of POAG.

Proteomic-based studies on hypolipidemic mechanism of total phenylpropanoid glycosides from ligustrum robustum (roxb. ) blume in hyperlipidemic hamsters / 中国药理学通报

Aim To explore the hypolipidemic mechanism of the total phenylpropanoid glycoside from Ligustrum robustum (Roxb. ) Blume (LRTPG) on hyperlipidemic hamsters using label-free quantitative proteomic technique. Methods The total protein was extracted from livers of model group and the group treated with LRTPG for label-free quantitative proteomics research. Results The proteomic data showed that a total of 2231 proteins were identified. And 549 proteins were found to be differentially expressed between model group and group treated with LRTPG. Among the 549 proteins, 93 proteins were up-regulated and 59 proteins were down-regulated, and 397 proteins had quantitative values only in model group or drug-administered group. Further, gene ontology (GO) analysis indicated that those differentially expressed proteins were primarily involved in an array of biological processes including metabolism, transport, oxidation-reduction, phosphorylation, signal transduction and lipid metabolism. KEGG pathway analysis revealed that these proteins were involved in several signal pathways including oxidative phosphorylation, non-alcoholic fatty liver dis-ease, PI3K-Akt, cAMP, and cGMP-PKG pathway. And some of these proteins were much related to the lipid metabolism, such as CD36, PK, HSS, GCK, ApoA I, Acly and FABP5. Conclusion The hypolipidemic effect of LRTPG may be related to CD36, PK, HSS, GCK, ApoA I, Acly and FABP5.

Plasma proteomics research in patients with diabetic retinopathy / 解放军医学杂志

Objective To screen potential DR-related plasma markers by profiling the plasma proteomics in patients with diabetic retinopathy (DR). Methods A total of 8 patients with moderate or higher non-proliferative diabetic retinopathy (NPDR) or proliferative diabetic retinopathy (PDR) enrolled in our hospital are included in the DR group and 6 patients without DR as NDR group. Plasma samples from these 14 patients were subjected to protein-labeled-free quantification and parallel liquid-phase tandem mass spectrometry (LC-MS/MS) in order to calculate the relative protein abundance of each protein detected in plasma. The results were statistically analyzed to find significant differences between the two groups of proteins. Further, bioinformatics analysis was performed, including gene ontology (GO) and Pathway annotation, GO and Pathway enrichment analysis, and protein interaction network analysis. Results A total of 41 differential proteins were identified with mean and median ratios ≥2.5(up/down). Among them, 26 were up-regulated and 15 were down-regulated. GO analysis showed that the binding protein in the differential protein was dominant, the single-organism process in the biological process accounted for the highest proportion, and the cell and cell part in the cell component accounted for the highest proportion. Pathway enrichment analysis indicated that the most significant up-regulated protein, tropomyosin 4 (TPM4), is associated with regulation of muscle contraction; while the most prominent down-regulated protein, platelet membrane glycoproteins V (GPV), plays an important role in extracellular matrix (ECM) receptor interactions. Protein interaction network analysis showed that there were potential interactions among glyceraldehyde-3-phosphate dehydrogenase (GAPDH), sulfhydryl oxidase 1 (QSOX1), immunoglobulin kappa locus (IGK@), TPM4, apolipoproteins C2 (APOC2), and immunoglobulin heavy variable 4-31(IGHV4-31); another potential interaction between pregnancy zone protein (PZP) and tissue inhibitor of metalloproteinase 2 (TIMP2). Conclusion Profiling the proteomics of DR vs NDR with label-free quantification technology successfully identified differentially expressed proteins in DR and NDR. These differential proteins are potential DR associated plasma markers and maybe a new target for early prevention and treatment of DR.

Comparative proteomics analysis of Leishmania infantum strains isolated from two epidemiological regions of visceral leishmaniasis in China / 中国血吸虫病防治杂志

Objective To analyze the protein abundance differences between two Leishmania infantum strains isolated from different epidemiological types of visceral leishmaniasis in China by comparative proteomics method. Methods Tryptic digests of total proteins were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free quantitative differential expression analysis. The MS data were analyzed with MaxQuant software (ver 1.3.0.5) against data base. Results This study resulted in the identification of 4 274 proteins across two strains (JIASHI-5 and SC6) . Of these, 1 219 differentially expressed proteins (ratio ＞ 2.0 or ＜ 0.5, P < 0.05) were identified. Considering the proteins differentially or uniquely expressed in the strains, 550 proteins were only found in the JIASHI-5 strain, and 174 proteins were only found in the SC6 strain. Totally 495 differentially proteins were expressed in the two groups, among which 328 proteins were down-regulated and 167 proteins were up-regulated in SC6 strain. Some of the identified differentially expressed proteins were demonstrated and they involved in energy metabolism, stress response, prolonging the lifetime of the infected host cell and survival and proliferation in virulent strains. Conclusion This study reveals a group of differentially expressed proteins and the related biologic function that may lay the foundation for screening and identification of the key Leishmania molecules relative to pathogenicity.

A multiscale Mueller polarimetry module for a stereo zoom microscope

Mueller polarimetry is a quantitative polarized light imaging modality that is capable of label-free visualization of tissue pathology, does not require extensive sample preparation, and is suitable for wide-field tissue analysis. It holds promise for selected applications in biomedicine, but polarimetry systems are often constrained by limited end-user accessibility and/or long-imaging times. In order to address these needs, we designed a multiscale-polarimetry module that easily couples to a commercially available stereo zoom microscope. This paper describes the module design and provides initial polarimetry imaging results from a murine preclinical breast cancer model and human breast cancer samples. The resultant polarimetry module has variable resolution and field of view, is low-cost, and is simple to switch in or out of a commercial microscope. The module can reduce long imaging times by adopting the main imaging approach used in pathology: scanning at low resolution to identify regions of interest, then at high resolution to inspect the regions in detail. Preliminary results show how the system can aid in region of interest identification for pathology, but also highlight that more work is needed to understand how tissue structures of pathological interest appear in Mueller polarimetry images across varying spatial zoom scales.