Quantitative Analysis Of Protein Expression Research Articles

Exploring Intra-Articular Administration of Monoclonal Antibodies as a Novel Approach to Osteoarthritis Treatment: A Systematic Review.

Biological drugs, including monoclonal antibodies, represent a revolutionary strategy in all fields of medicine, offering promising results even in the treatment of osteoarthritis (OA). However, their safety and efficacy have not been fully validated, highlighting the need for in-depth studies. Therefore, we provided a comprehensive systematic review of the intra-articular use of monoclonal antibodies for the treatment of OA in animal models, reflecting ongoing efforts to advance therapeutic strategies and improve patient outcomes. A systematic literature search was conducted in December 2023 following the PRISMA guidelines, using the Web of Science, Google Scholar, and PUBMED databases. Out of a total of 456, 10 articles were included in the study analyzing intra-articular antibodies and focusing on various targets, including vascular endothelial growth factor (VEGF), nerve growth factor (NGF), interleukin 4-10 (IL4-10), tumor necrosis factor α (TNF-α), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5), and matrix metalloproteinase 13 (MMP-13). Most studies administered the antibodies weekly, ranging from 1 to 10 injections. Animal models varied, with mean follow-up periods of 8.9 ± 4.1 weeks. The methods of assessing outcomes, including pain and morpho-functional changes, varied. Some studies reported only morphological and immunohistochemical data, while others included a quantitative analysis of protein expression. In conclusion, monoclonal antibodies represent a promising avenue in the treatment of OA, offering targeted approaches to modulate disease pathways. Further research and clinical trials are needed to validate their safety and efficacy, with the potential to revolutionize the management of OA and reduce reliance on prosthetic interventions.

Ubiquitous chromatin opening element enhance Darbepoetin alfa expression in CHO DG44 cell line

Despite extensive efforts in biotherapeutics production, there is a pressing need to enhance the biomanufacturing process to meet the growing demand for recombinant products. Ubiquitous Chromatin Opening Element (UCOE), chromatin-modifying elements derived from CpG islands, are crucial in regulating gene expression. Therefore, to explore the UCOE effect on gene expression, we cloned codon-optimized Darbepoetin alfa (DPO) sequence into the pOptiVEC then the Darbepoetin alfa-IRES-DHFR fragment was digested, and cloned into the UCOE vector. Both pOptiVEC-DPO and UCOE-DPO-IRES-DHFR cassettes were linearized and transfected into the two cell pools of CHO DG44 cells. The transfected cells were adapted with CD OptiCHO™ medium after 20 days. Recombinant DPO expression was evaluated by qRT-PCR technique. Qualitative and quantitative protein expression analysis was also carried out by western blotting and ELISA assays. Our findings showed a boosted DPO expression rate at both mRNA and protein levels in the UCOE-DPO-IRES-DHFR pool compared to pOptiVEC-DPO. These data suggest that UCOE and codon optimization result in high and stable gene expression in the CHO DG44 cell line.

Flow cytometry-based quantitative analysis of cellular protein expression in apoptosis subpopulations: A protocol

Flow cytometry techniques utilizing dual staining with annexin V and propidium iodide (PI) provide a robust method for quantitatively analyzing apoptosis induction. Annexin V binds phosphatidylserine exposed on the outer leaflet of the plasma membrane during early apoptosis, while PI permeates late apoptotic/necrotic cells. Simultaneous staining allows differentiation of viable, early apoptotic, and late apoptotic/necrotic populations. This approach can be enhanced by using fluorochrome-conjugated antibodies to stain specific proteins, enabling the simultaneous tracking of protein expression changes in defined cell subpopulations during apoptosis. This multiparametric approach provides key insights into signaling regulation and the mechanisms underlying the apoptotic response to cytotoxic treatments. Here we present a protocol that combines annexin V-FITC/PI staining with APC-conjugated antibody labeling in MDA-MB-231 breast cancer cells treated with doxorubicin. This protocol enables both the quantitative assessment of apoptosis induction and the tracking of decreased CD44 expression from viable to apoptotic cells. This protocol also provides guidelines for appropriate filter selection, compensation controls, gating strategies, and troubleshooting. This robust protocol holds significant potential for elucidating signaling networks involved in apoptosis and therapeutic resistance across various cellular models.

HIV-derived Proteins induce Hypertension, Sympatho-activation, and neuroinflammation via T cell-dependent mechanisms

Combination antiretroviral therapy (cART) has markedly increased life expectancy in people living with HIV (PLWH) and induced a switch in the cause of death from opportunistic diseases to cardiovascular disease (CVD). CVD is currently the leading cause of death in PLWH and hypertension, the primary risk factor for CVD, presents in more than 65% of them. However, the mechanisms whereby hypertension develops in PLWH remain unknown. In PLWH on cART, T lymphocytes (T cells) remain a viral reservoir for HIV and continue to secrete viral proteins. Viral proteins remain in circulation and are expressed in the central nervous system. Herein, we tested the hypothesis that expression of HIV-derived proteins increases blood pressure (BP), induces sympatho-activation and inflammation of the central nervous system. We utilized the Tg26 mouse model which expresses 7/9 viral proteins. Quantitative PCR analysis of viral protein expression showed that Tat, gp120, Nef, Vif, and Vpr are primarily expressed in spleen, lung, and brain tissues in Tg26 mice. Morphometric analysis revealed slight but significant increases and decreases in lean mass and fat weights respectively in Tg26. Flow cytometry analysis showed no reduction in CD4+ T cells in Tg26 mice indicating that this mouse model does not progress to AIDS and reproduce the conditions of virally suppressed PLWH. Blood pressure (BP) measurement via telemetry revealed a hypertensive phenotype in male and female Tg26 mice. Hypertension was associated with a larger drop in BP in response to ganglionic blockade but no alterations in heart rate responses to muscarinic and β-adrenergic receptor blockade in Tg26 mice, indicating that viral proteins expression increases neurogenic control of BP but did not alter cardiac autonomic control. β-adrenergic receptor blockade did not reduce BP in Tg26 mice. Tg26 mice also display increased aortic vasoconstriction to phenylephrine. To test the contribution of T cells in BP regulation and sympatho-activation, mice were subjected to a CTLA-4 inhibitor, abatacept, which inhibits T cell activation. Abatacept treatment significantly reduced BP, sympatho-activation, and phenylephrine constriction in Tg26 mice with no effects on wild-type mice. To determine whether the increase in sympatho-activation involves central inflammation, inflammatory cytokines and T cell markers levels were measured in the brain. Levels of pro-inflammatory cytokines including IL-1α, TNFα, IL-6, and CCL2 were increased in the brain of Tg26 mice as well as levels of the T-cell marker GATA3. Altogether, our data shows that the expression of HIV-derived proteins increases BP, sympatho-activation, and vascular adrenergic contractility via T cell-dependent mechanisms likely through central mechanisms involving neuro-inflammation. R01HL147639-01A1, R01HL15526501 and 19EIA34760167 (E.J.B). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Gastric Cancer Mesenchymal Stem Cells Trigger Endothelial Cell Functional Changes to Promote Cancer Progression.

Our previous studies have highlighted the pivotal role of gastric cancer mesenchymal stem cells (GCMSCs) in tumor initiation, progression, and metastasis. In parallel, it is well-documented that endothelial cells (ECs) undergo functional alterations in response to challenging tumor microenvironment. This study aims to elucidate whether functional changes in ECs might be induced by GCMSCs and thus influence cancer progression. Cell proliferation was assessed through CCK-8 and colony formation assays, while cell migration and invasion capabilities were evaluated by wound-healing and Transwell assays. Immunohistochemistry was employed to examine protein distribution and expression levels. Additionally, quantitative analysis of protein and mRNA expression was carried out through Western blotting and qRT-PCR respectively, with gene knockdown achieved using siRNA. Our findings revealed that GCMSCs effectively stimulate cell proliferation, migration, and angiogenesis of human umbilical vein endothelial cells (HUVECs), both in vitro and in vivo. GCMSCs promote the migration and invasion of gastric cancer cells by inducing the expression of Slit2 in HUVECs. Notably, the inhibition of phosphorylated AKT partially mitigates the aforementioned effects. In conclusion, GCMSCs may exert regulatory control over Slit2 expression in ECs via the AKT signaling pathway, thereby inducing functional changes in ECs that promote tumor progression.

Abstract 94: Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling

Abstract The STING (Stimulator of Interferon Genes) pathway is a pivotal player in the innate immune response, responsible for detecting cytosolic accumulation of endogenous DNA and initiating a cascade of events leading to the production of pro-inflammatory type I interferons, which promote an antitumor environment through activation of T cells, dendritic cells, and natural killer cells. Therefore, STING agonists are currently being explored as a potential therapeutic for cancer treatment, often in combination with immune checkpoint inhibitors. While the STING pathway can be stimulated in multiple cell types, activation in antigen presenting cells, such as dendritic cells (DC), is crucial for antitumor activity in the tumor microenvironment (TME). Additionally, the type 1 interferon family consists of multiple subtypes, including IFNb and IFNa, which can produce differential biological responses. Although the STING pathway is known to induce expression of multiple cytokines, combined spatial characterization of STING and type I interferons in the TME has not been performed. To characterize STING-interferon expression in the TME, we use the Integrated MultiOmyx-RNAscope platform. MultiOmyxTM (NeoGenomics Laboratories, Inc) is a proprietary multiplex immunofluorescence (mIF) platform for the visualization and characterization of up to 60 protein biomarkers in a single formalin-fixed paraffin-embedded (FFPE) section and offers high-resolution spatial and quantitative analysis of protein expression in tissue samples. RNAscopeTM (Bio-Techne) Multiplex is a highly sensitive fluorescent in-situ hybridization (ISH) assay that can detect up to 3 RNA markers in a single FFPE section. The Integrated MultiOmyx-RNAscope assay allows for simultaneous detection of both protein and RNA markers in a single sample. Herein we report the design and use of a novel panel of commercially-available antibodies and ISH probes broad enough to characterize various immune subpopulations and cytokine expressing cells, including DCs and interferons, in the TME of a variety of tumor indications including melanoma and head and neck squamous cell carcinoma. Quantification and analysis will be performed using NeoLYTXTM, the proprietary MultiOmyx Analytics pipeline, to examine the spatial distribution and expression levels of key STING pathway induced cytokines to elucidate the dynamic communication of signaling molecules and their localization within specific cell populations. Understanding of the variety and phenotype of STING/cytokine expressing cells in the TME is crucial to define the populations being targeted by therapies for cancer treatment. Citation Format: Sandra Lam, Courtney Todorov, Jiong Fei, Harry Nunns, Eric Leones, Marianne Thio, Flora Sahafi, Erinn Parnell, Qingyan Au. Interrogation of STING induced cytokines in the tumor microenvironment using an Integrated MultiOmyx-RNAscope panel for spatial and quantitative profiling [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 94.

Abstract C054: PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition

Abstract BACKGROUND: Ewing’s sarcoma (EWS) is a highly aggressive bone and soft tissue tumor with poor prognosis and an urgent need for novel therapies. We have identified that silencing of mono-ADP-ribosyltransferase PARP16 enhanced sensitivity of EWS cell lines to PARP1 inhibition. In this study, we explored the molecular mechanism of EWS vulnerability towards PARP16 targeting, alone and in conjunction with PARP1 inhibition, which may lead to development of PARP16-selective or dual targeting PARP1/16 inhibitors as new drugs for EWS. METHODS: The effect of PARP16 on cell viability was analyzed using RNA interference with various siRNAs and doxycycline-inducible PARP16 shRNAs, as well as CRISPR/Cas9-mediated genomic deletion. The changes on total protein expression upon silencing of PARP16 with or without PARP1 inhibition was determined by mass spectrometry (MS). Protein interaction partners and downstream effectors of PARP16 were determined by Co-IP/MS and ADP-ribosylation proteomics, respectively. RESULTS: Transient knockdown of PARP16 significantly reduced cell survival in EWS cell lines, particularly in combination with olaparib. Quantitative protein expression analysis by MS suggested doxycycline-induced shRNA-mediated PARP16 knockdown significantly reduced MYC protein expression in addition to PARP16 itself. Depletion of PARP16 in Cas9-expressing SK-N-MC EWS cells using different lentiviral sgRNAs further confirmed that targeting of PARP16 reduces cell survival and MYC expression, particularly in combination with PARP1 inhibition. Co-IP/MS using GFP-Trap agarose beads in mGFP/mGFP-PARP16 expressing cells suggested interaction of PARP16 with proteins that regulate post-translational modification of MYC in EWS cells. Further validation is ongoing. CONCLUSION: Our data suggest that targeting PARP16 confers vulnerability to EWS cells either alone or in conjunction with PARP1 inhibition and that it is involved in regulation of MYC. Identification of novel PARP16 substrates and interaction partners is expected to produce molecular markers that enable the development of novel PARP16 inhibitors. Citation Format: Ou Deng, Xueli Li, Vinayak C Palve, Bin Fang, Damon R Reed, Uwe Rix. PARP16 modulates MYC expression and susceptibility of Ewing’s Sarcoma cells to PARP1 inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C054.

Prognostic Implication of YY1 and CP2c Expression in Patients with Primary Breast Cancer.

Yin Yang 1 (YY1) is a transcription factor that regulates epigenetic pathways and protein modifications. CP2c is a transcription factor that functions as an oncogene to regulate cell proliferation. YY1 is known to interact with CP2c to suppress CP2c's transcriptional activity. This study aimed to investigate YY1 and CP2c expression in breast cancer and prognostic implications. In this study, YY1 and CP2c expression was evaluated using immunohistochemical staining, Western blot and RT-PCR assays. Of 491 patients with primary breast cancer, 138 patients showed YY1 overexpression. Luminal subtype and early stage were associated with overexpression (p < 0.001). After a median follow-up of 68 months, YY1 overexpression was found to be associated with a better prognosis (disease-free survival rates of 92.0% vs. 79.2%, p = 0.014). In Cox proportional hazards model, YY1 overexpression functioned as an independent prognostic factor after adjustment of hormone receptor/HER2 status and tumor size (hazard ratio of 0.50, 95% CI 0.26-0.98, p = 0.042). Quantitative analysis of YY1 and CP2c protein expression in tumors revealed a negative correlation between them. In conclusion, YY1 overexpression is a favorable prognostic biomarker in patients with breast cancer, and it has a negative correlation with CP2c at the protein level.

Single-cell HER2 quantification via instant signal amplification in microdroplets

Accurate and ultrasensitive evaluation of human epidermal growth factor receptor 2 (HER2) protein is key to early diagnosis and subtype differentiation of breast cancer. Single-cell analyses to reduce ineffective targeted therapies due to breast cancer heterogeneity and improve patient survival remain challenging. Herein, we reported a novel droplet microfluidic combined with an instant cation exchange signal amplification strategy for quantitative analysis of HER2 protein expression on single cells. In the 160 μm droplets produced by a tapered capillary bundle, abundant Immuno-CdS labeled on HER2-positive cells were replaced by Ag + to obtain Cd2+ that stimulated Rhod-5N fluorescence. This uniformly distributed and instantaneous fluorescence amplification strategy in droplets improves sensitivity and reduces signal fluctuation. Using HER2 modified PS microsphere to simulate single cells, we obtained a linear fitting of HER2-modified concentration and fluorescence intensity in microdroplets with the limit detection of 11.372 pg mL−1. Moreover, the relative standard deviation (RSD) was 4.2-fold lower than the traditional immunofluorescence technique (2.89% vs 12.21%). The HER2 protein on SK-BR-3 cells encapsulated in droplets was subsequently quantified, ranging from 9862.954 pg mL−1 and 205.26 pg mL−1, equivalent to 9.795 × 106 and 2.038 × 105 protein molecules. This detection system provides a universal platform for single-cell sensitive quantitative analysis and contributes to the evaluation of HER2-positive tumors.

Two-Dimensional Gel Electrophoresis and 2D-DIGE.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) continues to be one of the most versatile and widely used techniques to study the proteome of a biological system, particularly in the separation of intact proteins. A modified version of 2D-PAGE, two-dimensional difference gel electrophoresis (2D-DIGE), which uses differential labeling of protein samples with up to three fluorescent tags, offers greater sensitivity and reproducibility over conventional 2D-PAGE gels for differential quantitative analysis of protein expression between experimental groups. Both these methods have distinct advantages in the separation and identification of thousands of individual protein species including protein isoforms and post-translational modifications. This chapter discusses the principles of 2D-PAGE and 2D-DIGE including limitations to the methods. 2D-PAGE and 2D-DIGE continue to be popular methods in bioprocessing-related research, particularly on recombinant Chinese hamster ovary cells, which are also discussed in this chapter.

Tandem mass tag labeled quantitative proteomic analysis of differential protein expression on total alkaloid of Aconitum flavum Hand.-Mazz. against melophagus ovinus.

Melophagus ovinus disease is a common ectoparasitosis, which can lead to a decrease in animal production performance, product quality, and even death. Aconitum flavum Hand.-Mazz. has many pharmacological activities including insecticidal, heat-clearing, analgesic, and dehumidifying. However, there are few researches focused on the effects and related mechanism of Aconitum flavum Hand.-Mazz. in killing Melophagus ovinus. In this study, 11 alkaloids of Aconitum flavum Hand.-Mazz. were detected, and its total alkaloid activity was determined. The results showed when the total alkaloid concentration was 64 mg/ml and the treatment time was 16 h, the killing rate of Melophagus ovinus reached 100%. Through the observation of the differences in the surface of Melophagus ovinus in each experimental group, it was found that the morphology of the posterior end of the female Melophagus ovinus in the alkaloid treatment group was significantly different from that of the blank and positive control groups, and most of the epidermal tissue was obsessive and missing. Moreover, the enzyme activity determination results of 64 mg/ml group were significantly different when compared with the normal control group, while there was no significant difference in other groups. Then, the Melophagus ovinus gene library was established by the unreferenced genome transcriptome sequencing, the proteomic comparison was performed using tandem mass tag labeled protein detection technology, and finally, the samples were quantitatively analyzed by liquid chromatography-mass spectrometry tandem and bioinformatics methods. Based on the above experimental results, it was speculated that Aconitum flavum Hand.-Mazz. total alkaloids may cause the imbalance of protein disulfide isomerase expressions by affecting the regulation of Hsp40 cellular protein homeostasis and the oxidation of protein disulfide isomerase and related proteins. This would affect the selective recognition of signal sequence, the targeted transport of Sec 61, and the correct folding of the three-dimensional structure of amino acid chain, weakening the clearance of amino acid chains that cannot be correctly folded and eventually resulting in the killing of Melophagus ovinus. This study preliminarily revealed the mechanism of Aconitum flavum Hand.-Mazz. total alkaloids against Melophagus ovinus and provided a theoretical basis for the screening of Melophagus ovinus action targets and the development of new veterinary drugs.

Study on the Mechanism of Autophagy in Rats With Radiation Induced Heart Damage at Different Time Points

It is an important mode to combine local radiotherapy with systemic targeted therapy against tumor. MTOR is an ideal choice for tumor targeted therapy, which has been identified in the cardiovascular disease. Endoplasmic reticulum stress (ERS) was involved in the process of radiation induced heart damage (RIHD) in previous study, which could trigger autophagy by inhibiting mTOR. Here we established an animal experimental model to observe the performance of RIHD at different time points, and explored the mechanism of autophagy in a rat model of RIHD.56 adult male SD rats were randomly divided into four groups (n = 14): Control group (A), Radiation group (B), Rapamycin+radiation group (C), 3-MA + radiation Group (D). Animal echocardiography was performed on the 14th and 28th days after irradiation to record left ventricular function. Levels of IL-4, TNF-α, TGF-β1 and Beclin-1 and LC3 related to autophagy were assessed by quantitative analysis of protein expression.1. On the 14th day after irradiation, radiation exposure caused a decrease in LVFS, and the decrease was recovered in D group but was not changed in C group. On the 28th day after irradiation, radiation exposure caused an increase in LVEF and LVFS, accompanied by LVAW; s thickening, and except for the decrease of LVAW; s in C group there was no change of the other parameters in C and D group compared with B group. 2. On the 14th day after the heart of the rat is irradiated, edema of myocardial cells and apoptosis increased, myocardial interstitial and perivascular inflammation cells infiltrated, and myocardial interstitial collagen deposition increased. The above-mentioned injuries were aggravated in C group and were alleviated in D group compared with B group. With time extending to 28 days after irradiation, the above-mentioned histopathological changes were progressively exacerbated. Different from changes of the 14th day, these damages were alleviated in C group and were aggravated in D group on the 28th day. 3. On the 14th day, irradiation increased the levels of autophagy-related proteins Beclin-1 and LC3, inflammatory factors IL-4, TNF-α and TGF-β1. Enhancing autophagy furtherly increased the levels of inflammatory factors IL-4, TNF-α and TGF-β1. On the 28th day, irradiation reduced the expression of Beclin-1 and LC3 but still increased the levels of IL-4, TNF-α and TGF-β1. Enhancing autophagy decreased the levels of inflammatory factors IL-4, TNF-α and TGF-β1.RIHD were progressively exacerbated with the extension of observation time. Autophagy may play a bidirectional regulatory role in radiation- induced cardiac injury, and the mechanism of action is different at different time points. On the 14th day after irradiation, activated autophagy exacerbated acute radiation heart injury to a certain extent. As time prolonged to 28 days after irradiation, down-regulated autophagy exacerbated acute radiation heart injury to a certain extent.

Aberrant expression of PI3K/AKT signaling is involved in apoptosis resistance of hepatocellular carcinoma.

Phosphatidylinositol 3-kinase (PI3K)/AKT signaling is a crucial pathway for cell survival and proliferation, which are regulated by several growth factors and activated receptors. Upregulated PI3K/AKT signaling molecules were reported in several cancers and they are associated with altered cellular functions, leading to oncogenesis. Here, we have examined the implications of elevated PI3K/AKT expression in the apoptosis resistance of human hepatocellular carcinoma (HCC) Huh7 cells. We showed that PI3K/AKT signaling is significantly upregulated in Huh7 cells by quantitative polymerase chain reaction and protein expression analysis. Also, perversely upregulated PI3K/AKT signaling Huh7 cells are highly resistant to treatment with chemotherapy drugs (docetaxel and sorafenib) and acquired apoptosis resistance through downregulation of tumor suppressor protein PTEN (phosphatase and tensin homolog deleted on chromosome ten). Hence, we have investigated the effect of PTEN overexpression on apoptosis induction in Huh7 cells. We showed that PTEN overexpressed Huh7 cells became more sensitive toward the aforesaid drugs and induced apoptotic cell death due to intracellular reactive oxygen species (ROS) generation. Concurrently, the overexpression of PTEN leads to the activation of mitochondria facilitated intrinsic apoptosis, evidenced by upregulated cytochrome C, caspase 3, and caspase 9. Collectively, our data suggest that the aberrant expression of PI3K/AKT signaling contributes to apoptosis resistance in HCC.

Expression, clinical significance and correlation of RUNX3 and HER2 in colorectal cancer.

The incidence of colorectal cancer is high and on the rise. The genetic and protein expressions of RUNT-associated transcription factor 3 (RUNX3) and human epidermal growth factor receptor 2 (HER2) in colorectal cancer (CRC) and adjacent normal tissues were detected to preliminarily explore their correlation and clinical significance. CRC specimens excised during general surgery were selected for localization and quantitative analysis of protein and gene expression by SP (Streptavidin-peroxidase conjugated method) immunohistochemical staining, reverse transcription-polymerase chain reaction (RT-qPCR) and western blot. Combined with the patients' data, the relationship between the expression of the two genes and tumor characteristics was analyzed. Log-rank test was used to analyze the correlation between the two proteins and survival prognosis of CRC patients. The expression of RUNX3 in RKO and HCT-116 was knocked down, and the relative expression of HER2 in the two cell lines was detected. Immunohistochemical, RT-qPCR and Western blot results showed that the positive expression rate of RUNX3 in CRC was lower than in the normal group, and HER2 in CRC was higher than in the normal group. The positive expression of the two proteins correlated with the pT and pN stages of CRC. A significant negative correlation between the expression of the two genes in CRC. Follow-up results showed that the number of Runx3-positive patients was higher than negative ones, while HER2 positive were fewer than negative ones. In vitro experiments showed that RUNX3 protein and gene expression decreased, HER2 protein and gene expression increased in RUNX3 knockdown RKO and HCT-116 cells, respectively (P<0.05). The expression of RUNX3 and HER2 in CRC is related to the occurrence and development of CRC, and the two genes have a negative regulating effect.

An AIE‐based enzyme‐activatable fluorescence indicator for Western blot assay: Quantitative expression of proteins with reproducible stable signal and wide linear range

AbstractWestern blot is a commonly used experimental method to analyze the protein expression. However, the most commonly used chromogenic indicator based on chemiluminescence is limited by narrow linear range and unstable quantitative reproducibility, whereas the recently developed fluorescent indicator suffers from poor detection limit. Herein, we report an enzyme‐activatable fluorescence indicator to quantify proteins with reproducible stable signal and wide linear range, through introducing the hydrophilic alkaline phosphatase (ALP)‐triggered phosphoric acid moiety into our established aggregation‐induced emission (AIE) building block of quinoline‐malononitrile (QM). In this strategy, the indicator DQM‐ALP disperses well in both aqueous and lipid environments to exhibit initial “off” fluorescence, but when exposing to the ALP‐coupled secondary antibody on the PVDF membrane, the specific enzymatic turnover would liberate hydrophobic AIE luminogen (AIEgen) QM‐OH to emit strong luminescence, thereby achieving an ideal “off‐on” state for sensitively imaging proteins with high signal‐to‐noise (S/N) ratio. Moreover, benefiting from the excellent signal stability of AIE fluorophore, DQM‐ALP indicator exhibits superior quantitative analysis of protein expression with high reproducibility. Upon taking advantage of the AIEgens to reduce high concentration‐induced luminance quenching, the linear quantification range is extremely expanded. In contrast with the traditional chemiluminescent indicator, the AIE‐based enzyme‐activatable indicator DQM‐ALP not only greatly improves the signal stability for quantitative reproducibility, but also expands the linear quantification range, and further provides a practical alternative reagent for fluorescence Western blot assay.

Qualitative and quantitative analysis of protein expression in the caput and cauda regions of the rat epididymis

Background: The aim of this study was to identify the differentially expressed proteins in the sperm isolated from the caput and the cauda region of the rat epididymis. This is the first study on the quantitative nongel-based proteomics to have identified differentially expressed proteins in the cauda epididymal sperm. Material and Methods: This was achieved by isolation of sperm from the caput and the cauda of the rat epididymis followed by the tryptic digestion of the proteins and the resulted peptides were subjected to isobaric tags for relative and absolute quantitation-label and mass spectrometry (MS)/MS analysis. With the help of quantitative proteomics, we have been able to elucidate some of the major proteins involved in the process of sperm maturation. Results: In total, 999 proteins from the spermatozoa of caput and cauda region of the epididymis were identified. We have reported about 10 downregulated proteins and 15 upregulated proteins that have been in the sperm from the cauda region of the epididymis. HongrES1, the membrane of the SERPIN family specifically expressed in the principal cells of the cauda epididymis, have been found to be expressed three-fold higher. Conclusion: This study contributes to the understanding of the importance of different proteins at the different stages of the maturation during the transit of the spermatozoa. The higher and lower expression of different proteins in the epididymal region depicts their roles in priming the spermatozoa for normal fertilizing ability. Thus, the target proteins can be further studied for the possible development of male contraception.

Quantitative analysis of CDX2 protein expression improves its clinical utility as a prognostic biomarker in stage II and III colon cancer.

Better stratification of patients with stage II and stage III colon cancer for risk of recurrence is urgently needed. The present study aimed to validate the prognostic value of CDX2 protein expression in colon cancer tissue by routine immunohistochemistry and to evaluate its performance in a head-to-head comparison with tandem mass spectrometry-based proteomics. CDX2 protein expression was evaluated in 386 stage II and III primary colon cancers by immunohistochemical staining of tissue microarraysand by liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis using formalin-fixed paraffin-embeddedtissue sections of a matched subset of 23 recurrent and 23 non-recurrent colon cancers. Association between CDX2 expression and disease-specific survival (DSS) was investigated. Low levels of CDX2 protein expression in stage II and III colon cancer as determined by immunohistochemistry was associated with poor DSS (hazard ratio [HR]=1.97 (95% confidence interval [CI]: 1.26-3.06); p=0.002). Based on analysis of a selected sample subset, CDX2 prognostic value was more pronounced when detected by LC-MS/MS (HR=7.56 (95% CI: 2.49-22.95); p<0.001) compared to detection by immunohistochemistry (HR=1.60 (95% CI: 0.61-4.22); p=0.34). This study validated CDX2 protein expression as a prognostic biomarker in stage II and III colon cancer, conform previous publications. CDX2 prognostic value appeared to be underestimated when detected by routine immunohistochemistry, probably due to the semiquantitative and subjective nature of this methodology. Quantitative analysis of CDX2 substantially improved its clinical utility as a prognostic biomarker. Therefore, development of routinely applicable quantitative assays for CDX2 expression is needed to facilitate its clinical implementation.

Quantitative Proteomics in Drosophila with Holidic Stable-Isotope Labeling of Amino Acids in Fruit Flies (SILAF).

Protein-focused research has been challenging in Drosophila melanogaster due to few specific antibodies for Western blotting and the lack of effective labeling methods for quantitative proteomics. Herein, we describe the preparation of a holidic medium that allows stable-isotope labeling of amino acids in fruit flies (SILAF). Furthermore, in this chapter, we provide a protocol for mitochondrial enrichments from Drosophila larvae and flies together with a procedure to generate high-quality peptides for further analysis by mass spectrometry. Samples obtained following this protocol can be used for various functional studies such as comprehensive proteome profiling or quantitative analysis of posttranslational modifications upon enrichment. SILAF is based on standard fly routines in a basic wet lab environment and provides a flexible and cost-effective tool for quantitative protein expression analysis.

Traditional Chinese medicine classification of knee osteoarthritis with proteomics analysis

Osteoarthritis (OA) is a heterogeneous disease caused by the pathology of the synovial joint. About 10-12% of adults have symptomatic OA. In this study, the proteomics method was used to find differentially expressed proteins and to explore the material basis of traditional Chinese medicine (TCM) classification in knee OA patients. Fifteen patients of the treatment group with knee OA of three different classifications were divided into three groups according to the dialectical classification of TCM: kidney deficiency type (5 cases), Yang deficiency type (5 cases), blood stasis type (5 cases). Also, five patients with traumatic synovitis were enrolled as the control group. The joint fluids were drawn separately. The joint fluids before treatment were Aa, Ba, and Ca groups. After one week of medication, the three joint fluid types were drawn again, and they were Ab group, Bb, and Cb groups. Liquid from the control group was named group D. Quantitative analysis of protein expression was conducted to find out the differently expressed proteins. The treatment group was treated with prescriptions for syndromes according to the TCM classification. The proteomics analysis identified 251 differentially expressed protein groups, and the number of groups with quantitative information for all seven channels was 246. The three treatment groups and the control group had 32 differential proteins (P<0.01), of which nine might be differential proteins between OA and traumatic synovitis. Histone H4, histone H2A, S100 calbindin A8, fibrinogen γ, fiber protein α, cDNA (FLJ92148), C4b binding protein, and partial transketolase variants were down-regulation, and basement membrane glycan was up-regulation in the treatment group compared with the control group (P<0.01). HIST1H2BC and myeloperoxidase levels in the Aa group were less than those in the D and Ab groups. The serum amyloid P and apolipoprotein CI variants in the Ba group were less than those in the D and Bb groups. Histone H2A and C-reactive protein, Fibrinogen α levels in the Ca group were less than those in the D and Cb groups. Nine proteins are closely related to OA of different TCM classification.

Formation of a Photoelectrochemical Z-Scheme Structure with Inorganic/Organic Hybrid Materials for Evaluation of Receptor Protein Expression on the Membrane of Cancer Cells.

Quantitative analysis of receptor protein expression is essential to give new insights into tumor-related research. Benefitting from their high sensitivity and low background, photoelectrochemical (PEC) platforms are considered as powerful tools for evaluating the expression of receptor proteins. Herein, to reduce the cytotoxicity and facilitate the subsequent assembly, l-cysteine-modified Ag-ZnIn2S4 quantum dots (l-Cys AZIS QDs) are prepared and PEC responses under the irradiation of long wavelength light are obtained. To further improve the PEC behavior, iron phthalocyanine (FePc) is employed to form a Z-scheme structure with l-Cys AZIS QDs. The Z-scheme structure based on l-Cys AZIS QDs/FePc hybrid materials exhibits high photo-to-electric conversion efficiency and can be excited with near-infrared range light. Because hyaluronic acid linked to photoactive materials can recognize CD44 expressed on the membrane of cancer cells, cancer cells are immobilized onto l-Cys AZIS QDs/FePc hybrid materials, inducing a decrease of the photocurrent intensity. Consequently, a PEC cytosensor is constructed to quantify cancer cells expressing CD44. The PEC analytical platform is able to determine A549 cells in the range of 2 × 102 to 4.5 × 106 cells/mL, and a detection limit of 15 cells/mL is realized in the case of S/N = 3. In addition, the expression of CD44 in A549 and other five cancer cells is measured with this PEC method. Depending on our data, the expression of CD44 in different cancer cells is distinct, indicating great potential of this method in receptor protein-related studies.