Serum levels of tumor markers and their clinical significance in epithelial ovarian cancer. / ä¸Šçš®æ€§åµå·¢ç™Œè¡€æ¸ ä¸­è‚¿ç˜¤æ ‡å¿—ç‰©æ°´å¹³åŠå ¶ä¸´åºŠæ„ä¹‰ï¼ˆè‹±æ–‡ï¼‰.

OBJECTIVES: Tumor markers have been widely used clinically. Detection of serum CA125 is one of the commonly used clinical methods for early screening and early diagnosis of epithelial ovarian cancer, but it is difficult to diagnose epithelial ovarian cancer with a single specific tumor marker. In this study, the combinatorial tumor marker detection method was used to compare the value of each tumor marker alone and different combinations in the diagnosis of epithelial ovarian cancer. METHODS: The clinical data of patients with epithelial ovarian cancer (n=65) and ovarian benign disease (n=29) were collected. Multiple tumor marker protein chip was used to detect cancer antigen 125 (CA125), carbohydrate antigen 242 (CA242), alpha-fetoprotein (AFP), beta-human chorionic gonadotropin (ß-HCG), carcinoembryonic antigen (CEA), cancer antigen 199 (CA199), neuron-specific enolase (NSE), Ferritin, cancer antigen 153 (CA153), and human growth hormone (HGH) serum levels, and to compare the differences between the benign and malignant ovarian tumors. The correlation between tumor markers and clinicopathologic features for ovarian epithelial carcinoma was analyzed by χ2 test. Spearman rank analysis showed the correlation between CA125 expression level and other tumor markers in epithelial ovarian cancer and the correlation between age and the above 10 tumor markers. Sensitivity, specificity, positive predictive value, negative predictive value, Youden index, and diagnostic efficiency were used to evaluate the diagnostic value of single tumor marker and the combination of tumor markers. RESULTS: The levels of ß-HCG, NSE, CA153, and CA125 in the epithelial ovarian cancer group were higher than those in the ovarian benign disease group. The level of NSE in the serum of patients with epithelial ovarian cancer was related to the clinical stage of patients. In addition, the levels of CA242, ß-HCG, CEA, NSE, Ferritin, CA153 in the serum of patients with epithelial ovarian cancer were positively correlated with CA125 (rs=0.497, P<0.001; rs=0.612, P<0.001; rs=0.358, P=0.003; rs=0.680, P<0.001; rs=0.322, P=0.009; rs=0.609, P<0.001, respectively), and the levels of ß-HCG, Ferritin, CA153 were positively correlated with the patient's age (rs=0.256, P=0.040; rs=0.325, P=0.008; rs=0.249, P=0.046, respectively). In the diagnosis of epithelial ovarian cancer, the sensitivity, Youden index, and diagnostic efficiency of CA125 detection alone were higher than the results of the other 9 separate detections. When CA153, CA199, CA242, Ferritin, and CEA were combined with CA125, the sensitivity of the combined detection of different combinations was higher than that of CA125 alone. The combined detection sensitivities of CA125+CEA and CA125+Ferritin+CEA were 89.2% and 90.8%, respectively, and the diagnostic efficiencies were both 84.1%, which were higher than those of other combinations. The Youden index of CA125+CEA joint detection was 0.616, which was higher than those of other combinations. CONCLUSIONS: CA125 has a high diagnostic value in the diagnosis of epithelial ovarian cancer. The detection of combined tumor markers in serum has higher sensitivity and specificity in epithelial ovarian cancer.

Imbalance of Vaginal Microbiota and Immunity: Two Main Accomplices of Cervical Cancer in Chinese Women.

Objective: To explore the correlation of female vaginal microbiota and immune factors with cervical cancer. Methods: The distribution pattern difference of vaginal microbiota of four groups of women (cervical cancer, HPV-positive CIN, HPV-positive non-CIN, and HPV-negative groups) were compared by microbial 16S rDNA sequencing. The protein chip was used to detect the composition and changes of the immune factors in the four groups. Results: Alpha diversity analysis demonstrated that the diversity of the vaginal microbiota was increased as the disease develops. Among those bacteria abundant in the vaginal microbiota, Lactobacillus, Prevotella, and Gardnerella dominate at the genus level of vaginal flora. Compared with the HPV-negative group, the differentially dominant bacteria, such as Prevotella, Ralstonia, Gardnerella and Sneathia, are enriched in the cervical cancer group. Likewise, Gardnerella, Prevotella, and Sneathia are more in the HPV-positive CIN group, while Gardnerella and Prevotella in the HPV-positive non-CIN group, respectively. In contrast, Lactobacillus and Atopobium are dominant in the HPV-negative group (LDA>4log10). The concentration of inflammatory immune factors IP-10 and VEGF-A were increased in the cervical cancer group (P < 0.05), compared with other groups. Conclusion: The occurrence of cervical cancer is related to an increase of vaginal microbiota diversity and up-regulation of inflammatory immune factor proteins. The abundance of Lactobacillus was decreased while the one of Prevotella and Gardnerella were increased in the cervical cancer group, compared with other three groups. Moreover, the IP-10 and VEGF-A were also increased in the cervical cancer group. Thus, evaluation of changes in the vaginal microbiota and these two immune factor levels might be a potential non-invasive and simple method to predict cervical cancer. Furthermore, it is significant to adjust and restore the balance of vaginal microbiota and maintain normal immune function in preventing and treating cervical cancer.

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods.

OBJECTIVES: To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. METHODS: Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. RESULTS: The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. CONCLUSIONS: The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Construction and Evaluation of a Novel MAP Immunoassay for 9 Nutrition-and-Health-Related Protein Markers Based on Multiplex Liquid Protein Chip Technique.

We attempted to construct and evaluate a novel detection method to realize simultaneous detection based on a multiplex liquid protein chip technique for nine nutrition-and-health-related protein markers to meet the requirement of an accurate, simultaneous and comprehensive analysis of the proteomics of nutrition and health. The lower limits of detection, biological limits of detection and regression equations of serum ferritin (SF), soluble transferrin receptor (sTfR), c-reactive protein (CRP), retinol-binding protein4 (RBP4), apolipoprotein B (ApoB), alpha-fetoprotein (AFP), prealbumin (PA), carcino-embryonic antigen (CEA) and D-Dimmer (D-D) were determined after a series of optimal experiments. Then, the results of the methodological evaluation for this novel method indicated that the accuracies were between 70.12% and 127.07%, the within-run precisions were between 0.85% and 7.31%, the between-run precisions were between 3.53% and 19.07%, the correlation coefficients between this method and other methods were above 0.504 (p < 0.05), and the direct bilirubin (DBIL) of low concentration and the indirect bilirubin (IBIL) of high concentration could not interfere with the detected results of nine indicators. The novel multiplex detection method, which can increase accuracy and improve the ability of comprehensive analysis, can basically meet the requirement of detection and the diagnosis of the proteomics of nutrition and health.

CHIP Haploinsufficiency Exacerbates Hepatic Steatosis via Enhanced TXNIP Expression and Endoplasmic Reticulum Stress Responses.

TXNIP is a critical regulator of glucose homeostasis, fatty acid synthesis, and cholesterol accumulation in the liver, and it has been reported that metabolic diseases, such as obesity, atherosclerosis, hyperlipidemia, type 2 diabetes, and nonalcoholic fatty liver disease (NAFLD), are associated with endoplasmic reticulum (ER) stress. Because CHIP, an E3 ligase, was known to be involved in regulating tissue injury and inflammation in liver, its role in regulating ER stress-induced NAFLD was investigated in two experimental NAFLD models, a tunicamycin (TM)-induced and other diet-induced NAFLD mice models. In the TM-induced NAFLD model, intraperitoneal injection of TM induced liver steatosis in both CHIP+/+ and CHIP+/- mice, but it was severely exacerbated in CHIP+/- mice compared to CHIP+/+ mice. Key regulators of ER stress and de novo lipogenesis were also enhanced in the livers of TM-inoculated CHIP+/- mice. Furthermore, in the diet-induced NAFLD models, CHIP+/- mice developed severely impaired glucose tolerance, insulin resistance and hepatic steatosis compared to CHIP+/+ mice. Interestingly, CHIP promoted ubiquitin-dependent degradation of TXNIP in vitro, and inhibition of TXNIP was further found to alleviate the inflammation and ER stress responses increased by CHIP inhibition. In addition, the expression of TXNIP was increased in mice deficient in CHIP in the TM- and diet-induced models. These findings suggest that CHIP modulates ER stress and inflammatory responses by inhibiting TXNIP, and that CHIP protects against TM- or HF-HS diet-induced NAFLD and serves as a potential therapeutic means for treating liver diseases.

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods / 法医学杂志

OBJECTIVES@#To estimate postmortem interval (PMI) by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods.@*METHODS@#Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points (0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d) after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis (PCA) and orthogonal partial least squares (OPLS) were used for data analysis. Fisher discriminant model and back propagation (BP) neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis.@*RESULTS@#The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points (P<0.05) except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples.@*CONCLUSIONS@#The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats' and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

Nanocellulose Composites as Smart Devices With Chassis, Light-Directed DNA Storage, Engineered Electronic Properties, and Chip Integration.

The rapid development of green and sustainable materials opens up new possibilities in the field of applied research. Such materials include nanocellulose composites that can integrate many components into composites and provide a good chassis for smart devices. In our study, we evaluate four approaches for turning a nanocellulose composite into an information storage or processing device: 1) nanocellulose can be a suitable carrier material and protect information stored in DNA. 2) Nucleotide-processing enzymes (polymerase and exonuclease) can be controlled by light after fusing them with light-gating domains; nucleotide substrate specificity can be changed by mutation or pH change (read-in and read-out of the information). 3) Semiconductors and electronic capabilities can be achieved: we show that nanocellulose is rendered electronic by iodine treatment replacing silicon including microstructures. Nanocellulose semiconductor properties are measured, and the resulting potential including single-electron transistors (SET) and their properties are modeled. Electric current can also be transported by DNA through G-quadruplex DNA molecules; these as well as classical silicon semiconductors can easily be integrated into the nanocellulose composite. 4) To elaborate upon miniaturization and integration for a smart nanocellulose chip device, we demonstrate pH-sensitive dyes in nanocellulose, nanopore creation, and kinase micropatterning on bacterial membranes as well as digital PCR micro-wells. Future application potential includes nano-3D printing and fast molecular processors (e.g., SETs) integrated with DNA storage and conventional electronics. This would also lead to environment-friendly nanocellulose chips for information processing as well as smart nanocellulose composites for biomedical applications and nano-factories.

Vascular toxicity of multi-walled carbon nanotubes targeting vascular endothelial growth factor.

Multiwalled carbon nanotubes (MWCNTs) are currently widely used and are expected to be used as drug carriers and contrast agents in clinical practice. Previous studies mainly focused on their lung toxicity; therefore, their effects on the vascular endothelium are unclear. In this study, a human angiogenesis array was used to determine the effect of MWCNTs on the expression profile of angiogenic factors in endothelial cells and to clarify the role of vascular endothelial growth factor (VEGF) in MWCNT-induced endothelial cell injury at the cellular and animal levels. The results indicated that MWCNTs (20-30 nm and 30-50 nm) could enter endothelial cells and disrupt human umbilical vein endothelial cell (HUVECs) activity in a concentration-dependent manner. MWCNTs disrupted the tube formation ability and cell migration function of HUVECs. The results from a Matrigel Plug experiment in mice showed that angiogenesis in the MWCNT experimental group was significantly reduced. The results of a protein chip analysis indicated that VEGF expression in the MWCNT treatment group was decreased, a finding that was validated by ELISA results. The protein expression levels of AKT and eNOS in the MWCNT treatment group were significantly decreased; the administration of recombinant VEGF significantly alleviated the migration ability and tube formation ability of endothelial cells injured by MWCNTs, upregulated the protein expression of AKT and eNOS, and increased the number of neovascularization in mice in the MWCNT treatment group. This study demonstrated that MWCNTs affect angiogenesis via the VEGF-Akt-eNOS axis which can be rescued by VEGF endothelial treatment.

A poly(dimethylsiloxane)-based solid-phase microchip platform for dual detection of Pseudorabies virus gD and gE antibodies.

Pseudorabies caused by pseudorabies virus (PRV) infection is still a major disease affecting the pig industry; its eradication depends on effective vaccination and antibody (Ab) detection. For a more rapid and accurate PRV detection method that is suitable for clinical application, here, we established a poly(dimethylsiloxane)-based (efficient removal of non-specific binding) solid-phase protein chip platform (blocking ELISA) for dual detection of PRV gD and gE Abs. The purified gD and gE proteins expressed in baculovirus were coated into the highly hydrophobic nanomembrane by an automatic spotter, and the gray values measured by a scanner were used for the S/N (sample/negative) value calculation (gD and gE Abs standard, positive: S/N value ≤0.6; negative: S/N value >0.7; suspicious: 0.6 < S/N ≤ 0.7). The method showed an equal sensitivity in the gD Ab test of immunized pig serum samples compared to the neutralization test and higher sensitivity in the gE Ab test compared to the commercial gE Ab detection kit. In the clinical evaluation, we found an agreement of 100% (122/122) in the gD Ab detection compared to the neutralization test and an agreement of 97.5% (119/122) in the gE Ab detection compared to the commercial PRV gE Ab detection kit. In summary, the protein chip platform for dual detection of PRV gD and gE Abs showed high sensitivity and specificity, which is suitable for PRV immune efficacy evaluation and epidemic monitoring.

Chip-DSF: A rapid screening strategy for drug protein targets.

Identification of the drug target of lead compounds is an important means for rapid and efficient drug discovery. Protein chips are a high-throughput protein function analysis technology that has been widely used in screening drug protein targets in recent years. However, the verification of the results after high-throughput protein chip screening is still cumbersome. Based on our mature protein chip preparation platform, we prepared a protein chip containing 150 important high-frequency protein targets and used antibodies to prove the availability of the protein chip. To improve the accuracy of target screening, we combined the label-free differential scanning fluorimetry (DSF) with the protein chip, proposing the Chip-DSF strategy. Subsequently, we tested the method with small molecular ginsenoside-Rg2 (Rg2). The Chip-DSF strategy was used to successfully screen the potential target protein KRAS(G12C) of Rg2. Consistently, we found that Rg2 could inhibit NCI-H23 cell proliferation by inducing cell cycle arrest. Also, we found that Rg2 could reduce the amount of KRAS protein and inhibit the phosphorylation of KRAS downstream key signaling protein ERK1, RPS6, and P70S6K in NCI-H23 cells. Collectively, our Chip-DSF strategy could achieve rapid target verification which improved the accuracy and efficiency of target screening of protein chips.

[Identification of serological biomarkers for diagnosis of rheumatoid arthritis using a protein array-based approach].

OBJECTIVE: To study the cytokine patterns in patients with rheumatoid arthritis (RA) and healthy individuals and identify candidate serum biomarkers for clinical diagnosis of RA. METHODS: This study was conducted among 59 patients diagnosed with RA in our hospital from 2015 to 2019 with 46 age- and gender-matched healthy subjects who received regular physical examinations in our hospital as the control group. Serological autoimmune profiles of 5 RA patients and 5 healthy control subjects were obtained from human cytokine microarrays. We selected 4 differentially expressed cytokines (LIMPII, ROBO3, Periostin and IGFBP-4) and 2 soluble cytokine receptors of interest (2B4 and Tie-2) and examined their serum levels using enzyme-linked immunosorbent assay in 54 RA patients and 41 healthy control subjects. Spearman correlation test was performed to assess the correlation of serum cytokine and soluble receptor expression levels with the clinical features including rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), disease activity score (DAS28) and health assessment questionnaire (HAQ). Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic capability of these cytokines. RESULTS: We identified 6 dysregulated cytokines and soluble receptors (2B4, LIMPII, Tie-2, ROBO3, periostin and IGFBP-4) in RA patients (P < 0.01). The serum levels of LIMPII, ROBO3 and periostin were significantly correlated with the disease activity indicators including RF (P < 0.001), CRP (P < 0.001), DAS28 (P < 0.001) and HAQ (P < 0.001) in RA patients. Among the 6 candidate cytokines, 2B4 showed the largest area under the curve (AUC) of 0.861 for RA diagnosis (P < 0.001), followed then by LIMPII, ROBO3, periostin, Tie-2 and IGFBP-4. CONCLUSION: Serum levels of LIMPII, ROBO3 and periostin can be indicative of the disease activity of RA, and serum 2B4, LIMPII, periostin, ROBO3, IGFBP-4 and Tie-2 levels may serve as biomarkers for the diagnosis of RA.

Neutrophil peptide-1 promotes the repair of sciatic nerve injury through the expression of proteins related to nerve regeneration.

Objectives Small-molecule polypeptide neutrophil peptide 1 (NP-1) was reported to promote the regeneration of the sciatic nerve after denervation, but the mechanisms underlying this effect of NP-1 are unclear. Here, we established a Sprague-Dawley rat model of crush injury to study the effect of a single intermuscular injection of NP-1 on the repair of injured peripheral nerves and elucidate the possible underlying mechanism.Methods 39 rats were randomly selected to join this study and divided into the blank control group (normal group, n=9), experimental group (NP-1 group, n=15), and negative control group (NS group, n=15). The dynamic expression of cytokines in different groups of nerve tissues during Wallerian degeneration was observed using protein chips at different time points after injury. Recovery of injured nerves was determined based on the general condition, local gross morphology of the nerve suture site, sciatic nerve function index, neuroelectrophysiology, and osmic acid staining at 6 weeks after the surgery. The recovery of effector function was determined based on wet weight, hematoxylin-eosin staining, modified Gomori staining, and nicotinamide adenine dinucleotide-tetrazolium reductase staining at 6 weeks after the surgery.Results It was found that a single topical administration of NP-1 promoted sciatic nerve regeneration after crush injury and affected the expression of proteins related to neurotrophy, inflammation, cell chemotaxis, and cell generation pathways.

Identification of serological biomarkers for diagnosis of rheumatoid arthritis using a protein array-based approach / 南方医科大学学报

OBJECTIVE@#To study the cytokine patterns in patients with rheumatoid arthritis (RA) and healthy individuals and identify candidate serum biomarkers for clinical diagnosis of RA.@*METHODS@#This study was conducted among 59 patients diagnosed with RA in our hospital from 2015 to 2019 with 46 age- and gender-matched healthy subjects who received regular physical examinations in our hospital as the control group. Serological autoimmune profiles of 5 RA patients and 5 healthy control subjects were obtained from human cytokine microarrays. We selected 4 differentially expressed cytokines (LIMPII, ROBO3, Periostin and IGFBP-4) and 2 soluble cytokine receptors of interest (2B4 and Tie-2) and examined their serum levels using enzyme-linked immunosorbent assay in 54 RA patients and 41 healthy control subjects. Spearman correlation test was performed to assess the correlation of serum cytokine and soluble receptor expression levels with the clinical features including rheumatoid factor (RF), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), disease activity score (DAS28) and health assessment questionnaire (HAQ). Receiver operating characteristic (ROC) curve was used to evaluate the diagnostic capability of these cytokines.@*RESULTS@#We identified 6 dysregulated cytokines and soluble receptors (2B4, LIMPII, Tie-2, ROBO3, periostin and IGFBP-4) in RA patients (P < 0.01). The serum levels of LIMPII, ROBO3 and periostin were significantly correlated with the disease activity indicators including RF (P < 0.001), CRP (P < 0.001), DAS28 (P < 0.001) and HAQ (P < 0.001) in RA patients. Among the 6 candidate cytokines, 2B4 showed the largest area under the curve (AUC) of 0.861 for RA diagnosis (P < 0.001), followed then by LIMPII, ROBO3, periostin, Tie-2 and IGFBP-4.@*CONCLUSION@#Serum levels of LIMPII, ROBO3 and periostin can be indicative of the disease activity of RA, and serum 2B4, LIMPII, periostin, ROBO3, IGFBP-4 and Tie-2 levels may serve as biomarkers for the diagnosis of RA.

[Mechanism of PI3K/AKT/mTOR signaling pathway for mediating anti-inflammatory and anti-oxidant effects of chrysin: a protein microarray-based study].

OBJECTIVE: To investigate the mechanism of PI3K/AKT/mTOR signaling pathway for mediating the anti-inflammatory and anti-oxidant effects of chrysin. METHODS: RAW264.7 cells were treated with different concentrations of chrysin for 24 h, and the changes in cell viability were detected using CCK-8 method. The cells with or without chrysin pretreatment for 2 h were stimulated with lipopolysaccharide (LPS) for different lengths of time, and the related signal molecules were screened using protein chip technique. In cells pretreated with chrysin for 2 h followed by LPS stimulation for 18 h, the release of IL-6, MCP-1 and TNF-α by the cells was detected with ELISA, and NO production was examined using Griess method, and ROS level was determined using DCFH-DA. The effects of chrysin, LPS, and their combination on the mRNA expressions of iNOS and COX-2 were detected using RT-PCR; Western blotting was performed to examine the changes in cellular expressions of p-AKT, p-PRAS40, p-mTOR, mTOR, p-P70S6k, p-S6RP and S6RP following the treatments with LPS, N-Acetyl-L-cysteine, and chrysin, alone or in combinations. RESULTS: Chrysin below 60 µg/mL did not significantly affect the viability of RAW264.7 cells (P>0.05). Chrysin treatment significantly reduced the release of IL-6, MCP-1, and TNF-α and the level of NO (P < 0.01), and inhibited the mRNA and protein expressions of iNOS and COX-2 (P < 0.01) in the cells. The results of protein chip screening suggested that LPS could activate the AKT/mTOR pathway, which was significantly inhibited by chrysin pretreatment, and the results were verified by Western blotting (P < 0.01). Chrysin treatment significantly reduced the generation of endogenous ROS, and treatment with N-Acetyl-L-cysteine to eliminate intracellular ROS obviously reduced the expressions of iNOS and COX-2 (P < 0.05) and blocked the AKT/mTOR pathway (P < 0.05). CONCLUSION: Chrysin can inhibit the synthesis of the upstream signaling molecule ROS to inhibit the activation of AKT/mTOR signaling pathway, regulate the translation process of ribosomes, down-regulate the synthesis and release of pro-inflammatory cytokines and inflammatory mediators, and thus produce anti-inflammatory effects.

Evaluation and Differential Diagnosis of a Genetic Marked Brucella Vaccine A19ΔvirB12 for Cattle.

Brucella abortus is an important zoonotic pathogen that causes severe economic loss to husbandry and poses a threat to human health. The B. abortus A19 live vaccine has been extensively used to prevent bovine brucellosis in China. However, it is difficult to distinguish the serological response induced by A19 from that induced by natural infection. In this study, a novel genetically marked vaccine, A19ΔvirB12, was generated and evaluated. The results indicated that A19ΔvirB12 was able to provide effective protection against B. abortus 2308 (S2308) challenge in mice. Furthermore, the safety and protective efficacy of A19ΔvirB12 have been confirmed in natural host cattle. Additionally, the VirB12 protein allowed for serological differentiation between the S2308 challenge/natural infection and A19ΔvirB12 vaccination. However, previous studies have found that the accuracy of the serological detection based on VirB12 needs to be improved. Therefore, we attempted to identify potential supplementary antigens with differential diagnostic functions by combining label-free quantitative proteomics and protein chip technology. Twenty-six proteins identified only in S2308 were screened; among them, five proteins were considered as potential supplementary antigens. Thus, the accuracy of the differential diagnosis between A19ΔvirB12 immunization and field infection may be improved through multi-antigen detection. In addition, we explored the possible attenuation factors of Brucella vaccine strain. Nine virulence factors were downregulated in A19ΔvirB12. The downregulation pathways of A19ΔvirB12 were significantly enriched in quorum sensing, ATP-binding cassette transporter, and metabolism. Several proteins related to cell division were significantly downregulated, while some proteins involved in transcription were upregulated in S2308. In conclusion, our results contribute to the control and eradication of brucellosis and provide insights into the mechanisms underlying the attenuation of A19ΔvirB12.

[Application of SPR protein chip in screening for imported malaria].

Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.

Neurotrophic factor levels in the serum and cerebrospinal fluid of neonates infected with human cytomegalovirus.

Human cytomegalovirus (HCMV) is most likely to damage the central nervous system (CNS) during early embryonic development; however, the early neurodevelopmental abnormalities caused by HCMV infection and the regulation of cytokines remain unclear. Therefore, we investigated neuronal factors in the serum and cerebrospinal fluid (CSF) of newborns infected with HCMV using protein microarray technology with a view to elucidating the changes in specific neuronal factors for use in the development of a reliable index for predicting CNS injury caused by HCMV infection. Serum and CSF were collected from four newborns with HCMV infection and CNS injury (HCMV-infected group) and from four newborns without CNS infection (control group). A protein microarray containing 29 kinds of CNS-related cytokines was used to identify differentially expressed neuronal factors in the serum and CSF of the HCMV-infected and control groups. The levels of the differentially expressed proteins were verified further in 30 CSF samples from an HCMV-infected group using enzyme-linkedimmunosorbent assay (ELISA). Between newborns in the HCMV-infected and control groups, the protein microarray analysis identified three differentially expressed neurotrophic factors in the CSF samples: Acrp30, MMP-3, and interleukin-1 alpha (IL-1α). No differential cytokine expression was seen in the serum. ELISA showed significantly higher expression levels of Acrp30 and MMP-3 in the CSF of the 30 newborns with HCMV infection and CNS injury than in those in the control group, whereas the expression of IL-1α was significantly lower. Our results demonstrate that changes in the expression levels of Acrp30, MMP-3, and IL-1α in the CSF of newborns infected with HCMV may be related to the pathogenesis of CNS infection.

Application of SPR protein chip in screening for imported malaria / 生物工程学报

Imported malaria has become a major risk factor for malaria prevention and control in China. How to screen malaria quickly for people entering China is an urgent problem to be solved. Protein microarrays are widely used in high-throughput screening and diagnosis. In this study, surface plasmon resonance (SPR) technique for malaria detection was established by using the specific adsorption surface treated by polyethylene glycol polymer, and the malaria specific antigen HRP2 was used as capture probe. The optimal concentration of antigen, sensitivity and specificity of detection, as well as anti-interference ability of the chip were analyzed. The SPR protein chip was applied to detect specific antibodies of malignant malaria in serum with the advantage of label-free, instant and fast. Compared with fluorescence quantitative PCR, there were no significant difference in sensitivity and specificity between the two methods. This study lays a foundation for further development of protein microarray for malaria typing identification, and it is conducive to the rapid screening of malaria for people entering.

Prognostic Significance of CHIP and RIPK3 in Non-Small Cell Lung Cancer.

RIPK3 is a key regulator of necroptosis, which plays a double-edged sword role in tumor progression. CHIP is an E3 ubiquitin ligase that regulates necroptosis by degrading RIPK3. Here, we investigated the prognostic value of RIPK3 and CHIP expression in 404 patients with non-small cell lung cancer (NSCLC). Expressions of CHIP and RIPK3 showed opposite correlations with survival. CHIP expression was associated with the longer overall survival (OS), whereas RIPK3 expression was associated with the shorter OS. RIPK3 positivity showed marginal association with shorter OS and disease-free survival (DFS) in adjuvant radiotherapy recipients but not in non-recipients, suggesting that necroptosis may induce radioresistance. In multivariate analysis, CHIP expression was associated with longer OS. Compared with other patients, CHIP(-)/RIPK3(+) patients had shorter OS and DFS. In summary, in patients with NSCLC, the expression of CHIP was an independent favorable prognostic factor while that of RIPK3 was an adverse prognostic factor.

Two novel protein chips for the detection of antibodies against porcine parvovirus.

BACKGROUND: PPV is one of the most important pathogens causing porcine reproductive disorder. It has been shown in clinical cases to be a commonly mixed infection with other important swine diseases which can aggravate the severity of the disease and bring serious economic losses to the pig industry. Serological methods, such as hemagglutination inhibition assays (HAI), serum neutralization (SN), and the modified direct complement-fixation (MDCF) test were utilized earlier, whereas the enzyme-linked immunosorbent assay (ELISA) is the most frequently applied assay to detect PPV-specific antibodies. RESULTS: We establish the visible protein chip and the cyanine dye 3 (Cy3)-labeled protein chip to detect the clinical serum from pigs. In this study, the recombinant protein VP2 of PPV was expressed in E.coli, purified with nickel magnetic beads, and then printed onto epoxy-coated glass slides for preparation of the protein chip. After a series of experiments, the conditions of antigen protein concentration, incubation time of primary antibody or secondary antibody, and optimal serum dilution fold were optimized, resulting in a successful visible protein chip and Cy3-labeled protein chip. The results showed that the positive serum, diluted up to 6000-fold, can be detected by the visible protein chip, and the positive serum, diluted up to 12,800-fold, can be detected by the Cy3-labeled protein chip, suggesting the high sensitivity of these protein chips. Moreover, the positive detection ratio, sensitivity, and specificity of these two kinds of protein chips were higher than those of commercial ELISA antibody detection kits. CONCLUSION: Overall, these two protein chips can be used to rapidly diagnose clinical samples with high throughput.