T-cell activation is associated with high-grade serous ovarian cancer survival.

AIM: High-grade serous ovarian cancer (HGSOC) is an aggressive disease that is largely resistant to today's immunotherapies. Here, we aimed to investigate the prognostic significance of CTLA4, PD-1, and T-cell activation status in HGSOC. METHODS: Using a publicly accessed microarray dataset including 260 HGSOC samples, we calculated Kaplan-Meier survival curves for overall survival (OS), evaluated associations with multivariate Cox regression models to evaluate the associations, and summarized using a hazard ratio (HR). The correlations between PD-1 gene expression and that of other genes were calculated by Pearson correlation. RESULTS: Multivariate survival analyses showed that high PD-1 expression but not CTLA4 was associated with longer OS (HR = 0.69; 95% confidence interval [CI] = 0.52-0.91; p = 0.01), and that higher T-cell activation score was associated with better outcome (HR = 0.74; 95% confidence interval [CI] = 0.58-0.95; p = 0.02). The top three PD-1 highly correlated genes were SIRPG (r = 0.90, p < 2E-16), FASL (r = 0.89, p < 2E-16), and CD8a (r = 0.87, p < 2E-16). HGSOC patients' OS is positively associated T-cell activation score and PD-1 expression but not CTLA4. CONCLUSION: T cell activation score may serve as a candidate for personalized immunotherapy in HGSOC. The application of anti-PD-1 therapy to HGSOC should be cautious.

Detection of catecholamine metabolites in urine based on ultra-high-performance liquid chromatography-tandem mass spectrometry.

The excretion of neurotransmitter metabolites in normal individuals is of great significance for health monitoring. A rapid quantitative method was developed with ultra-performance liquid chromatography-tandem mass spectrometry. The method was further applied to determine catecholamine metabolites vanilymandelic acid (VMA), methoxy hydroxyphenyl glycol (MHPG), dihydroxy-phenyl acetic acid (DOPAC), and homovanillic acid (HVA) in the urine. The urine was collected from six healthy volunteers (20-22 years old) for 10 consecutive days. It was precolumn derivatized with dansyl chloride. Subsequently, the sample was analyzed using triple quadrupole mass spectrometry with an electrospray ion in positive and multireaction monitoring modes. The method was sensitive and repeatable with the recoveries 92.7-104.30%, limits of detection (LODs) 0.01-0.05 µg/mL, and coefficients no less than 0.9938. The excretion content of four target compounds in random urine samples was 0.20 ± 0.086 µg/mL (MHPG), 1.27 ± 1.24 µg/mL (VMA), 3.29 ± 1.36 µg/mL (HVA), and 1.13 ± 1.07 µg/mL (DOPAC). In the urine, the content of VMA, the metabolite of norepinephrine and adrenaline, was more than MHPG, and the content of HVA, the metabolite of dopamine, was more than DOPAC. This paper detected the levels of catecholamine metabolites and summarized the characteristics of excretion using random urine samples, which could provide valuable information for clinical practice.

Gene Co-Expression Analysis Identified Preserved and Survival-Related Modules in Severe Blunt Trauma, Burns, Sepsis, and Systemic Inflammatory Response Syndrome.

BACKGROUND: Severe trauma and burns accompanied by sepsis are associated with high morbidity and mortality. Little is known about the transcriptional similarity between trauma, burns, sepsis, and systemic inflammatory response syndrome (SIRS). Uncovering key genes and molecular networks is critical to understanding the biology of disease. Conventional analysis of gene changes (fold change) analysis is difficult for time-serial data such as post-injury blood transcriptome. METHODS: Weighted gene co-expression network analysis (WGCNA) was applied to the trauma dataset to identify modules and hub genes. Module stability was tested by half sampling. Module preservations of burns, sepsis, and SIRS were calculated using trauma as reference. Module functional enrichment was analyzed in gProfiler server. Candidate drugs were screened using Connectivity Map based on hub genes. The modules were visualized in Cytoscape. RESULTS: Seventeen modules were identified. The modules were robust to the exclusion of half the sample. They were involved in lymphocyte and platelet activation, erythrocyte differentiation, cell cycle, translation, and interferon signaling. In addition, highly connected hub genes were identified in each module, such as GUCY1B1, BCL11B, HMMR, and CEACAM6. High BCL11B (M13) or low CEACAM6 (M27) expression indicates better survival prognosis in sepsis patients regardless of endotype class and age. Network preservation in burns, sepsis, and SIRS showed a general similarity between trauma and burns. M4, M5, M13, M16, M20, and M27 were significantly associated with injury time in trauma and burns. High M13 (T cell activation), low M15 (cell cycle), and low M27 (neutrophil activation) indicate better survival of sepsis patients, regardless of endotype class and age. Moreover, the modules can efficiently separate patients with different diseases. Some modules and hub genes have good prognostic performance in sepsis. Based on the hub genes of each module, six candidate drugs were screened. CONCLUSION: This study first compared the gene co-expression modules in trauma, burns, sepsis, and SIRS. The identified modules are useful for disease prognosis and drug discovery. BCL11B and CEACAM6 may be promising biomarkers for sepsis risk assessment.

Combining the advantages of prokaryotic expression and T7 phage display systems to obtain antigens for antibody preparation.

The gene encoding the phage major capsid protein 10A was cloned into the prokaryotic expression vector pET24a, and a 6XHis-tag was fused to the 3'-end of the 10A gene to verify complete expression. The recombinant plasmid was transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and 10A expression was induced by IPTG. SDS-PAGE and Western blot were used to confirm the target protein expression. The T7Select10-3b vector was added to the cultured bacteria expressing 10A at a multiplicity of infection (MOI) ranging from 0.01 to 0.1, and complete lysis of the bacteria was monitored by absorbance changes in the medium. The recombinant phage (reP) was harvested by PEG/NaCl sedimentation and resuspended in PBS. ELISA was performed to verify the presence of the 6XHis-tag on the surface of reP. The 10A-fusion expression vectors (pET10A-flag, pET10A-egfp, and pET10A-pct) were constructed, and fusion proteins were expressed and detected by the same method. The corresponding rePs (reP-Flag, reP-EGFP, and reP-PCT) were prepared by T7Select10-3b infection. After the expression of the peptides/proteins on the reP surfaces was confirmed, reP-Flag and reP-PCT were used to immunize mice to prepare anti-Flag and anti-PCT antibodies. The results showed that rePs prepared using the 10A-fusion vector and T7Select10-3b can be used as antigens to immunize mice and prepare antibodies. This method may be able to meet the rapid antigen preparation requirements for antibody production. Notably, the recombinant phage (reP) described in this study was obtained by the sedimentation method from T7Select10-3b-infected E. coli BL21 (DE3) cells carrying the major capsid protein 10A expression vector or 10A-fusion protein vector.

Saikosaponin a increases interleukin-10 expression and inhibits scar formation after sciatic nerve injury.

Nerve scarring after peripheral nerve injury can severely hamper nerve regeneration and functional recovery. Further, the anti-inflammatory cytokine, interleukin-10, can inhibit nerve scar formation. Saikosaponin a (SSa) is a monomer molecule extracted from the Chinese medicine, Bupleurum. SSa can exert anti-inflammatory effects in spinal cord injury and traumatic brain injury. However, it has not been shown whether SSa can play a role in peripheral nerve injury. In this study, rats were randomly assigned to three groups. In the sham group, the left sciatic nerve was directly sutured after exposure. In the sciatic nerve injury (SNI) + SSa and SNI groups, the left sciatic nerve was sutured and continuously injected daily with SSa (10 mg/kg) or an equivalent volume of saline for 7 days. Enzyme linked immunosorbent assay results demonstrated that at 7 days after injury, interleukin-10 level was considerably higher in the SNI + SSa group than in the SNI group. Masson staining and western blot assay demonstrated that at 8 weeks after injury, type I and III collagen content was lower and nerve scar formation was visibly less in the SNI + SSa group compared with the SNI group. Simultaneously, sciatic functional index and nerve conduction velocity were improved in the SNI + SSa group compared with the SNI group. These results confirm that SSa can increase the expression of the anti-inflammatory factor, interleukin-10, and reduce nerve scar formation to promote functional recovery of injured sciatic nerve.

A systematic investigation on animal models of cyclosporine A combined with Escherichia coli to simulate the immunosuppressive status of sepsis patients before onset.

Immunosuppression is an important mechanism for the development of sepsis pathology, and is the key to the high mortality of sepsis. However, patients appear to be immunocompromised before sepsis onset due to lack of enough attention. Present sepsis models cannot fully mimic the onset of sepsis in patients. Hence, effective treatments in animal experiments could not be transformed into clinical application. In the present study, we improved the animal model of sepsis and used cyclosporine A immunosuppressive mice to make it closer to immune status before the onset of sepsis, followed by the intraperitoneal injection of Escherichia coli (E. coli) CMCC (B) 44,102 standard strain to produce the immunocompromised sepsis model. This trial systematically evaluates the new immunosuppressive sepsis model. Compared with routine sepsis models, the release of inflammatory factors in the new sepsis model was insufficient, blood bacteria were more cultured, diffuse intravascular coagulation (DIC) was more severe, lung, liver and kidney damage were heavier, and mortality rate was higher. In conclusion, the new sepsis model can mimic the patient's pre-onset immunocompromised state, is suitable for the development and evaluation of new methods of sepsis, and solves the controversy of sepsis treatment, providing new ideas and direction.

Exendin-4 Plays a Protective Role in a Rat Model of Spinal Cord Injury Through SERCA2.

BACKGROUND/AIMS: Current therapies for spinal cord injury (SCI) have limited efficacy, and identifying a therapeutic target is a pressing need. Sarcoplasmic/endoplasmic reticulum Ca2+ ATPase 2 (SERCA2) plays an important role in regulating calcium homeostasis, which has been shown to inhibit apoptosis. Exendin-4 has been shown to inhibit the apoptosis of nerve cells in SCI, which can also improve SERCA2 expression. In this study, we sought to determine whether exendin-4 plays a protective role in a rat model of SCI via SERCA2. METHODS: To investigate the effects of exendin-4 on SCI, a rat model of SCI was induced by a modified version of Allen's method. Spinal cord tissue sections from rats and western blot analysis were used to examine SERCA2 expression after treatment with the long-acting glucagon-like peptide 1 receptor exendin-4 or the SERCA2 antagonist 5(6)-carboxyfluorescein diacetate N-succinimidyl ester (CE). Locomotor function was evaluated using the Basso Beattie Bresnahan locomotor rating scale and slanting board test. RESULTS: Cell apoptosis was increased with CE treatment and decreased with exendin-4 treatment. Upregulation of SERCA2 in female rats with SCI resulted in an improvement of motor function scores and histological changes. CONCLUSION: These findings suggest that exendin-4 plays a protective role in a rat model of SCI through SERCA2 via inhibition of apoptosis. Existing drugs targeting SERCA2 may be an effective therapeutic strategy for the treatment of SCI.

Modified enzyme-linked immunosorbent assay strategy using graphene oxide sheets and gold nanoparticles functionalized with different antibody types.

Gold nanoparticles (GNPs) and graphene oxide (GO) sheets are excellent nano carriers in many analytical methods. In this study, a modified enzyme-linked immunosorbent assay (ELISA) strategy was developed using antibody-functionalized GO sheets and GNPs. This modification significantly reduced the limit of detection (LOD) and cost greatly of this assay. The applicability of the method was demonstrated by detecting HSP70 in a human serum sample. This result suggests that the 3G-ELISA method is feasible to detect an antigen in a complex mixture, and the LOD is up to 64-fold and the cost is as low as one-tenth of the conventional ELISA method.

A sensitive dual signal amplification method for western blotting based on antibody-functionalised graphene oxide and gold nanoparticles.

In this study, we report an ultrasensitive western blotting method using antibody-functionalised graphene oxide sheets and gold nanoparticles. Additionally, the cost is reduced greatly by conjugating two different primary antibodies on gold nanoparticles (C. Welinder and L. Ekblad, J. Proteome Res., 2011, 10, 1416-1419).

[Preparation and characterization of monoclonal antibody against HCCR protein].

AIM: Preparation of monoclonal antibody (mAb) to HCCR, which is a candidate biomarker for human hepatocellular carcinoma (HCC). METHODS: The recombinant protein HCCR-1(167-360); was expressed and was used as immunogen to immunize mouse for generation of mAb against HCCR. The protein Ep-HCCR, which displayed a epitope of HCCR, was also expressed and purified to use to detect serum antibody titer and to screen the positive clones of hybridmas. The properties of HCCR antibody were analyzed by ELISA, Western blot, immunofluorescence and immunohistochemistry. RESULTS: A hybridmas clone, which secreted anti-HCCR mAb, was obtained. The affinity constant (Kaff) of the mAb is 5.4×10(6); L/mol analyzed by ELISA; Western blot showed that the mAb could specifically recognize HCCR-1 and HCCR-2 expressed in HepG2 cells; The mAb was also used to detect the expression of HCCR proteins in hepatoma cells and HCC tissues. The results of immunofluorescence indicated that HCCR proteins mainly localized on the plasma membrane and cytoplasm of HepG2 cells. In addition, HCCR was found high-expressed in HCC tissues but not in normal liver tissue detected by Immunohistochemistry. CONCLUSION: A specific mAb against HCCR was successfully generated, which laid the foundation for establishing HCC detection method based on HCCR.

[Generation of monoclonal antibody to GP73 based on antigen epitope].

AIM: Preparation of monoclonal antibody (mAb) against GP73 protein. METHODS: The N-terminal peptide (AAAERGAVELK) of GP73 protein was displayed on T7 phage, the recombinant phage was amplified and used as the immunogen to immunize mouse to produce antibody. The titer of the antiserum and the positive hybridoma clones which secreted the mAb against GP73 protein were detected by ELISA. The mAb specificity was assayed by ELISA and Western blot. RESULTS: The high specificity mAb against GP73 protein was selected from the mouse immunized with the recombinant T7 phages displaying the epitope of GP73 by cell fusion and screening. CONCLUSION: The appropriate protein epitope displayed on T7 phage could be used as alternative antigen to immunize animals to make specific antibody against the corresponding native protein.

[Production and identification of a monoclonal antibody against human HPPCn].

AIM: To make monoclonal antibody(mAb) against human HPPCn for the use in research on HPPCn's function and its relationship with liver diseases. METHODS: The female BALB/c mice were immunized with the recombinant HPPCn proteins. Splenocytes and Sp2/0 cells were fused with PEG-1500. The positive clone was identified through indirect ELISA and then subcloned by limited dilution. Indirect ELISA, Western blot and Ig sub-class identification kit were used to identify the mAb's properties. By immunofluorescence experiments, we studied the cellular localization of HPPCn. The mAb epitope was also analyzed using peptide phage display technology. RESULTS: An anti-HPPCn mAb, named W2-D5, was obtained. It belongs to IgG1 subclass. It could specially bind to human HPPCn. Furthermore, by immunofluorescence results, wo confirmed HPPCn located in the nucleus and our mAb could combined with the natural protein. With the mAb, the minimal detectable concentration was 0.1 µg/L for HPPCn; The peptide sequence of HPPCn7₋13;(IHLELRN)was identified as the epitope of the mAb. CONCLUSION: An anti-HPPCn mAb with high specificity and high affinity was successfully obtained.