TCR repertoire intratumor heterogeneity of CD4+ and CD8+ T cells in centers and margins of localized lung adenocarcinomas.

Intratumor heterogeneity (ITH) of T cell receptor (TCR) repertoire in different T-cell subsets and locations in lung adenocarcinomas was unclear. Here, we investigated percentages and TCR repertoire of freshly isolated CD4+ and CD8+ tumor infiltrating lymphocytes (TILs) in tumor centers and margins by flow cytometry on 80 tumor samples from 20 patients and high-throughput TCR sequencing on 27 and 25 samples of CD4+ and CD8+ TILs from seven patients. Our results demonstrated that amount and TCR repertoire diversity of CD4+ TILs were significantly higher than those of CD8+ TILs and moreover substantial ITH regarding amount and TCR repertoire of CD4+ and CD8+ TILs were observed. Additionally, ITH of CD4/CD8 T-cell ratio and CD8+ TIL repertoire across center regions was lower than that across margin regions. The amount and TCR repertoire ITH of CD4+ and CD8+ TILs and mean clonality of CD8+ TILs in tumor centers were associated with relapse. Our study provides insights into amount and TCR repertoire ITH of CD4+ and CD8+ TILs in tumor centers and margins as well as corresponding association with prognosis in lung adenocarcinoma patients, suggesting potential clinical significance of TCR repertoire.

Effectiveness of Intensive Endoscopic Screening for Esophageal Cancer in China: A Community-Based Study.

Evidence is required to evaluate the effectiveness of population-level endoscopic screening for esophageal cancer (EC). In this study, 5,632 permanent residents aged 25-65 years from 6 villages in Hua County, Henan Province, China, were defined as the screening cohort and were offered intensive endoscopic screening. Residents of all 914 remaining villages in Hua County were included as the control cohort, and age-sex standardization was used to calculate the expected numbers of EC and upper gastrointestinal (GI) tract cancer cases and deaths in the screening cohort. The effectiveness of screening was assessed by comparing observed numbers of cases and deaths with expected numbers after 9-year follow-up of these screened subjects (2007-2016). In the screening cohort, 23 upper GI cancers (including 16 ECs) and 10 upper GI cancer deaths (including 5 EC deaths) were identified, and 47% (standardized incidence ratio = 0.53, 95% confidence interval (CI): 0.33, 0.87) and 66% (standardized mortality ratio = 0.34, 95% CI: 0.14, 0.81) reductions in cumulative EC incidence and mortality were found. For upper GI cancers, incidence and mortality were lowered by 43% (standardized incidence ratio = 0.57, 95% CI: 0.38, 0.86) and 53% (standardized mortality ratio = 0.47, 95% CI: 0.25, 0.88), respectively. This study showed that upper GI tract endoscopy is an effective population-level screening test for EC in high-risk regions.

Enhancement of gefitinib-induced growth inhibition by Marsdenia tenacissima extract in non-small cell lung cancer cells expressing wild or mutant EGFR.

BACKGROUND: Non-small cell lung cancer (NSCLC) expressed high levels of epidermal growth factor receptor (EGFR). Gefitinib (Iressa) has demonstrated clinical efficacy in NSCLC patients harboring EGFR mutations or refractory to chemotherapy. However, most of NSCLC patients are with wild type EGFR, and showed limited response to gefitinib. Therefore, to develop new effective therapeutic interventions for NSCLC is still required. Our previous study showed Marsdenia tenacissima extract (MTE) restored gefitinib efficacy in the resistant NSCLC cells, but whether MTE acts in the gefitinib-sensitive NSCLC cells is the same as it in the resistant one is unknown. METHODS: Dose response curves for gefitinib and MTE were generated for two sensitive NSCLC cell lines with mutant or wild type EGFR status. Three different sequential combinations of MTE and gefitinib on cell growth were evaluated using IC50 and Combination Index approaches. The flow cytometric method was used to detect cell apoptosis and cell cycle profile. The impact of MTE combined with gefitinib on cell molecular network response was studied by Western blotting. RESULTS: Unlike in the resistant NSCLC cells, our results revealed that low cytotoxic dose of MTE (8 mg/ml) combined gefitinib with three different schedules synergistically or additively enhanced the growth inhibition of gefitinib. Among which, MTE→MTE+gefitinib treatment was the most effective one. MTE markedly prompted cell cycle arrest and apoptosis caused by gefitinib both in EGFR mutant (HCC827) and wild type of NSCLC cells (H292). The Western blotting results showed that MTE→MTE+gefitinib treatment further enhanced the suppression of gefitinib on cell growth and apoptosis pathway such as ERK1/2 and PI3K/Akt/mTOR. This combination also blocked the activation of EGFR and c-Met which have cross-talk with each other. Unlike in gefitinib-resistant NSCLC cells, MTE alone also demonstrated certain unexpected modulation on EGFR related cell signal pathways in the sensitive cells. CONCLUSION: Our results suggest that MTE is a promising herbal medicine to improve gefitinib efficacy in NSCLC regardless of EGFR status. However, why MTE acted differently between gefitinib-sensitive and -resistant NSCLC cells needs a further research.

Single nucleotide polymorphisms of CD20 gene and their relationship with clinical efficacy of R-CHOP in patients with diffuse large B cell lymphoma.

BACKGROUND: R-CHOP has significantly improved survival rates of patients with diffuse large B cell lymphoma (DLBCL) by ~20% as compared to CHOP. CD20 antigen, highly expressed on more than 80% of B-cell lymphomas, is the target for rituximab. The goal of our study was to examine polymorphism in the CD20 gene in Chinese DLBCL population and whether CD20 gene polymorphism is associated with clinical response to R-CHOP. METHOD: CD20 gene polymorphism was detected in the entire coding regions including 6 exons by polymerase chain reaction (PCR)-sequencing assay in 164 patients with DLBCL. Among them, 129 patients treated with R-CHOP as frontline therapy (R ≥ 4 cycles) were assessable for the efficacy. RESULTS: Polymorphisms at three single nucleotides (SNP) were identified in the entire coding regions of the CD20 gene in the 164 patients. One of them, CD20 Exon2 [216] was found to be highly correlated with response to R-CHOP. Patients with homozygous C genotype showed a trend toward higher overall response rate than others with CT plus TT genotype (90.6% vs. 79.5%; P =0.166). A trend toward higher complete remission (CR) rate was observed in patients with homozygous C genotype (67.4%) compared with CT plus TT genotype (47.1%) (P = 0.091). CONCLUSION: These results suggest that there are 3 SNPs in CDS of the CD20 gene in Chinese DLBCL population. The CC genotype at Exon2 [216] appears to be associated with favourable response to R-CHOP.

Assessing patient safety culture in obstetrics ward: A pilot study using a modified Manchester Patient Safety Framework in China.

AIM: The primary objective of this study was to assess the patient safety culture in a general hospital in Shanghai, China, through a modified Manchester Patient Safety Framework (MaPSaF). DESIGN: This study has a qualitative interview design. Data were collected through group interviews and analyses performed through content analysis. METHODS: The MaPSaF was translated into Chinese and used to assess the patient safety culture in a large general hospital in Shanghai, China. Group interviews using the MaPSaF were conducted with 15 nurses in the obstetric ward. Participants rated their safety practice individually on each of the nine MaPSaF safety culture dimensions. The dimensions and scores were then collectively discussed and a practice-wide consensus score for each dimension was agreed. Discussions were recorded, transcribed and analysed to assess patient safety in the obstetric ward. RESULTS: It took about 2 hr to complete the discussion focusing on patients' safety employing the MaPSaF. Most participants recognized the process as acceptable and useful. The MaPSaF directed team discussion about patient safety issues and facilitated communication, prompting some practice changes. All participants responded positively to the discussion and perceived MaPSaF as a good safety culture assessment tool, with clear, comprehensive and understandable entries. The process demonstrated that the department of obstetrics in the hospital already had a positive patient safety culture, but certain areas were highlighted as still needing improvement. Based on participants' positive experience and perception of the MaPSaF, it can be concluded that there is potential benefit in its adaptation and use in obstetrics wards of Chinese hospitals. The MaPSaF has the potential to strengthen existing safety cultures and improve general safety through collaborative measures.

Research on the Internal Flow Field of Left Atrial Appendage and Stroke Risk Assessment with Different Blood Models.

Extant clinical research has underscored that patients suffering from atrial fibrillation (AF) bear an elevated risk for stroke, predominantly driven by the formation of thrombus in the left atrial appendage (LAA). As such, accurately identifying those at an increased risk of thrombosis becomes paramount to facilitate timely and effective treatment. This study was designed to shed light on the mechanisms underlying thrombus formation in the LAA by employing three-dimensional (3D) left atrium (LA) models of AF patients, which were constructed based on Computed Tomography (CT) imaging. The distinct benefits of Computational Fluid Dynamics (CFD) were leveraged to simulate the blood flow field within the LA, using three distinct blood flow models, both under AF and sinus rhythm (SR) conditions. The potential risk of thrombus formation was evaluated by analyzing the Relative Residence Time (RRT) and Endothelial Cell Activation Potential (ECAP) values. The results gleaned from this study affirm that all three blood flow models align with extant clinical guidelines, thereby enabling an effective prediction of thrombosis risk. However, noteworthy differences emerged when comparing the intricacies of the flow field and thrombosis risk across the three models. The single-phase non-Newtonian blood flow model resulted in comparatively lower residence times for blood within the LA and lower values for the Oscillatory Shear Index (OSI), RRT, and ECAP within the LAA. These findings suggest a reduced thrombosis risk. Conversely, the two-phase non-Newtonian blood flow model exhibited a higher residence time for blood and elevated RRT value within the LAA, suggesting an increased risk for thrombosis.

Left atrial appendage closure in patients with reversed chicken-wing morphology: Anatomical features and procedural strategy.

Background: Left atrial appendage (LAA) closure (LAAC) in atrial fibrillation (AF) patients with the reversed chicken-wing (RCW) LAA is challenging. Aims: To elucidate the LAAC strategy of the RCW-LAA. Methods: A total of 802 AF patients who were enrolled in the LAACablation registry for LAAC procedure were included, 55 of whom presented with the RCW-LAA. The WATCHMAN device was implanted using the standard protocol when the sheath depth was no less than the device depth (the simple group). For those with a sheath depth of less than the device depth (the complex group), device deployment was attempted with acceptable protrusion or after a repeated atrial transseptal puncture (re-ATP) at a more inferior and anterior position. The anatomical and procedural features were compared between groups and before and after the re-ATP. Results: The success rate of LAAC was significantly lower in patients with the RCW-LAA than with the other morphologies (92.7% vs. 98.8%, p = 0.001). Compared with the simple group, the complex group had shorter root depth and shorter neck length, and more LAAs in the complex group were at lower position (all p < 0.05). The sheath depth after the re-ATP was significantly greater than that before the re-ATP (18.8 ± 3.4 mm vs. 14.7 ± 2.6 mm, p < 0.001). For the patients who underwent re-ATP, the sheath went significantly deeper in successful procedures than in aborted procedures (19.7 ± 3.3 mm vs. 15.8 ± 1.8 mm, p = 0.040). Conclusions: The anatomical features of the RCW-LAA were related to the complexity of the LAAC procedure. The re-ATP at an inferior and anterior location could increase the success rate of LAAC. ClinicalTrialsgov: NCT03788941.

Translation, Cultural Adaptation, Reliability, and Validity Testing of a Chinese Version of the Self-Administered Mediterranean Diet Scale.

Mediterranean Diet management for people with cardiovascular disease (CVD) or CVD risk is supported by evidence. However, there is no valid Chinese language instrument for the measurement of adherence to this diet. The objective of this study was to generate a Chinese version of the Mediterranean Diet Scale (MDS-Chinese) and to validate a self-administered version with Chinese participants with CVD or CVD risk. The MDS-Chinese was created by translation and cultural adaptation and tested for psychometric properties. A panel of 10 experts in the field, who evaluated the MDS-Chinese content, showed that the content validity index ranged from 0.88 to 1.00. Sixteen native Chinese speakers with CVD or CVD risk evaluated the clarity of the MDS-Chinese, and the resulting instruction and items clarity scores ranged from 9.2 to 10.0. A total of 326 participants completed the MDS-Chinese and a Chinese version of the Coronary Artery Disease Education Questionnaire-Short Version (CADE-Q SV). Analysis indicated that the MDS-Chinese has 4 factors, and the Pearson's correlation between the MDS-Chinese and CADE-Q SV was 0.73. Fifty randomly selected participants completed the MDS-Chinese again with a 1-week interval to assess reliability. Internal consistency was acceptable (Cronbach's α was 0.62) and the inter-class correlation reliability coefficients (ICC) for each item ranged from 0.73 to 0.88. This study showed that the MDS-Chinese has acceptable reliability and validity for use among those in the Chinese population with CVD or CVD risk. Given that diet is one of the key secondary prevention strategies for management in cardiac rehabilitation, the MDS-Chinese instrument may be a useful and convenient tool for use with those in the Chinese population with CVD or with high risk of CVD, to monitor the level of Mediterranean diet (MD) adherence, information which is important for clinical practice. In addition, the establishment of the MDS-Chinese gives a fundamental tool for diet-related CVD research in the Chinese population. Moreover, employment of the MDS-Chinese in the Chinese community may improve awareness of the importance of a healthy diet in CVD prevention and management. Clinical Trial Registration: http://www.chictr.org.cn/enIndex.aspx, identifier: ChiCTR2000032810.

Clonal Distribution and Intratumor Heterogeneity of the TCR Repertoire in Papillary Thyroid Cancer With or Without Coexistent Hashimoto's Thyroiditis.

The intratumor heterogeneity (ITH) of the amount and TCR repertoires of tumor infiltrating lymphocytes (TILs) in PTC with and without coexistent Hashimoto's thyroiditis (HT) are unclear. Here, we investigated the amount of T cells in tumor and corresponding normal tissues by immunohistochemical staining on 80 tumor samples and 40 normal samples from 40 patients. The immune repertoire of T cells was identified on 24 tumor samples and 12 normal samples from 12 patients using TCR high-throughput sequencing. The results demonstrated that the numbers of CD3+, CD4+ and CD8+ T cells in PTC without coexistent HT (PTC-WO) were significantly lower than those in PTC with existing HT (PTC-W). In PTC-W, the density of CD4+ TILs were generally higher when compared with CD8+ TILs. Furthermore, we found that the numbers of CD3+ T cells and their CD4+, CD8+ subtypes in tumor samples were generally higher than those in normal tissue in PTC-WO and moreover, the number of CD3+ T cells was negatively associated with TCR clonality in PTC-WO. In addition, although ITH of the TCR repertoire truly existed in PTC-W and PTC-WO, the TCR repertoires between distinct regions of the non-adjacent tumor foci were presented with a higher degree of similarity than those between tumor and matched normal tissue in PTC-WO, yet the similarity of intratumor repertoires was not significantly higher than those between tumor and corresponding normal samples in PTC-W. This research comprehensively delineated the quantity and TCR repertoire ITH of T cells in PTC-W and PTC-WO, suggesting that TILs might be reactive to tumor antigens in PTC-WO. Moreover, multiregion biopsies should be performed to precisely identify the immune background in PTC-W and PTC-WO.

Research on the difference between patients with coronary heart disease and healthy controls by surface enhanced Raman spectroscopy.

Coronary heart disease (CHD) is one of the primary causes of death globally. There are several diagnostic techniques for CHD at present, but they are invasive and with limited accuracy. In the work, measurement of human urine based on surface-enhanced Raman spectroscopy (SERS) was proposed to diagnose CHD. Urine samples of 157 CHD patients and 63 healthy controls (HC) were investigated by SERS. Statistical analysis of the measured data was then performed. It was found that there were intensity differences in nine Raman peaks (1223/1243/1272/1463/1481/1516/1536/1541/1550 cm-1) between CHD and HC in their average SERS spectrum. Furthermore, principal component analysis (PCA)-linear discriminant analysis (LDA) was then utilized to establish a prediction model to classify CHD and HC. It revealed that the accuracy, specificity and sensitivity of the prediction model validated by leave-one-patient-out cross validation (LOPOCV) were 84.09%, 92.06% and 80.89%, respectively. Therefore, the proposed method can be employed as a non-invasive, rapid and accurate tool for CHD diagnosis in clinical application.

Numerical study of the risk of thrombosis in the left atrial appendage of chicken wing shape in atrial fibrillation.

Atrial fibrillation (AF) is a common and life-threatening disease. For the patients with AF, more than 90% of the thrombi are formed in the left atrial appendage (LAA), thrombus dislodgement can cause vascular embolism, making them is becoming a high-risk group for stroke. Therefore, identifying the patients with high risk of thrombosis is crucial for advanced stroke warning. To better investigate the mechanism behind thrombus formation in the LAA, this study reconstructed the 3-D Left Atrium (LA) models of six AF volunteer patients by corresponding Computed Tomography (CT) images. Combine the advantages of Computational Fluid Dynamics (CFD), the blood flow field in LA both in AF and sinus heart rate states were studied. The risk of thrombus was evaluated based on the blood viscosity, shear rate thrombus prediction model and Time Average Wall Shear Stress (TAWSS), Oscillatory Shear Index (OSI), and Relative Residence Time (RRT) values. The results showed that the left atrium had lower blood flow velocity and TAWSS values at the LAA in both AF and sinus rhythm, thus the LAA is the most thrombogenic region in the LA. Besides, the RRT value of LAA was generally higher in AF than in sinus rhythm. Therefore, AF carries a higher risk of thrombosis.

Latexin expression is downregulated in human gastric carcinomas and exhibits tumor suppressor potential.

BACKGROUND: Latexin, also known as endogenous carboxypeptidase inhibitor (CPI), has been found to inhibit mouse stem cell populations and lymphoma cell proliferation, demonstrating its potential role as a tumor suppressor. Our previous study also suggested a correlation between latexin expression and malignant transformation of immortalized human gastric epithelial cells. Here, we examined latexin expression in human gastric carcinomas and investigated the effect of differential latexin expression on proliferation of gastric cancer cells in vitro and in vivo. METHODS: Monoclonal antibody against human latexin was prepared and immunohistochemical analysis was performed to detect latexin expression in 41 paired gastric carcinomas and adjacent normal control tissues. Human gastric cancer cells MGC803 (latexin negative) stably transfected with LXN gene and BGC823 cells (latexin positive) stably transfected with antisense LXN gene were established for anchorage-dependent colony formation assay and tumorigenesis assay in nude mice. Differentially expressed genes in response to exogeneous latexin expression were screened using microarray analysis and identified by RT-PCR. Bisulfite sequencing was performed to analyze the correlation of the methylation status of LXN promoter with latexin expression in cell lines. RESULTS: Immunohistochemical analysis showed significantly reduced latexin expression in gastric carcinomas (6/41, 14.6%) compared to control tissues (31/41, 75.6%) (P < 0.05). Overexpression of LXN gene in MGC803 cells inhibited colony formation and tumor growth in nude mice. Conversely, BGC823 cells transfected with antisense LXN gene exhibited enhanced tumor growth and colony formation. Additionally, several tumor related genes, including Maspin, WFDC1, SLPI, S100P, and PDGFRB, were shown to be differentially expressed in MGC803 cells in response to latexin expression. Differential expression of Maspin and S100P was also identified in BGC823 cells while latexin expression was downregulated. Further bisulfite sequencing of the LXN gene promoter indicated CpG hypermethylation was correlated with silencing of latexin expression in human cells. CONCLUSIONS: Latexin expression was reduced in human gastric cancers compared with their normal control tissues. The cellular and molecular evidences demonstrated the inhibitory effect of latexin in human gastric cancer cell growth and tumorigenicity. These results strongly suggest the possible involvement of latexin expression in tumor suppression.

[A methodology study on flow cytometric analysis of cell DNA stained with DAPI and Hoechst33342].

OBJECTIVE: To discuss a simple method for flow cytometric analysis of cell DNA stained with DAPI and Hoechst33342. METHODS: HT 29 cells stained with DAPI, Hoechst33342 or PI were measured by BD FACSAria and the percentages of cells in G0/G1, S and G2/M phases with three staining methods, then the results were analyzed and compared. Before measurement we monitored the quality of DNA analysis of flow cytometer through UV beads QC experiment and analyzed the standard chicken erythrocyte nuclei (CEN) and calf thymocyte nuclei (CTN) stained with DAPI and Hoechst33342. RESULTS: CV value of UV peak was 2.4 after QC experiments. There were 4 peaks on CEN histograms and the ratios of peak channel mean of G2/G1, G3/G1, and G4/G1 were about 2, 3, and 4 respectively. Both CV values of the first peak were 2.4. There were 2 peaks on CTN histograms and the ratio of peak channel mean of G2/G1 was 1.97, and CV value of G0/G1 2.4. The complete cell cycle of HT29 cells stained with DAPI, Hoechst33342 or PI was showed entirely, CV values were 3.40, 3.02 and 4.42, respectively, and the percentages of cells in G0/G1 were 60.86%, 60.22% and 60.81%,respectively, in S, 28.85%, 29.70% and 29.82%,respectively, and in G2/M, 10.29%, 9.09% and 9.37%, respectively. The results by the three methods showed no difference. CONCLUSION: This method for measurement of cellular DNA content is a simple and efficient approach to determining cell cycle and can be the first choice when using flow cytometer with 355 nm UV.

TfR1 binding with H-ferritin nanocarrier achieves prognostic diagnosis and enhances the therapeutic efficacy in clinical gastric cancer.

H-ferritin (HFn) nanocarrier is emerging as a promising theranostic platform for tumor diagnosis and therapy, which can specifically target tumor cells via binding transferrin receptor 1 (TfR1). This led us to investigate the therapeutic function of TfR1 in GC. The clinical significance of TfR1 was assessed in 178 GC tissues by using a magneto-HFn nanoparticle-based immunohistochemistry method. The therapeutic effects of doxorubicin-loaded HFn nanocarriers (HFn-Dox) were evaluated on TfR1-positive GC patient-derived xenograft (GC-PDX) models. The biological function of TfR1 was investigated through in vitro and in vivo assays. TfR1 was upregulated (73.03%) in GC tissues, and reversely correlated with patient outcome. TfR1-negative sorted cells exhibited tumor-initiating features, which enhanced tumor formation and migration/invasion, whereas TfR1-positive sorted cells showed significant proliferation ability. Knockout of TfR1 in GC cells also enhanced cell invasion. TfR1-deficient cells displayed immune escape by upregulating PD-L1, CXCL9, and CXCL10, when disposed with IFN-γ. Western blot results demonstrated that TfR1-knockout GC cells upregulated Akt and STAT3 signaling. Moreover, in TfR1-positive GC-PDX models, the HFn-Dox group significantly inhibited tumor growth, and increased mouse survival, compared with that of free-Dox group. TfR1 could be a potential prognostic and therapeutic biomarker for GC: (i) TfR1 reversely correlated with patient outcome, and its negative cells possessed tumor-aggressive features; (ii) TfR1-positive cells can be killed by HFn drug nanocarrier. Given the heterogeneity of GC, HFn drug nanocarrier combined with other therapies toward TfR1-negative cells (such as small molecules or immunotherapy) will be a new option for GC treatment.

E3 ubiquitin ligase E6AP-mediated TSC2 turnover in the presence and absence of HPV16 E6.

We previously found that HPV16 E6 causes the degradation of the tumor suppressor protein TSC2, resulting in the phosphorylation of S6 kinase and S6 even in the absence of insulin. In the present study, we investigated the role of E6-associated protein (E6AP) in HPV16 E6-induced TSC2 degradation. Our results demonstrated that TSC2 was targeted for degradation in the presence or absence of HPV16 E6. Over-expression of E6AP enhanced the degradation of TSC2 by HPV16 E6, while expression of a dominant negative E6AP (C833A) inhibited the E6-induced degradation. Additionally, by using shRNAs to block E6AP expression in HPV16 positive and negative cells, we found a significantly prolonged TSC2 half-life. An in vivo ubiquitination assay was done to reveal that E6AP promoted the ubiquitination of TSC2 independent of HPV16 E6. We further found that TSC2 bound E6AP in the presence as well as in the absence of HPV16 E6. The binding regions on E6AP and TSC2 have been identified as amino acid (aa) 260-316, aa 428-500 and aa 1-175, aa 1251-1807, respectively. Taken together, degradation of TSC2 is mediated by E6AP ubiquitin ligase.

Influence of drugs on the prospective diagnostic method for coronary heart disease with urine.

The morbidity of coronary heart disease (CHD) with high risks has been rising in recent years. A novel and noninvasive method based on surface-enhanced Raman spectroscopy (SERS) was proposed by Yang et al. (Analyst 143: 2235, 2018) to prospectively diagnose the arterial blockage by detecting platelet-derived growth factor-BB (PDGF-BB) in urine. Clinically, anti-platelet drugs (such as aspirin, statins and clopidogrel) are often used for ordinary CHD patients or patients with percutaneous coronary intervention (PCI). Therefore, whether the previous developed method can be applied to the CHD patients on long-term medication (more than 6 months) or post-PCI patients was investigated here. Firstly, urine samples of 13 CHD patients on long-term medication (aspirin, rosuvastatin, clopidogrel bisulfate) and 13 post-PCI patients were measured by the proposed method. Clinical data of coronary angiography results provided by Xin Hua Hospital and Yangpu District Central Hospital Antu Branch revealed that these 26 patients were with serious arterial blockage, however, characteristic Raman peak at 1509 cm-1 attributed to PDGF-BB was not observed in the SERS spectra of these 26 patients. In addition, an eight-day follow-up investigation was performed on a CHD patient with PCI three years ago and on long-term medication. It was found that the Raman peak at 1509 cm-1 could be only observed in the third and fourth day after suspending the drugs. Furthermore, SERS spectra of mixed solutions of PDGF-BB and aspirin, rosuvastatin, mixed solutions of these two drugs and clopidogrel bisulfate were analyzed. The Raman peak at 1509 cm-1 was not found in all these spectra, it indicated that all the three kinds of drugs could influence on the SERS signal of PDGF-BB. Therefore, the previous developed method is not suitable for CHD patients on long-term medication and post-PCI patients.

Downregulation of TfR1 promotes progression of colorectal cancer via the JAK/STAT pathway.

Background: Colorectal cancer (CRC) is one of the most prevalent gastrointestinal malignancies. The incidence of CRC has been rapidly increasing in China. Transferrin receptor 1 (TfR1) is a key regulator of cellular iron homeostasis. Several studies have demonstrated TfR1 overexpression in a variety of human tumors, but the association between TfR1 and CRC remains unclear. Methods: TfR1 expression was evaluated in six CRC cell lines and tumor tissues. A total of 201 CRC patients were included for immunohistochemistry and 19 pairs of frozen tissues were used for real-time PCR. Cell proliferation, cell cycle, cell migration and invasion, and in vivo carcinogenesis were tested after downregulation of TfR1 by lentivirus. Protein microarray and Western blot analyses were used to explore the underlying mechanisms of TfR1 in CRC. Results: TfR1 expression was higher in CRC tissues than in normal tissues (57.2% vs 22.9%, P＜0.001). TfR1 expression was obviously higher in CRC tissues with well differentiation (P＝0.008), no lymph node metastasis (P＝0.002), no distant metastasis (P＝0.006), no vascular invasion (P＜0.001) and early TNM stage (P＝0.013). CRC patients with TfR1-positive expression had a better survival than those with TfR1-negative expression (P＝0.044). Downregulation of TfR1 expression inhibited cell proliferation, promoted cells from G1 phase to S phase and facilitated cell migration and invasion. Knockdown of TfR1 also suppressed tumor growth in BALB/C-nu mice. Protein microarray and Western blot analyses showed that the Janus protein tyrosine kinase/signal transducer and activator of transcription pathway was activated along with downregulation of TfR1 expression. Conclusion: Though TfR1 was overexpressed in colorectal cancer tissues, there was evidence that downregulation of TfR1 could promote cancer progression.

Interactions between human hemoglobin subunits and peroxiredoxin 2.

Red blood cells (RBCs) are exposed to exogenous reactive oxygen species in the circulatory system. To this end, the interactions between the different hemoglobin (Hb) subunits and peroxiredoxin 2, which is a ubiquitous member of the antioxidant enzymes that also controls the cytokine-induced peroxide levels, were assessed. We predicted by the increment of diversity with quadratic discriminant analysis (IDQD) that peroxiredoxin2 (Prx2) could interact with the hemoglobin alpha, beta and gamma subunits but not with the delta subunit. Coimmunoprecipitation (co-IP), electrospray ionization quadrupole time of flight (ESI-Q-TOF) mass spectrometry, Western blotting and X-ray absorption fine structure (XAFS) spectroscopy were performed to verify these predictions. The results showed that Prx2 was a member of the beta-globin immunoprecipitating complex that existed in hemoglobin A, hemolysate-hemoglobin A, hemoglobin A-hemoglobin A2, hemolysate-hemoglobin A-hemoglobin A2 and hemoglobin A2 but not in hemolysate-hemoglobin A2. Adding Prx2 to hemoglobin A altered the second shell of iron embedded in hemoglobin A. Therefore, Prx2 interacts with hemoglobin A (Alpha2Beta2) and hemoglobin F (Alpha2Gamma2) but not with hemoglobin A2 (Alpha2Delta2).

Plasma CAMK2A predicts chemotherapy resistance in metastatic triple negative breast cancer.

BACKGROUND: Chemotherapy resistance is a great obstacle in effective treatment for metastatic triple negative breast cancer (TNBC). The ability to predict chemotherapy response would allow chemotherapy administration to be directed toward only those patients who would benefit, thus maximizing treatment efficiency. Differentially expressed plasma proteins may serve as putative biomarkers for predicting chemotherapy outcomes. PATIENTS AND METHODS: In this study, 26 plasma samples (10 samples with partial response (S) and 16 samples with progression disease (R)) from patients with metastatic TNBC were measured by Tandem Mass Tag (TMT)-based proteomics analysis to identify differentially expressed proteins between the S and R group. Potential proteinswere validated with enzyme-linked immunosorbent assay (ELISA) in another 67 plasma samples. RESULTS: A total of 320 plasma proteins were identified, and statistical analysis showed that 108 proteins were significantly dysregulated between R and S groups in the screening stage. Bioinformatics revealed relevant pathways and regulatory networks of the differentially expressed proteins. Three differentially expressed proteins were validated by ELISA with 67 samples from TNBC patients. The R group had significantly higher plasma CAMK2A level than the S group (P=0.0074). The ROC curve analysis showed an AUC of 0.708, with sensitivity 48.4% and specificity 86.1%. In multivariate logistic regression analysis, the level of plasma CAMK2A was also significant for chemotherapeutic response (P=0.009, OR=0.152). Furthermore, the patients with higher CAMK2A level had shorter OS than those with lower CAMK2A level, which amounted to 13.9 and 28.9 months, respectively (P=0.034). In the multivariate Cox regression analysis, CAMK2A level still had significant effect on OS (P=0.031, HR=1.865). CONCLUSION: TMT-based proteomic analysis was able to identify potential biomarkers in plasma that predicted chemotherapy resistance in the metastatic TNBC. The plasma of CAMK2A level may serve as apotential predictive and prognostic biomarker for chemotherapy in metastatic TNBC.

Antitumor effects of Xi Huang pills on MDA­MB­231 cells in vitro and in vivo.

The management of patients with triple­negative breast cancer is challenging due to the lack of effective therapeutic options, aggressive behavior and relatively poor prognosis. Xi Huang pills (XHP) are a well­known traditional Chinese medicine that demonstrate anticancer activities. The aim of the present study was to investigate the antitumor effects of XHP on MDA­MB­231 cells in vitro and in vivo, and its potential underlying molecular mechanisms. In the present study, an MTT assay was used to evaluate the antiproliferative activity of XHP on MDA­MB­231 cells. In order to investigate the effects further, cell cycle distribution, apoptosis and mitochondrial membrane potential assays were performed, as well as western blot analyses. In addition, a tumor xenograft model was employed to investigate the effects of XHP in vivo. The results of the MTT assay demonstrated that the viability of MDA­MB­231 cells was markedly inhibited by XHP in a dose­ and time­dependent manner. The inhibitory effect of XHP on the viability of MDA­MB­231 cells was greater when compared with MCF­10A cells. An increase in apoptosis and loss of mitochondrial membrane potential was observed following 4, 8 and 12 mg/ml XHP treatment of MDA­MB­231 cells. The protein expression levels of cleaved caspase­3 were increased by 1.62­, 2.13­ and 2.19­fold, respectively, when compared with the untreated controls, whereas no effects on the expression of B­cell lymphoma 2 (Bcl­2) or Bcl­2­associated X protein (Bax) were observed. The results of the cell cycle distribution assay analysis demonstrated that XHP treatment arrested cells at the G2/M phase. In addition, XHP treatment decreased the expression of cyclin A and increased the expression of p21Cip1. In vivo experiments revealed that XHP inhibited the growth of MDA­MB­231 xenograft tumors without body weight loss, and demonstrated similar effects on the protein expression levels of cleaved caspase 3, cyclin A and p21Cip1 as observed in vitro. In conclusion, the viability of MDA­MB­231 cells was inhibited by XHP in a dose­dependent, time­dependent and cell­selective manner in vitro, and the potential underlying mechanisms may involve apoptosis and cell cycle arrest at the G2/M phase. XHP may induce apoptosis in MDA­MB­231 cells via the intrinsic pathway, which does not involve the Bcl­2/Bax ratio. G2/M phase arrest may have been due to the integrated action of decreased cyclin A expression and increased p21Cip1 expression. In addition, XHP inhibited the growth of xenograft tumors in the absence of body weight loss in vivo.