PLOD3 regulates the expression of YAP1 to affect the progression of non-small cell lung cancer via the PKCδ/CDK1/LIMD1 signaling pathway.

Procollagen-lysine, 2-oxoglutarate 5-dioxygenase (PLOD3) is a crucial oncogene in human lung cancer, whereas protein kinase C δ (PKCδ) acts as a tumor suppressor. In this study, we aimed to explore the regulation by PLOD3 on the expression of YAP1 to affect the progression of non-small cell lung cancer (NSCLC) via the PKCδ/CDK1/LIMD1 signaling pathway. We found that PLOD3, CDK1, and YAP1 were highly expressed, while LIMD1 was poorly expressed in NSCLC tissues. Mechanistic investigation demonstrated that silencing PLOD3 promoted the cleavage of PKCδ in a caspase-dependent manner to generate a catalytically active fragment cleaved PKCδ, enhanced phosphorylation levels of CDK1, and LIMD1 but suppressed nuclear translocation of YAP1. Furthermore, functional experimental results suggested that loss of PLOD3 led to increased phosphorylation levels of CDK1 and LIMD1 and downregulated YAP1, thereby suppressing the proliferation, colony formation, cell cycle entry, and resistance to apoptosis of NSCLC cells in vitro and inhibiting tumor growth in vivo. Taken together, these results show that PLOD3 silencing activates the PKCδ/CDK1/LIMD1 signaling pathway to prevent the progression of NSCLC, thus providing novel insight into molecular targets for treating NSCLC.

Role of thioredoxin nitration in bleomycin-induced pulmonary fibrosis in rats.

Oxidant stimulation has been suggested to play an important role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Our study aimed to investigate the role and mechanisms of thioredoxin (Trx) nitration during the development of IPF. A rat model of IPF was established by intratracheal instillation of bleomycin (BLM). Male Wistar rats were randomly distributed among the control group and BLM-treated group, in which rats were intratracheally instilled with a single dose of BLM (5.0 mg/kg body mass in 1.0 mL phosphate-buffered saline). At 7 or 28 days after instillation the rats were euthanized. Histopathological and biochemical examinations were performed. The activity and protein level of thioredoxin were assessed. The thioredoxin nitration level was determined using immunoprecipitation and immunoblotting techniques. Our results demonstrated that protein tyrosine nitration increased in the BLM-treated group compared with the control group. Trx activity decreased in the BLM group compared with control group, whereas Trx expression and nitration level increased dramatically in the BLM group compared with the control group. Our results indicated that Trx nitration might be involved in the pathogenesis of IPF.

MiR-195 targets HDGF to inhibit proliferation and invasion of NSCLC cells.

Non-small cell lung cancer (NSCLC) is one of the most common causes of cancer-related death worldwide. MicroRNAs (miRNAs) play critical roles in the development and progression of NSCLC. miR-195 acts as a tumor suppressor in several cancers, however, its role in NSCLC is not well understood. Herein, we found that miR-195 was significantly decreased in both NSCLC tissues and cell lines. Forced expression of miR-195 significantly suppressed proliferation, migration, and invasion of NSCLC cells. Hepatoma-derived growth factor (HDGF) was identified as a target of miR-195 in NSCLC cells. Overexpression of HDGF dramatically abolished the tumor suppressive role of miR-195 in NSCLC cells. Our results demonstrated a tumor suppressive role of miR-195 in NSCLC, and suggested a potential therapeutic target for NSCLC.

MiR-186 targets ROCK1 to suppress the growth and metastasis of NSCLC cells.

MicroRNAs (miRNAs) act as oncogenes or tumor suppressors in human cancers. Increasing evidence shows that deregulation of miRNAs contributes to the development and progression of human non-small cell lung cancer (NSCLC). Here, we identified miR-186 as a tumor suppressor in NSCLC, which was decreased in NSCLC. Overexpression of miR-186 significantly inhibited proliferation, migration, and invasion of NSCLC cells. In addition, Rho-associated protein kinase 1 (ROCK1) was identified as a target of miR-186 in NSCLC cells. Restoration of ROCK1 remarkably reversed the tumor-suppressive effects of miR-186 on cell proliferation, migration, and invasion in NSCLC cells. Furthermore, ROCK1 was inversely correlated with miR-186 expression in NSCLC. Collectively, our data indicate that miR-186 functions as tumor suppressor in NSCLC by targeting ROCK1.

MiR-96 downregulates RECK to promote growth and motility of non-small cell lung cancer cells.

MicroRNAs play critical roles in the development and progression of non-small cell lung cancer (NSCLC). miR-96 acts as an oncogene in some malignancies, while its role in NSCLC is unclear. Here, we validated that miR-96 was significantly increased in both human NSCLC tissues and cell lines. Inhibition of miR-96 expression remarkably reduced cell proliferation, colony formation, migration, and invasion of NSCLC cells. Reversion-inducing-cysteine-rich protein with kazal motifs (RECK) was identified as a target of miR-96 in NSCLC cells. In addition, the expression of RECK was found to be negatively correlated with the expression of miR-96 in NSCLC tissues. Our data suggest that miR-96 might promote the growth and motility of NSCLC cells partially by targeting RECK.

Inhibition of non-small-cell lung cancer cell proliferation by Pbx1.

Lung cancer is one of the most deadly human cancers and continues to be a major unsolved health problem worldwide. Here, we evaluate the function of Pbx1 in the proliferation of non-small-cell lung cancer (NSCLC). In contrast with its known proliferative function, we found that Pbx1 inhibits the proliferation of lung cancer cells. In particular, Pbx1-specific RNA interference resulted in increased proliferation in lung cancer cells. In addition, histone H3 phosphorylation was also increased following inhibition of Pbx1 expression. In contrast, Pbx1 overexpression repressed the proliferation of lung cancer cells and inhibited DNA synthesis. Collectively, our data indicate that Pbx1 inhibits proliferation in lung cancer cells, suggesting a complex role for Pbx1 in modulating the proliferation of cancer cells and making this protein a potential new target for lung cancer therapy.

Intermittent hypoxia-induced downregulation of microRNA-320b promotes lung cancer tumorigenesis by increasing CDT1 via USP37.

Obstructive sleep apnea-hypopnea (OSAH) is correlated with an increased incidence of lung cancer. In our study, we explored the functional roles of microRNAs (miRNAs) in lung cancer patients that were complicated with OSAH involving the deubiquitination enzyme. The miR-320b expression pattern in lung cancer tissues and cells was determined. The interactions between ubiquitin-specific peptidase 37 (USP37) and miR-320b were evaluated by a dual-luciferase reporter gene assay, whereas USP37 and Cdc10-dependent transcript 1 (CDT1) was assessed by co-immunoprecipitation and immunofluorescence. After the induction of intermittent hypoxia (IH), a gain-of function approach was performed to investigate roles of miR-320b, USP37, and CDT1 in lung cancer cell proliferation and invasion. In addition, nude mouse xenograft models were used to study their effects on tumor growth in vivo. miR-320b was poorly expressed in lung cancer patients with OSAH. IH treatment downregulated the expression of miR-320b but promoted the proliferation and invasion capabilities of lung cancer cells, both of which were suppressed by the overexpression of miR-320b through decreasing USP37. USP37 interacted with and deubiquitinated CDT1 to protect it from proteasomal degradation. Our study uncovered that IH-induced downregulation of miR-320b promoted the tumorigenesis of lung cancer by the USP37-mediated deubiquitination of CDT1.

LINC00184 silencing inhibits glycolysis and restores mitochondrial oxidative phosphorylation in esophageal cancer through demethylation of PTEN.

BACKGROUND: Total lesion glycolysis has been reported to be a satisfactory predictor of survival in patients with locally advanced esophageal cancer (EC). The aim of the present study is to investigate the function of long intergenic non-protein coding RNA 184 (LINC00184) on the EC cell glycolysis and mitochondrial oxidative phosphorylation (OXPHOS). METHODS: The expression of LINC00184 was determined to be highly expressed and PTEN was poorly expressed in EC tissues and cells by RT-qPCR. In order to evaluate the effects of LINC00184 on cellular process in vitro and in vivo, gain- and loss-of-function approaches were performed to alter the expression of LINC00184 and PTEN in EC cells. RESULTS: Silencing of LINC00184 was observed to inhibit the proliferation, migration, invasion, colony formation, and glycolysis of EC cells and tumour growth, while the mitochondrial OXPHOS was restored. By recruiting DNMT1, LINC00184 enhanced the promoter methylation of PTEN. Inhibition of PTEN promoter methylation suppressed EC glycolysis, whereas, improved mitochondrial OXPHOS. Mechanically, LINC00184 modulated glycolysis and mitochondrial OXPHOS in EC cells through induction of the Akt phosphorylation. After blockage of Akt signaling pathway by an Akt inhibitor, LY294002, the regulatory effects of LINC00184 on the glycolysis and mitochondrial OXPHOS of EC cells were reversed. CONCLUSION: Taken together, the LINC00184/PTEN/Akt axis mediates glycolysis and mitochondrial OXPHOS in EC cells. This study highlighted a potential intervention target for treating EC.

Analysis of the effect of laparoscopy and hysteroscopy on ovarian function, immune function and quality of sexual life of patients with hysteromyoma at different ages.

This study aims to comparatively analyze the impact of laparoscopic myomectomy (LM) and transcervical resection of myoma (TCRM) on ovarian function, immune function and quality of sexual life of patients with hysteromyoma. Two hundred and forty patients with hysteromyoma admitted into the Second Affiliated Hospital of Zhengzhou University from June 2014 to June 2016 were divided into laparoscopic myomectomy group (LM group, n=120) and transcervical resection of myoma group (TCRM group, n=120) according to random figure table. The difference between the two surgical methods were compared among hysteromyoma patients of different age in the following aspects: perioperative indicators, ovarian function, immune function and quality of sexual life before treatment and after operation. 1) there was no significant difference in age structure between LM and TCRM group (P>0.05). 2) There were no significant differences in the amount of bleeding, operative time, analgesics usage rate between the groups (P>0.05), but the anal exhaust time, postoperative ambulation time, hospitalization time and hospitalization costs in TCRM group were significantly less than that in LM group, the difference was statistically significant (P<0.01). 3) Before treatment and in the 3rd, and 6th month after operation, there was no significant difference on follicle stimulating hormone (FSH), luteinizing hormone (LH) or estradiol (E2) level between the two groups (P>0.05); but in the 3rd month after operation, the level of E2 decreased remarkably, with FSH and LH level increased significantly compared with the 6th month after operation and before treatment (P<0.05). 4) Compared with the preoperative status, there was no significant decrease in humoral immunity (IgG, IgA, IgM) and cellular immune function (CD4+, CD8+) in TCRM group at the 3rd and 6th month after operation (P>0.05). While at the 3rd month after operation, the levels of CD4+, CD8+ were decreased significantly in comparison to those at the 6th month after operation (P<0.05), but the levels of IgG, IgA and IgM were not decreased significantly (P>0.05). 5) There was no significant difference in the quality of sexual life between the two groups before operation or at the 3rd and 6th month after operation (P>0.05). In recent years, endoscopic treatment has become the main treatment evolution of hysteromyoma, patients of all ages are paying attention to the preservation of the uterus. This study showed that TCRM is approaching through the natural cavity, which has the features of less damage to body, faster recovery and lower cost of hospitalization than LM. The two surgical methods have no obvious influence on ovarian function, immune function and quality of sexual life.

[Surgical treatment of 120 high risk elderly patients with lung cancer].

BACKGROUND: Lung cancer is easy to occur in patients older than 70 years, whose special health condition makes their treatment more difficult. The aim of this study is to investigate the best surgical treatment and perioperative management for high risk elderly patients with lung cancer. METHODS: After 5 years' follow-up, 120 high risk elderly patients with lung cancer were analyzed retrospectively. RESULTS: Of 120 patients, there was no operative death. Four patients died of respiration failure and 2 died of myocardial infarction. Perioperational death rate was 5.0%. The overall 1-, 3-and 5-year survival rate was 67.7%, 41.7% and 28.1%, respectively. CONCLUSIONS: Appropriate management of complications is the key point to decrease perioperative death rate, and it may influence the quality of life of patients. To resect tumor maximally may be not the best choice for elderly patients. Lobectomy or segmental resection combined with positive lymph node dissection may be the considerable patterns.

Genetic variation of lncRNA GAS5 contributes to the development of lung cancer.

Lung cancer remains the leading cause of cancer-related deaths throughout the world. In spite of great effort for the research of carcinogenesis, the molecular mechanisms of lung cancer remain unclear. In current study, we investigated the possible association between susceptibility of lung cancer and GAS5 rs145204276, which showed contradictory roles in carcinogenesis of colorectal cancer and hepatocellular carcinoma. We found that the del allele was significantly associated with 21% decreased risk of lung cancer (OR=0.79; 95% CI=0.66-0.93; P value = 0.006). Compared with the genotype ins/ins, both the genotype ins/del (OR=0.78; 95% CI=0.62-0.99) and del/del (OR=0.59;95% CI=0.39-0.89) showed decreased susceptibility of lung cancer. Real-time PCR analysis found that the expression levels of lncRNA GAS5 in lung cancer tissues were significantly lower than those in the corresponding normal tissues (P<0.01). Also the relative GAS5 expression level in samples with del/del genotype was significantly higher than that in samples with ins/del and ins/ins genotype (P<0.01). Taken together, our findings provided strong evidence for the hypothesis that GAS5 rs145204276 were significantly associated with the susceptibility of lung cancer, and GAS5 functions as a tumor suppressor in carcinogenesis of lung cancer.

Down-regulation of miR-30a-3p/5p promotes esophageal squamous cell carcinoma cell proliferation by activating the Wnt signaling pathway.

AIM: To investigate the potential role of microRNA-30a (miR-30a) in esophageal squamous cell carcinoma (ESCC). METHODS: Expression of miR-30a-3p/5p was analyzed using microarray data and fresh ESCC tissue samples. Both in vitro and in vivo assays were used to investigate the effects of miR-30a-3p/5p on ESCC cell proliferation. Furthermore, Kyoto Encyclopedia of Genes and Genomes analysis was performed to explore underlying mechanisms involved in ESCC, and then, assays were carried out to verify the potential molecular mechanism of miR-30a in ESCC. RESULTS: Low expression of miR-30a-3p/5p was closely associated with advanced ESCC progression and poor prognosis of patients with ESCC. Knock-down of miR-30a-3p/5p promoted ESCC cell proliferation. Increased miR-30a-3p/5p expression inhibited the Wnt signaling pathway by targeting Wnt2 and Fzd2. CONCLUSION: Down-regulation of miR-30a-3p/5p promotes ESCC cell proliferation by activating the Wnt signaling pathway through inhibition of Wnt2 and Fzd2.

[Protective mechanism of ischemic preconditioning to the lung ischemia-reperfusion injury].

OBJECTIVE: To determine the protective mechanism of preconditioning to the lung injury induced by ischemia-reperfusion. METHODS: Twelve pigs were randomly divided into 2 groups: control group ( Group C) and ischemic preconditioning group ( Group IP). The concentration of superoxide distrautase (SOD) and malondialdehyde (MDA) in circuit were checked before and after the perfusion to reflect the lipid peroxidation in the lungs. Left lung biopsies were performed immediately after the perfusion and 1 hour postperfusion for histologic examination. The ICAM-1 expression was assessed by immunohistochemical analysis with Envision method and the mRNA expression of ICAM-1 was analyzed by RT-PCR. RESULTS: SOD in Group IP was much higher than that in Group C (P < 0.01 ). MDA in Group IP was much lower than that in Group C ( P < 0.01 ). The lung histologic examination showed that Group C was significantly more serious than Group IP in pulmonary edema, inflammatory cell infiltration, mild focal hemorrhage, and alveolar disruption. The expression of ICAM-1 of lung tissue obviously decreased in Group IP than that in Group C (P <0.01 ). The expression of ICAM-1 mRNA of lung tissue was significantly lower in Group IP than that in Group C (P < 0.01 ). CONCLUSION: Lung ischemic preconditioning can reduce the lung injury. The mechanisms of the protective effects of the IP may be related to the increase of SOD and the decrease of MDA. The preconditioning down-regulated the ICAM-1 expression is one of the mechanisms in reducing the lung injury.

Galectin-3 knockdown increases gefitinib sensitivity to the inhibition of EGFR endocytosis in gefitinib-insensitive esophageal squamous cancer cells.

Esophageal cancer (EC) is one of the most aggressive malignancies with a distinctly high incidence and mortality rate. Esophageal squamous cell carcinoma (ESCC) is the major histologic subtype of EC, with 40-70 % of tumors overexpressing the epidermal growth factor receptor (EGFR). Blockade of EGFR signal transduction may be a promising and effective strategy for EC therapy. However, the therapeutic efficacy of EGFR-tyrosine kinase inhibitors is clinically limited because of drug resistance. Galectin-3, a member of the animal lectin family, has been associated with a variety of biological functions and the progression of multiple tumors, including ESCC. In this study, we investigated the role of galectin-3 involved in potential gefitinib-resistance mechanisms in EGFR-positive ESCC cell lines. The results revealed that gefitinib treatment induced different inhibitory effects on cell viability, cell cycle progression and cell invasion in gefitinib-sensitive KYSE-450 and gefitinib-insensitive TE-8 cells with different levels of galectin-3 expression. Interestingly, we further found that EGF-induced EGFR endocytosis and EGFR signaling were different between gefitinib-sensitive and gefitinib-insensitive ESCC cell lines. Galectin-3 inhibition in combination with gefitinib treatment induced greater inhibitory effects on cell viability, cell cycle progression and cell invasion in gefitinib-insensitive TE-8 cells. Moreover, galectin-3 inhibition increased the gefitinib sensitivity of TE-8 cells in terms of EGFR endocytosis in vitro and anti-tumor effects in vivo. Taken together, galectin-3 knockdown increased gefitinib sensitivity through the inhibition of EGFR endocytosis in gefitinib-insensitive ESCC cells and galectin-3 may be a rational therapeutic target in ESCC with gefitinib resistance.

Meis1 regulates proliferation of non-small-cell lung cancer cells.

As one of the most common cancers, lung cancer remains to be a major public health problem. Non-small-cell lung adenocarcinoma cancer exhibits higher resistance to chemotherapy than small cell lung cancer, which requires novel strategies. To further understand underlying mechanisms for non-small-cell lung adenocarcinoma cancer cell proliferation, we explored the role of Meis1 in non-small-cell lung adenocarcinoma cancer cells. The results show that Meis1 inhibits non-small-cell lung cancer (NSCLC) cell proliferation. Specific knockdown of Meis1 resulted in strengthened proliferative ability of non-small-cell lung adenocarcinoma cancer cells. Cell cycle analysis indicated that DNA synthesis was increased when Meis1 was down-regulated specifically. As well as histone H3 phosphorylation, which is indicative of mitosis. More importantly, forced Meis1 expression repressed the proliferation of non-small-cell lung adenocarcinoma cancer cell. These data demonstrated Meis1 limits the proliferation of non-small-cell lung adenocarcinoma cancer cell and could potentially represent a therapeutic strategy that may control non-small-cell lung adenocarcinoma cancer cell proliferation.

Prognostic value of C-reactive protein in esophageal cancer: a meta-analysis.

BACKGROUND: The classical inflammatory biomarker, C-reactive protein (CRP), has been identified to be related to progression of esophageal cancer. Some research showed that elevated pretreatment serum CRP indicated a poor prognosis, but results have been inconsistent. MATERIALS AND METHODS: We searched the Medline, Embase and the Cochrane Central Search Library for suitable studies and a meta-analysis of eleven (1,886 patients) was conducted to examine the relationship between elevated serum CRP level and overall survival (OS) in esophageal cancer cases. Moreover, correlation analyses were conducted to assess links between pretreatment serum CRP level and tumor node metastasis (TNM) stage as well as T, N, M grade, respectively. RESULTS: The pooled analysis showed that elevated pretreatment serum CRP level was significantly associated with poorer overall survival (HR 2.09, 95%CI 1.52-2.87, p<0.01). Subgroup analyses were conducted by "country", "cut-off value", "treatment" and "number of patients", and no single factor could alter the result. Elevated pretreatment serum CRP was significantly correlated with more advanced TNM stage and T, N, M grade respectively. CONCLUSIONS: Elevated pretreatment serum CRP levels are associated with poorer prognosis in esophageal cancer patients, and could serve as a useful biomarker for outcome prediction.

Improvements of surgical techniques in a rat model of an orthotopic single lung transplant.

BACKGROUND: Rats are widely used in modeling orthotopic lung transplantation. Recently the introduction of the cuff technique has greatly facilitated the anastomosing procedure used during the transplant. However, the procedure is still associated with several drawbacks including twisting of blood vessels, tissue injury and the extensive time required for the procedure. This study was performed to optimize the model of rat lung transplantation (LT) with the cuff technique. METHODS: A total of 42 adult Lewis rats received orthotopic LT with our newly modified procedures. The modified procedures were based on the traditional procedure and incorporated improvements involving orotracheal intubation; a cuff without a tail; conservative dissection in the hilum; preservation of the left lung during anastomosis; successive anatomizing of the bronchus, the pulmonary vein, and the pulmonary artery; and one operator. RESULTS: Transplants were performed in 42 rats with a successful rate of 95.23% (40/42). The mean duration for the complete procedure was 82.93 ± 14.56 minutes. All anastomoses were completed in one attempt without vessel laceration, twisting or angulation. In our study, two animals died within three days and one animal died ten days after the operation. All grafts were well inflated with robust blood perfusion and functioned normally as demonstrated by blood gas analysis. CONCLUSIONS: We have developed a modified orthotopic LT technique that can be easily performed while overcoming major drawbacks. The modified technique has many advantages, such as easy graft implanting, shortened operation time, fewer complications and high reproducibility.

Orthotopic and heterotopic tracheal transplantation model in studying obliterative bronchiolitis.

BACKGROUND: Several animal models have been established to investigate the mechanisms of obliterative bronchiolitis after lung transplantation. In this study, we compared three prevalent murine models of obliterative bronchiolitis in terms of several basic pathologic changes in a relatively short span of time after transplantation. METHODS: Each of the recipient mice simultaneously received orthotopic, intra-omental and subcutaneous tracheal transplantation in both syngeneic and allogeneic settings. No immunosuppressive treatment was administered. Tracheal grafts were harvested on Day 14, 21 and 28 after transplantation for histological and immunohistochemical analyses. RESULTS: Syngeneic tracheal grafts from different transplant sites retained normal histologic structures, while their corresponding allografts demonstrated more occlusion of the airway lumen as well as more infiltration of CD4(+)/CD8(+) mononuclear cells and myofibroblasts, but less regenerative epithelium and neovascularized vessels at indicated times (P<0.05). Compared with two heterotopic allografts, orthotopic allografts had less occlusion of the tracheal lumen as well as less infiltration of CD4(+)/CD8(+) mononuclear cells and myofibroblasts, but more regenerative epithelium and neovascularized vessels (P<0.05). CONCLUSIONS: Orthotopic tracheal transplantation in mice can be considered as a model to study early stages of obliterative bronchiolitis, and heterotopic tracheal transplantation can be a model for late stages of obliterative bronchiolitis.

T cell immunohistochemistry refines lung transplant acute rejection diagnosis and grading.

OBJECTIVE: Lung transplant volume has been increasing. However, inaccurate and uncertain diagnosis for lung transplant rejection hurdles long-term outcome due to, in part, interobserver variability in rejection grading. Therefore, a more reliable method to facilitate diagnosing and grading rejection is warranted. METHOD: Rat lung grafts were harvested on day 3, 7, 14 and 28 post transplant for histological and immunohistochemical assessment. No immunosuppressive treatment was administered. We explored the value of interstitial T lymphocytes quantification by immunohistochemistry and compared the role of T cell immunohistochemistry with H&E staining in diagnosing and grading lung transplant rejection. RESULTS: Typical acute rejection from grade A1 to A4 was found. Rejection severity was heterogeneously distributed in one-third transplanted lungs (14/40): lesions in apex and center were more augmented than in the base and periphery of the grafts, respectively. Immunohistochemistry showed profound difference in T lymphocyte infiltration among grade A1 to A4 rejections. The coincidence rate of H&E and immunohistochemistry was 77.5%. The amount of interstitial T lymphocyte infiltration increased gradually with the upgrading of rejection. The statistical analysis demonstrated that the difference in the amount of interstitial T lymphocytes between grade A2 and A3 was not obvious. However, T lymphocytes in lung tissue of grade A4 were significantly more abundant than in other grades. CONCLUSIONS: Rejection severity was heterogeneously distributed within lung grafts. Immunohistochemistry improves the sensitivity and specificity of rejection diagnosis, and interstitial T lymphocyte quantitation has potential value in diagnosing and monitoring lung allograft rejection. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1536075282108217.