Bidimensional Tandem Mass Spectrometry for Selective Identification of Nitration Sites in Proteins.

Nitration of protein tyrosine residues is very often regarded as a molecular signal of peroxynitrite formation during development, oxidative stress, and aging. However, protein nitration might also have biological functions comparable to protein phosphorylation, mainly in redox signaling and in signal transduction. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Existing methodologies essentially rely on immunochemical techniques either using 2D-PAGE fractionation in combination with western blot analyses or exploiting immunoaffinity procedures to selectively capture nitrated proteins. Here we report a totally new approach involving dansyl chloride labeling of the nitration sites that rely on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and an MS3 analysis. This new procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixtures

A rapid and selective mass spectrometric method for the identification of nitrated proteins.

The nitration of protein tyrosine residues represents an important posttranslational modification during development, oxidative stress, and biological aging. The major challenge in the proteomic analysis of nitroproteins is the need to discriminate modified proteins, usually occurring at substoichiometric levels, from the large amount of nonmodified proteins. Moreover, precise localization of the nitration site is often required to fully describe the biological process. Identification of the specific targets of protein oxidation was previously accomplished using immunoprecipitation techniques followed by immunochemical detection. Here, we report a totally new approach involving dansyl chloride labeling of the nitration sites which relies on the enormous potential of MSn analysis. The tryptic digest from the entire protein mixture is directly analyzed by MS on a linear ion trap mass spectrometer. Discrimination between nitro- and unmodified peptide is based on two selectivity criteria obtained by combining a precursor ion scan and a MS3 analysis. The novel labeling procedure was successfully applied to the identification of 3-nitrotyrosine residues in complex protein mixture

Cl-IB-MECA Inhibits Human Thyroid Cancer Cell Proliferation Independently of A3 Adenosine Receptor Activation

A3 adenosine receptor (A3AR) agonists have been reported to modulate cellular proliferation. This work was aimed to investigate the expression and the possible implication of A3AR in the human thyroid carcinomas. Normal thyroid tissue samples did not express A3 adenosine receptor, while primary thyroid cancer tissues expressed high level of A3AR, as determined by immunohistochemistry analysis. In human papillary thyroid carcinoma cell line, NPA, at concentrations > or =10 microM, the A3AR-selective agonist 2-chloro-N(6)-(3-iodobenzyl)adenosine-5'-N-methylcarboxamide (Cl-IB-MECA) produced inhibition of cell growth, by blocking the G(1) cell cycle phase in a concentration- and time-dependent manner. This effect was well correlated with a reduction of protein expression of cyclins D1 and E2 after 24 hours of Cl-IB-MECA treatment. Moreover Cl-IB-MECA induced dephosphorylation of ERK1/2 in a time- and concentration-dependent manner, which in turn inhibits cell proliferation. The effect of Cl-IB-MECA was not prevented by A3AR antagonists, MRS1191 or MRS1523 or FA385. Furthermore, neither nucleoside transporter inhibitors, Dypiridamole and NBTI, nor the A1, A2A and A2B receptors antagonists were able to block the response to Cl-IB-MECA. Although Cl-IB-MECA has been shown to influence cell death and survival in other systems through an A3AR-mediated mechanism, in NPA cells the growth inhibition induced by micromolar concentrations of Cl-IB-MECA is not related to A3AR activation and hence that its effects on human papillary carcinoma cell line seem to be independent of the presence of this receptor subtype

Detection of high mobility group A2 specific mRNA in the plasma of patients affected by epithelial ovarian cancer

Ovarian cancer is the most lethal gynecological malignancy and the high mortality rate is associated with advanced-stage disease at the time of the diagnosis. In order to find new tools to make diagnosis of Epithelial Ovarian Cancer (EOC) at early stages we have analyzed the presence of specific HMGA2 mRNA in the plasma of patients affected by this neoplasm. HMGA2 overexpression represents a feature of several malignances including ovarian carcinomas. Notably, we detected HMGA2 specific mRNA in the plasma of 40 out 47 patients with EOC, but not in the plasma of healthy donors. All cases found positive for HMGA2 mRNA in the plasma showed HMGA2 protein expression in EOC tissues. Therefore, on the basis of these results, the analysis of circulating HMGA2 specific mRNA might be considered a very promising tool for the early diagnosis of EOC

FRA-1 protein overexpression is a feature of hyperplastic and neoplastic breast disorders

BACKGROUND: Fos-related antigen 1 (FRA-1) is an immediate early gene encoding a member of AP-1 family of transcription factors involved in cell proliferation, differentiation, apoptosis, and other biological processes. fra-1 gene overexpression has an important role in the process of cellular transformation, and our previous studies suggest FRA-1 protein detection as a useful tool for the diagnosis of thyroid neoplasias. Here we investigate the expression of the FRA-1 protein in benign and malignant breast tissues by immunohistochemistry, Western blot, RT-PCR and qPCR analysis, to evaluate its possible help in the diagnosis and prognosis of breast neoplastic diseases. METHODS: We investigate the expression of the FRA-1 protein in 70 breast carcinomas and 30 benign breast diseases by immunohistochemistry, Western blot, RT-PCR and qPCR analysis. RESULTS: FRA-1 protein was present in all of the carcinoma samples with an intense staining in the nucleus. Positive staining was also found in most of fibroadenomas, but in this case the staining was present both in the nucleus and cytoplasm, and the number of positive cells was lower than in carcinomas. Similar results were obtained from the analysis of breast hyperplasias, with no differences in FRA-1 expression level between typical and atypical breast lesions; however the FRA-1 protein localization is mainly nuclear in the atypical hyperplasias. In situ breast carcinomas showed a pattern of FRA-1 protein expression very similar to that observed in atypical hyperplasias. Conversely, no FRA-1 protein was detectable in 6 normal breast tissue samples used as controls. RT-PCR and qPCR analysis confirmed these results. Similar results were obtained analysing FRA-1 expression in fine needle aspiration biopsy (FNAB) samples. CONCLUSION: The data shown here suggest that FRA-1 expression, including its intracellular localization, may be considered a useful marker for hyperplastic and neoplastic proliferative breast disorders

Gene expression analysis of pancreatic cell lines reveals genes overexpressed in pancreatic cancer

Background: Pancreatic cancer is one of the leading causes of cancer-related death. Using DNA gene expression analysis based on a custom made Affymetrix cancer array, we investigated the expression pattern of both primary and established pancreatic carcinoma cell lines. Methods: We analyzed the gene expression of 5 established pancreatic cancer cell lines (AsPC-1, BxPC-3, Capan-1, Capan-2 and HPAF II) and 5 primary isolates, 1 of them derived from benign pancreatic duct cells. Results: Out of 1,540 genes which were expressed in at least 3 experiments, we found 122 genes upregulated and 18 downregulated in tumor cell lines compared to benign cells with a fold change > 3. Several of the upregulated genes (like Prefoldin 5, ADAM9 and E-cadherin) have been associated with pancreatic cancer before. The other differentially regulated genes, however, play a so far unknown role in the course of human pancreatic carcinoma. By means of immunohistochemistry we could show that thymosin [β-10 (TMSB10), upregulated in tumor cell lines, is expressed in human pancreatic carcinoma, but not in non-neoplastic pancreatic tissue, suggesting a role for TMSB10 in the carcinogenesis of pancreatic carcinoma. Conclusion: Using gene expression profiling of pancreatic cell lines we were able to identify genes differentially expressed in pancreatic adenocarcinoma, which might contribute to pancreatic cancer development.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

PATZ1 acts as a tumor suppressor in thyroid cancer via targeting p53-dependent genes involved in EMT and cell migration

PATZ1, a POZ-Zinc finger protein, is emerging as an important regulator of development and cancer, but its cancer-related function as oncogene or tumor-suppressor is still debated. Here, we investigated its possible role in thyroid carcinogenesis. We demonstrated PATZ1 is down-regulated in thyroid carcinomas compared to normal thyroid tissues, with an inverse correlation to the degree of cell differentiation. In fact, PATZ1 expression was significantly further down-regulated in poorly differentiated and anaplastic thyroid cancers compared to the papillary histotype, and it resulted increasingly delocalized from the nucleus to the cytoplasm proceeding from differentiated to undifferentiated thyroid carcinomas. Restoration of PATZ1 expression in three thyroid cancer-derived cell lines, all characterized by fully dedifferentiated cells, significantly inhibited their malignant behaviors, including in vitro proliferation, anchorage-independent growth, migration and invasion, as well as in vivo tumor growth. Consistent with recent studies showing a role for PATZ1 in the p53 pathway, we showed that ectopic expression of PATZ1 in thyroid cancer cells activates p53-dependent pathways opposing epithelial-mesenchymal transition and cell migration to prevent invasiveness. These results provide insights into a potential tumor-suppressor role of PATZ1 in thyroid cancer progression, and thus may have potential clinical relevance for the prognosis and therapy of thyroid cancer

YY1 overexpression is associated with poor prognosis and metastasis-free survival in patients suffering osteosarcoma

<p>Abstract</p> <p>Background</p> <p>The polycomb transcription factor Yin Yang 1 (YY1) overexpression can be causally implicated in experimental tumor growth and metastasization. To date, there is no clinical evidence of YY1 involvement in outcome of patients with osteosarcoma. Prognosis of osteosarcoma is still severe and only few patients survive beyond five years. We performed a prospective immunohistochemistry analysis to correlate YY1 immunostaining with metastatic development and survival in a selected homogeneous group of patients with osteosarcoma.</p> <p>Methods</p> <p>We studied 41 patients suffering from osteosarcoma (stage II-IVa). Multivariate analysis was performed using Cox proportional hazard regression to evaluate the correlation between YY1 expression and both metastasis development and mortality.</p> <p>Results</p> <p>YY1 protein is not usually present in normal bone; in contrast, a high number of patients (61%) showed a high score of YY1 positive cells (51-100%) and 39% had a low score (10-50% positive cells). No statistical difference was found in histology, anatomic sites, or response to chemotherapy between the two degrees of YY1 expression. Cox regression analysis demonstrated that the highest score of YY1 expression was predictive of both low metastasis-free survival (HR = 4.690, 95%CI = 1.079-20.396; p = 0.039) and poor overall survival (HR = 8.353, 95%CI = 1.863-37.451 p = 0.006) regardless of the effects of covariates such as age, gender, histology and chemonecrosis.</p> <p>Conclusion</p> <p>Overexpression of YY1 in primary site of osteosarcoma is associated with the occurrence of metastasis and poor clinical outcome.</p

Acute diverticulitis management: evolving trends among Italian surgeons. A survey of the Italian Society of Colorectal Surgery (SICCR)

Acute diverticulitis (AD) is associated with relevant morbidity/mortality and is increasing worldwide, thus becoming a major issue for national health systems. AD may be challenging, as clinical relevance varies widely, ranging from asymptomatic picture to life-threatening conditions, with continuously evolving diagnostic tools, classifications, and management. A 33-item-questionnaire was administered to residents and surgeons to analyze the actual clinical practice and to verify the real spread of recent recommendations, also by stratifying surgeons by experience. CT-scan remains the mainstay of AD assessment, including cases presenting with recurrent mild episodes or women of child-bearing age. Outpatient management of mild AD is slowly gaining acceptance. A conservative management is preferred in non-severe cases with extradigestive air or small/non-radiologically drainable abscesses. In severe cases, a laparoscopic approach is preferred, with a non-negligible number of surgeons confident in performing emergency complex procedures. Surgeons are seemingly aware of several options during emergency surgery for AD, since the rate of Hartmann procedures does not exceed 50% in most environments and damage control surgery is spreading in life-threatening cases. Quality of life and history of complicated AD are the main indications for delayed colectomy, which is mostly performed avoiding the proximal vessel ligation, mobilizing the splenic flexure and performing a colorectal anastomosis. ICG is spreading to check anastomotic stumps' vascularization. Differences between the two experience groups were found about the type of investigation to exclude colon cancer (considering the experience only in terms of number of colectomies performed), the size of the peritoneal abscess to be drained, practice of damage control surgery and the attitude towards colovesical fistula

How future surgery will benefit from SARS-COV-2-related measures: a SPIGC survey conveying the perspective of Italian surgeons

COVID-19 negatively affected surgical activity, but the potential benefits resulting from adopted measures remain unclear. The aim of this study was to evaluate the change in surgical activity and potential benefit from COVID-19 measures in perspective of Italian surgeons on behalf of SPIGC. A nationwide online survey on surgical practice before, during, and after COVID-19 pandemic was conducted in March-April 2022 (NCT:05323851). Effects of COVID-19 hospital-related measures on surgical patients' management and personal professional development across surgical specialties were explored. Data on demographics, pre-operative/peri-operative/post-operative management, and professional development were collected. Outcomes were matched with the corresponding volume. Four hundred and seventy-three respondents were included in final analysis across 14 surgical specialties. Since SARS-CoV-2 pandemic, application of telematic consultations (4.1% vs. 21.6%; p &lt; 0.0001) and diagnostic evaluations (16.4% vs. 42.2%; p &lt; 0.0001) increased. Elective surgical activities significantly reduced and surgeons opted more frequently for conservative management with a possible indication for elective (26.3% vs. 35.7%; p &lt; 0.0001) or urgent (20.4% vs. 38.5%; p &lt; 0.0001) surgery. All new COVID-related measures are perceived to be maintained in the future. Surgeons' personal education online increased from 12.6% (pre-COVID) to 86.6% (post-COVID; p &lt; 0.0001). Online educational activities are considered a beneficial effect from COVID pandemic (56.4%). COVID-19 had a great impact on surgical specialties, with significant reduction of operation volume. However, some forced changes turned out to be benefits. Isolation measures pushed the use of telemedicine and telemetric devices for outpatient practice and favored communication for educational purposes and surgeon-patient/family communication. From the Italian surgeons' perspective, COVID-related measures will continue to influence future surgical clinical practice