Maria da Gloria da Costa Carvalho

XDIA: improving on the label-free data-independent analysis

SUMMARY XDIA is a computational strategy for analyzing multiplexed spectra acquired using electron transfer dissociation and collision-activated dissociation; it significantly increases identified spectra (approximately 250%) and unique peptides (approximately 30%) when compared with the data-dependent ETCaD analysis on middle-down, single-phase shotgun proteomic analysis. Increasing identified spectra and peptides improves quantitation statistics confidence and protein coverage, respectively. AVAILABILITY The software and data produced in this work are freely available for academic use at http://fields.scripps.edu/XDIA CONTACT: paulo@pcarvalho.com SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

The frequency of human papillomavirus findings in normal oral mucosa of healthy people by PCR

UNLABELLED The human papillomavirus (HPV) is a DNA virus, which belongs to papillomaviridae family, being of low and high risk, which infect the skin and mucous membranes and can induce benign and malign tumor formation. In the oral mucosa they have been associated with oral papilloma, focal epithelial hyperplasia, leucoplakia and oral neoplasia. AIM to study the frequency of HPV finding in oral mucosa of normal people. MATERIALS AND METHODS Prospective study, cross-sectional cohort. One hundred volunteers, young adults, healthy, aged between 20 and 31 years, university students with no history, no complains, without oral or oropharyngeal lesions. They were submitted to a questionnaire with questions regarding HPV infection epidemiology. The samples were harvested by brushing and analyzed by PCR. RESULTS The results were negative for HPV in all samples. CONCLUSION It seems we had high social and economical class individuals, with nutrition rich in carotenoyds and vitamin C, low smoking and alcohol consumption and heterosexual habits with predominant monogamy and regular use of condoms.

Detection of DNA in the Plasma of Septic Patients

Abstract: Small amounts of plasma‐free DNA have been observed both in healthy individuals and in patients with various diseases such as systemic lupus erythematosus, rheumatoid arthritis, viral hepatitis, and cancer. This communication demonstrates that septic patients also release DNA in plasma. After DNA extraction from plasma, exon 1 of the K‐ras gene was amplified by PCR and products were analyzed by dot‐blot hybridization. Plasmas from polytraumatic patients and control healthy individuals were used for comparisons with septic patients. Our results show that septic patients present DNA in their plasma. As far as we know, this is the first evidence of circulating DNA in septic patients.

Chemo-resistant protein expression pattern of glioblastoma cells (A172) to perillyl alcohol

Glioblastoma multiform (GBM) is by far the most malignant glioma. We have introduced a new treatment for GBMs that comprises the inhalation of a naturally occurring terpene with chemotherapeutic properties known as perillyl alcohol (POH). Clinical trial results on recurrent GBM patients showed that POH extends the average life by more than eight months, temporarily slows tumor growth, and in some cases even decreases tumor size. After approximately seven months, the tumor continues to grow and leads to a dismal prognosis. To investigate how these tumors become resistant to POH, we generated an A172 human glioblastoma cell culture tolerant to 0.06 mM of POH (A172r). We used Multidimensional Protein Identification Technology (MudPIT) to compare the protein expression profile of A172r cells to the established glioblastoma A172 cell line. Our results include a list of identified proteins unique to either the resistant or the nonresistant cell line. These proteins are related to cellular growth, negative apoptosis regulation, Ras pathway, and other key cellular functions that could be connected to the underlying mechanisms of resistance.

Detection of free circulating Epstein-Barr virus DNA in plasma of patients with Hodgkin's disease

CONTEXT AND OBJECTIVE Free circulating Epstein-Barr virus (EBV) DNA is often present in the plasma of Hodgkins disease patients. The aim here was to evaluate the prevalence of this finding, its correlation with the immunohistochemical expression of LMP-1 (latent membrane protein 1) and the influence of other clinical factors. DESIGN AND SETTING Prospective study in two public tertiary institutions: Hematology Service, Universidade Federal do Rio de Janeiro, and Oncology Service, Instituto Nacional do Câncer, Rio de Janeiro. METHODS A cohort of 30 patients with newly diagnosed Hodgkins disease was studied. The control group consisted of 13 healthy adult volunteers. EBV DNA was determined by conventional polymerase chain reaction (PCR). RESULTS The median age was 28 years, and 16 patients were women. Advanced disease was present in 19 patients, and six were HIV-positive. EBV DNA was present in the plasma of 13 patients and one control (43% versus 8%, p = 0.03). EBV DNA prevalence was higher in HIV-positive patients (100% versus 29%, p = 0.0007) and those with advanced disease (63% versus 9%, p = 0.006). Among HIV-negative patients alone, EBV DNA prevalence remained higher in those with advanced disease. EBV DNA was found in 10/11 patients with LMP-1 expression in the lymph nodes, and in 3/19 without LMP-1 expression (kappa coefficient = 0.72). CONCLUSION EBV DNA was present in 91% of patients with EBV-associated Hodgkins disease, and in all patients with HIV-associated Hodgkins disease. EBV DNA prevalence was higher in patients with advanced disease, irrespective of HIV status.

The prevalence of human cytomegalovirus DNA in gliomas of Brazilian patients

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Association between human papillomavirus and Epstein - Barr virus DNA and gene promoter methylation of RB1 and CDH1 in the cervical lesions: a transversal study

BackgroundHuman papillomavirus (HPV) inactivates the retinoblastoma 1 (RB1) gene by promoter methylation and reduces cellular E-cadherin expression by overexpression of DNA methyltransferase 1 (DNMT1). The Epstein-Barr virus (EBV) is an oncogenic virus that may be related to cervical carcinogenesis. In gastric cancer, it has been demonstrated that E-cadherin gene (CDH1) hypermethylation is associated with DNMT1 overexpression by EBV infection. Our aim was to analyze the gene promoter methylation frequency of RB1 and CDH1 and verify the association between that methylation frequency and HPV and EBV infection in cervical lesions.MethodsSixty-five samples were obtained from cervical specimens: 15 normal cervices, 17 low-grade squamous intraepithelial lesions (LSIL), 15 high-grade squamous intraepithelial lesions (HSIL), and 18 cervical cancers. HPV and EBV DNA testing was performed by PCR, and the methylation status was verified by MSP.ResultsHPV frequency was associated with cervical cancer cases (p = 0.005) but not EBV frequency (p = 0.732). Viral co-infection showed a statistically significant correlation with cancer (p = 0.027). No viral infection was detected in 33.3% (5/15) of controls. RB1 methylated status was associated with cancer (p = 0.009) and HPV infection (p = 0.042). CDH1 methylation was not associated with cancer (p = 0.181). Controls and LSIL samples did not show simultaneous methylation, while both genes were methylated in 27.8% (5/18) of cancer samples. In the presence of EBV, CDH1 methylation was present in 27.8% (5/18) of cancer samples. Only cancer cases presented RB1 promoter methylation in the presence of HPV and EBV (33.3%).ConclusionsThe methylation status of both genes increased with disease progression. With EBV, RB1 methylation was a tumor-associated event because only the cancer group presented methylated RB1 with HPV infection. HPV infection was shown to be significantly correlated with cancer conditions. The global methylation frequency was higher when HPV was present, showing its epigenetic role in cervical carcinogenesis. Nevertheless, EBV seems to be a cofactor and needs to be further investigated.Virtual SlidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1159157579149317.

Charge prediction machine: tool for inferring precursor charge states of electron transfer dissociation tandem mass spectra.

Electron transfer dissociation (ETD) can dissociate highly charged ions. Efficient analysis of ions dissociated with ETD requires accurate determination of charge states for calculation of molecular weight. We created an algorithm to assign the charge state of ions often used for ETD. The program, Charge Prediction Machine (CPM), uses Bayesian decision theory to account for different charge reduction processes encountered in ETD and can also handle multiplex spectra. CPM correctly assigned charge states to 98% of the 13,097 MS2 spectra from a combined data set of four experiments. In a comparison between CPM and a competing program, Charger (ThermoFisher), CPM produced half the mistakes.

DNA Release by Line-1 (L1) Retrotransposon Could It Be Possible?

Abstract: We have verified the presence of line‐1 retrotransposon (L1) in plasma DNA in 15/17 brain tumor (glioma) patients and in 6/6 healthy people by applying PCR amplification of part of the L1 5′ end. The same samples were separately amplified for K‐ras. Results suggested that L1 sequences are circulating throughout the body. We hypothesized the participation of transposable elements such as L1 in a putative DNA release mechanism.

A strategy to identify genes associated with circulating solid tumor cell survival in peripheral blood.

Efforts in metastasis research have centered on the phenotypic and genetic differences between primary site and metastatic site tumors. However, genes that may be used as molecular markers of metastasis in circulating tumor cells remain unidentified. Genes regulating the dissemination and survival of solid tumor cells in the blood, as well as their adaptation to new environments, could be candidates for unique metastatic tumor markers. Differential display (DD) was conducted to compare the blood of tumor-free individuals with the blood of patients with lung, breast, and colon cancers. Twenty-one up-expressed genes in the tumor patient blood samples but none in the tumor-free donor blood samples were identified. Nine of these samples were isolated, amplified, and directly sequenced. A gene AB-1 homologous to a Bcl-2 family member, which might function as an apoptosis inhibitor, was identified. The overexpression of an apoptosis inhibitor in blood from patients with metastatic tumors might be correlated with the capability of solid tumor cells to survive in peripheral blood. This is the first demonstration of the usefulness of comparing control and patient blood samples by DD to find novel potential genetic markers identifying metastasis in the blood.