81 research outputs found
Microarray Bioinformatics and Applications in Oncology
Bioinformatics’ is one of the newest fields of biological research, and should be viewed broadly as
the use of mathematical, statistical, and computational methods for the processing and analysis
of biologic data [1]. Over the last decade, the rapid growth of information and technology in
both ‘genomics1’ and ‘omics2’ era’s has been overwhelming for the laboratory scientists to process
experimental results. Traditional gene-by-gene approaches in research are insufficient to meet
the growth and demand of biological research in understanding the true biology. The massive
amounts of data generated by new technologies as genomic sequencing and microarray chips
make the management of data and the integration of multiple platforms of high importance;
this is then followed by data analysis and interpretation to achieve biological understanding
and therapeutic progress. Global views of analyzing the magnitude of information are necessary
and traditional approaches to labwork have steadily been changing towards a bioinformatic
era. Research is moving from being restricted to a laboratory environment to working with
computers in a ‘virtual lab’ environment
High-resolution genome profiling differentiated Staphylococcus epidermidis isolated from patients with ocular infections and normal individuals
Purpose: To investigate the potential phenotypic and genetic differences among the Staphylococcus epidermidis isolates obtained from control subjects (lower conjunctival sac; n = 14) with those from patients with keratitis (corneal scrapings; n = 18) or endophthalmitis (vitreous; n = 24). Methods: Biofilm-forming capacity was detected by PCR for the icaAB gene and phenotyping by microtiter plate assay and congo red agar plate. Genotyping was performed by using fluorescence-amplified fragment length polymorphism (FAFLP) and in silico analysis of the FAFLP profiles. Results: Biofilm phenotyping (congo red agar/microtiter plate) differentiated disease-causing strains from control subjects. PCR assays (mecA, icaAB) were not useful in differentiating disease-causing strains from that of control subjects. The biofilm-forming capability appeared more critical in the pathogenesis of keratitis than in that of endophthalmitis. Cluster analysis of FAFLP data generated 11 clusters comprising 4 major clusters (I, II, III, and V) and 7 minor ones. FAFLP analysis clearly showed clustering of most of the commensal isolates in cluster I, separate from keratitis and endophthalmitis isolates. In silico analysis mapped signature bands to genes such as ebh, tagD, ptsI, and sepA, which might have a significant role in transforming less virulent populations of S. epidermidis to more virulent ones. Conclusions: The population dynamics of S. epidermidis revealed that there are significant genetic variations that can be detected through FAFLP between ocular disease causing isolates and the commensal population
Retroviral Integration Mutagenesis in Mice and Comparative Analysis in Human AML Identify Reduced PTP4A3 Expression as a Prognostic Indicator
Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e., Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML
Development and validation of a liquid chromatography coupled to mass spectrometer (LC-MS) method for the simultaneous quantification of estrone-3-sulfate, progesterone, estrone and estradiol in serum of mares and American bisons
peer reviewedSteroid concentrations in serum are fluctuating during pregnancy of many mammal species. The current
knowledge about endocrinology of gestation is mainly based on immunoassays. However, the lack of specificity
of these assays hampers the reliability of the results. In the present work, we developed and validated a methodology associating liquid chromatography (LC) and mass spectrometry (MS) to simultaneously quantify, with
high specificity and accuracy, estrone-3-sulfate (E3S), progesterone (PRO), estrone (E1) and estradiol (E2) in
serum of two different mammal species.
The sample preparation procedure is based on a simple protein precipitation and a derivatization with dansyl
chloride. After the chromatographical separation, compounds were analyzed with a triple-quadrupole mass
spectrometer operating in multiple reaction monitoring. Mare and American bison serum samples were analyzed
with the validated method and results were compared with concentrations measured with commercial radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA) and chemiluminescent microparticle immunoassay (CMIA).
Following these criterions: relative standard deviation <15% and relative bias <15%, lower limits of quantification of 0.5 ng/mL (E3S), 0.1 ng/mL (PRO) and 2 pg/mL (E1 and E2) were achieved. Most of the comparison
between immunoassays and LC-MS showed poor correlation and proportional differences.
Our LC-MS method is able to simultaneously quantify several steroid hormones with high specificity, accuracy
and sensitivity in serum of two different mammal species. Our method constitutes a useful and performant tool
for veterinary clinicians and LC-MS should thus be used to update and refine the current knowledge about the
endocrinology of pregnancy in mammals
Evaluation de la digestion in-situ du collagène de type I dans les matrices biologiques
Simultaneous detection and quantification of angiotensin I, II, 1-7 and 1-9 by LC-MS/MS in human plasma
peer reviewedRecent studies showed that angiotensin-converting 2 (ACE2) is used by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as a cellular entry receptor. SARS-CoV-2 causes down regulation of ACE2 leading to renin-angiotensin-aldosterone system (RAAS) major imbalance. This is an essential element of unfavourable evolution in patients with COVID-19. With lower level of ACE2, cleavage of And I and And II is decrease and therefore, And 1-7 and And 1-9 levels are decreased. The development of a quantitative method for these angiotensins is particularly interesting in the context of the prognosis/follow-up of patients with COVID-19. Based on this, this work aims at assessing angiotensins profile variations occurring over time with patients COVID-19 + using a LC-MS/MS method. In this project, plasma samples are prepared with an microelution MAX plate before injection on a Vexera X2 UPLC (Shimadzu Corporation, Kyoto, Japan) coupled to a QT5500 mass spectrometer (Scion, CA, USA) fitted with an IonDriveTMTurbo V ion source and using electro spray ionisation in positive mode. The mobile phase are composed of water (+0,4% formic acid) and of acetonitrile (+0,4% formic acid)
Détection et quantification simultanée de la gastrine 17 et 34 sous forme sulfatée et non-sulfatée par LC-MS/MS dans le plasma humain
editorial reviewedGastrin, secreted by G cells, plays a crucial role in digestion and has diverse functions including regulation of the intestinal epithelium and stomach growth.
Gastrin peptides are derived from progastrin. Peptides G17 and G34 are the most abundant in the blood. Both of them may be sulfated.
Current gastrin measurement relies on the DIAsource RIA kit, however it displays cross-reactivity issues. Therefore, we developed a LC-MS/MS method to quantify both sulfated and non-sulfated G17 and G34 forms
Iohexol quantitation and possible degradation kinetics in human urine using mass spectrometry coupled to liquid chromatography (LC)
peer reviewedIohexol is a well-known marker used to evaluate glomerular filtration rate (GFR) which is one indicator of kidney function. This GFR in often calculated using Iohexol intensity decay calculated using LC-MS/MS approaches on human plasma and urines. In these approaches, urines or plasma are taken from patients who were administered Iohexol at different timepoints and Iohexol is quantified at each time using one MRM approach. Once those value obtained, kinetics can be performed and GFR is calculated. However, some discrepancies can occur between urine and plasma results from the same patient and no study clearly explained this. Based on this, this work aims at assessing molecule profile variations occurring over time with patients that took Iohexol using LC coupled with high resolution mass spectrometry.
In this project, urine samples are taken at given timepoints from patients who received Iohexol. The samples are first centrifuged, and the supernatant is diluted 100 times with water before injection in a NanoACQUITY UPLC system coupled with a SYNAPT XS instrument operating in positive ion mode. The mobile phases are composed of water (+0.1% formic acid) and of acetonitrile (+0.1% formic acid). Standard samples (commercial Iohexol drug) are also analyzed as quality control.
The Iohexol LC-MS/MS method on a triple quadripole instrument has successfully been implemented on the SYNAPT XS – NanoACQUITY UPLC system. The first results on different patient urines show the presence of other peaks that Iohexol in patient samples. Interestingly, those peaks are not present in standards and in urines of patients who did not receive the drug. Investigation of all mass spectra is in progress and these results open the possibility for a large screening over time. Comparison with data obtained using LC-MS/MS is also in progress and the next step is the comparison with data obtained on plasma samples
BRCA1-like signature in triple negative breast cancer: Molecular and clinical characterization reveals subgroups with therapeutic potential.
Triple negative (TN) breast cancers make up some 15% of all breast cancers. Approximately 10-15% are mutant for the tumor suppressor, BRCA1. BRCA1 is required for homologous recombination-mediated DNA repair and deficiency results in genomic instability. BRCA1-mutated tumors have a specific pattern of genomic copy number aberrations that can be used to classify tumors as BRCA1-like or non-BRCA1-like. BRCA1 mutation, promoter methylation, BRCA1-like status and genome-wide expression data was determined for 112 TN breast cancer samples with long-term follow-up. Mutation status for 21 known DNA repair genes and PIK3CA was assessed. Gene expression and mutation frequency in BRCA1-like and non-BRCA1-like tumors were compared. Multivariate survival analysis was performed using the Cox proportional hazards model. BRCA1 germline mutation was identified in 10% of patients and 15% of tumors were BRCA1 promoter methylated. Fifty-five percent of tumors classified as BRCA1-like. The functions of genes significantly up-regulated in BRCA1-like tumors included cell cycle and DNA recombination and repair. TP53 was found to be frequently mutated in BRCA1-like (PÂ <Â 0.05), while PIK3CA was frequently mutated in non-BRCA1-like tumors (PÂ <Â 0.05). A significant association with worse prognosis was evident for patients with BRCA1-like tumors (adjusted HRÂ =Â 3.32, 95% CIÂ =Â 1.30-8.48, PÂ =Â 0.01). TN tumors can be further divided into two major subgroups, BRCA1-like and non-BRCA1-like with different mutation and expression patterns and prognoses. Based on these molecular patterns, subgroups may be more sensitive to specific targeted agents such as PI3K or PARP inhibitors
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