CGHPRO – A comprehensive data analysis tool for array CGH

BACKGROUND: Array CGH (Comparative Genomic Hybridisation) is a molecular cytogenetic technique for the genome wide detection of chromosomal imbalances. It is based on the co-hybridisation of differentially labelled test and reference DNA onto arrays of genomic BAC clones, cDNAs or oligonucleotides, and after correction for various intervening variables, loss or gain in the test DNA can be indicated from spots showing aberrant signal intensity ratios. Now that this technique is no longer confined to highly specialized laboratories and is entering the realm of clinical application, there is a need for a user-friendly software package that facilitates estimates of DNA dosage from raw signal intensities obtained by array CGH experiments, and which does not depend on a sophisticated computational environment. RESULTS: We have developed a user-friendly and versatile tool for the normalization, visualization, breakpoint detection and comparative analysis of array-CGH data. CGHPRO is a stand-alone JAVA application that guides the user through the whole process of data analysis. The import option for image analysis data covers several data formats, but users can also customize their own data formats. Several graphical representation tools assist in the selection of the appropriate normalization method. Intensity ratios of each clone can be plotted in a size-dependent manner along the chromosome ideograms. The interactive graphical interface offers the chance to explore the characteristics of each clone, such as the involvement of the clones sequence in segmental duplications. Circular Binary Segmentation and unsupervised Hidden Markov Model algorithms facilitate objective detection of chromosomal breakpoints. The storage of all essential data in a back-end database allows the simultaneously comparative analysis of different cases. The various display options facilitate also the definition of shortest regions of overlap and simplify the identification of odd clones. CONCLUSION: CGHPRO is a comprehensive and easy-to-use data analysis tool for array CGH. Since all of its features are available offline, CGHPRO may be especially suitable in situations where protection of sensitive patient data is an issue. It is distributed under GNU GPL licence and runs on Linux and Windows

The Relationship of Markers With Carotid Artery Stenosis and Lesion Hardness: Superiority of C-Reactive Protein and Uric Acid

Background : Atherosclerosis is a disease that cholesterol plaque builds up inside arteries. The process of atherosclerosis starts when certain substances such as cholesterol, fats, and cellular waste products accumulate in the walls of arteries, and the immune system responds to these substances, triggering inflammation. Over time, this inflammation can cause the plaque to grow and harden, narrowing the artery and reducing blood flow. Carotid artery disease (CAD) is a conclusion of plaques in carotid artery. CAD can increase the risk of stroke, a potentially life-threatening condition that occurs when blood flow to the brain is interrupted. Objectives: The objectives of this study were to detect the association between carotid artery stenosis and inflammatory markers. Methods: This study was designed prospectively and included 109 and 100 patients having mild carotid stenosis and severe carotid stenosis, respectively. Further, 101 patients were included in the control group. The carotid ultrasonography was evaluated in all patients. After classifying the plaques into(severe stenosis) categories, they were also grouped into echogenicity plaques, namely, echolucent (soft) and echogenic (hard) plaques. Results: The uric acid (UA) values of the mild and severe stenosis groups were higher than that of the control group (P<0.01). The mean C-reactive protein (CRP) value was the highest in the severe stenosis group, and the lowest CRP value was found in the control group (P<0.01). A one-unit increase in UA could increase the risk by 2.203 times. The CRP value was higher in the soft lesion group without calcification than in the hard lesion group with calcification. Conclusion: Our findings demonstrated that age, UA, and CRP values were identified as predictors independent of each other in the development of carotid stenosis. Regarding plaque classification, our results identified CRP, mean platelet volume (MPV), white blood cell, and lymphocyte values as negative predictors. The findings of our study indicate that CRP and UA are valuable in predicting the severity of stenosis and the formation of soft plaque

Correlation of SHOX2 Gene Amplification and DNA Methylation in Lung Cancer Tumors

<p>Abstract</p> <p>Background</p> <p>DNA methylation in the <it>SHOX2 </it>locus was previously used to reliably detect lung cancer in a group of critical controls, including 'cytologically negative' samples with no visible tumor cell content, at a high specificity based on the analysis of bronchial lavage samples. This study aimed to investigate, if the methylation correlates with <it>SHOX2 </it>gene expression and/or copy number alterations. An amplification of the <it>SHOX2 </it>gene locus together with the observed tumor-specific hypermethylation might explain the good performance of this marker in bronchial lavage samples.</p> <p>Methods</p> <p><it>SHOX2 </it>expression, gene copy number and DNA methylation were determined in lung tumor tissues and matched morphologically normal adjacent tissues (NAT) from 55 lung cancer patients. Quantitative HeavyMethyl (HM) real-time PCR was used to detect <it>SHOX2 </it>DNA methylation levels. <it>SHOX2 </it>expression was assayed with quantitative real-time PCR, and copy numbers alterations were measured with conventional real-time PCR and array CGH.</p> <p>Results</p> <p>A hypermethylation of the <it>SHOX2 </it>locus in tumor tissue as compared to the matched NAT from the same patient was detected in 96% of tumors from a group of 55 lung cancer patients. This correlated highly significantly with the frequent occurrence of copy number amplification (p < 0.0001), while the expression of the <it>SHOX2 </it>gene showed no difference.</p> <p>Conclusions</p> <p>Frequent gene amplification correlated with hypermethylation of the <it>SHOX2 </it>gene locus. This concerted effect qualifies <it>SHOX2 </it>DNA methylation as a biomarker for lung cancer diagnosis, especially when sensitive detection is needed, i.e. in bronchial lavage or blood samples.</p

Typisierung biallelischer Marker (SNPs) mit DNS-Mikrorastern

In this study we describe a novel protocol for the detection of SNPs using oligonucleotide microarrays. The method relies on the allele-specific elongation of immobilized oligonucleotide primers during which a fluorescently labeled nucleotide(Cy3-dUTP) is incorporated and detected by confocal laser scanning. Cycled reactions consisting of a denaturation, annealing and elongation step are employed to increase the yield of elongated product. Using human mitochondrial DNA as a model system, we tested two different means of generating single-stranded DNA targets used in the reactions: asymmetric PCR products and exonuclease-treated PTO-modified PCR products. Both proved to be suitable in this SNP detection system. Using asymmetric PCR products, we demonstrated that a single PCR primer upstream of 9 SNPs in a 426 bp template is sufficient. 46 of 48 SNPs could be detected in this way using 3 multiplexed asymmetric PCR reactions. The disadvantage of asymmetric PCR reactions is the difficult judgement of multiplexed reactions since asymmetric PCR products appear as a smear of bands in agarose gels. The application of exonuclease-treated PTOmodified PCR products as targets is therefore advantageous. More importantly, however, we demonstrate that it is possible to type 44 out of 46 randomly distributed mitochondrial SNPs by using 5 targets of 2.5kb to 4.4kb which together cover the entire mitochondrial genome. The size limit of such targets was found to lie between 4.4kb and 5.7kb. The approach described here simplifies PCR-amplification of SNP loci, which is a major problem in transforming microarray-based SNP typing into a high-throughput method

Typing of bi-allelic marker (SNPs) with DNA microarrays

Titelblatt Widmung I. Inhaltsverzeichnis I II. Abkürzungen VI 1\. Einleitung 1 2\. Materialien 19 3\. Methoden 38 4\. Ergebnisse 73 5\. Diskussion 103 6\. Zusammenfassung 121 7\. Summary 122 8\. Literatur 123 9\. Anhang 139 9.1 Danksagung 139 9.2 Publikationen 140 9.3 Lebenslauf 141In dieser Arbeit wurde eine neue Methode zur Detektion von SNPs mit Hilfe von Oligonukleotid-Mikroarraysbeschrieben. Diese Methode stützt sich auf die Allel-spezifische Elongation von immobilisierten Oligonukleotiden,bei der ein Fluoreszenz-markiertes Nukleotid (Cy3-dUTP) eingebaut und mittels konfokalem Laser Scanner detektiert wird. Um den Ertrag an verlängertem Produkt zu steigern, wurden mehrere Zyklen bestehend aus einem Denaturierungs-, Annealing- und Elongationsschritt durchgeführt. Humane mitochondriale DNS wurde als Modellsystem verwendet und in der Reaktion verwendete Einzelstrang DNS Sonden wurden auf zwei verschiedenen Wegen hergestellt. Hierbei handelte es sich um asymmetrische PCR Produkte und Exonuklease-behandelte PTO- modifizierte PCR Produkte. Beide Sonden erwiesen sich als brauchbar für den Einsatz im SNP Detektions System. Es wurde zeigt, dass mit einem asymmetrischen 426 bp PCR Produkt neun SNPs parallel detektiert werden können. Zur Herstellung des asymmetrischen Produktes reichte der Einsatz eines einzigen Oligonukleotides in der PCR Reaktion aus. Als Matrize diente ein 426 bp PCR Produkt. 46 von 48 SNPs konnten durch den Einsatz von 3 asymmetrischen multiplex PCR Produkten nachgewiesen werden. Der Nachteil von asymmetrischen PCR Reaktionen ist die schwierige Beurteilung von multiplex Reaktionen, weil die asymmetrischen PCR Produkte als ein Schmier von Banden im Agarosegel erscheinen. Die Verwendung von Exonuklease-behandelten PTO-modifizierte PCR Produkten als Sonde ist deshalb vorteilhaft. Mit dem Einsatz 5 solcher Produkte einer Länge von 2,5 bis 4,4 kb konnten 44 von 46 zufällig verteilten mitochondrialen SNPs typisiert werden. Diese 5 DNS-Sonden decken zusammen das mitochondriale Genom vollständig ab. Es konnte ermittelt werden, dass Einzelstrang-Sonden dieser Art bis zu einer Grösse zwischen 4,4kb und 5,7kb in der Primer-Elongations-Reaktion eingesetzt werden können. Das hier beschriebene Verfahren vereinfacht ausserdem die PCR Amplifikation von SNP Loci, welche einen entscheidenden Engpaß bei Microarray-basierten SNP- Typisierungs Methoden in hohem Durchsatz dargestellt.In this study we describe a novel protocol for the detection of SNPs using oligonucleotide microarrays. The method relies on the allele-specific elongation of immobilized oligonucleotide primers during which a fluorescently labeled nucleotide (Cy3-dUTP) is incorporated and detected by confocal laser scanning. Cycled reactions consisting of a denaturation, annealing and elongation step are employed to increase the yield of elongated product. Using human mitochondrial DNA as a model system, we tested two different means of generating single-stranded DNA targets used in the reactions: asymmetric PCR products and exonuclease-treated PTO-modified PCR products. Both proved to be suitable in this SNP detection system. Using asymmetric PCR products, we demonstrated that a single PCR primer upstream of 9 SNPs in a 426 bp template is sufficient. 46 of 48 SNPs could be detected in this way using 3 multiplexed asymmetric PCR reactions. The disadvantage of asymmetric PCR reactions is the difficult judgement of multiplexed reactions since asymmetric PCR products appear as a smear of bands in agarose gels. The application of exonuclease- treated PTO-modified PCR products as targets is therefore advantageous. More importantly, however, we demonstrate that it is possible to type 44 out of 46 randomly distributed mitochondrial SNPs by using 5 targets of 2.5kb to 4.4kb which together cover the entire mitochondrial genome. The size limit of such targets was found to lie between 4.4kb and 5.7kb. The approach described here simplifies PCR-amplification of SNP loci, which is a major problem in transforming microarray-based SNP typing into a high-throughput method

Synthesis & characterization of heterocyclic disazo - azomethine dyes and investigating their molecular docking & dynamics properties on acetylcholine esterase (AChE), heat shock protein (HSP90 alpha), nicotinamide N-methyl transferase (NNMT) and SARS-CoV-2 (2019-nCoV, COVID-19) main protease (M-pro)

In the study, by using 5-amino-4-arylazo-3-methyl-1H-pyrazole derivatives and 2-hydroxy-5(phenyldiazenyl) benzaldehyde; eight novel heterocyclic disperse disazo-azomethine dyes were synthesized, their chemical structures were characterized via FT-IR and 1 H NMR studies. Synthesized compounds were also investigated computationally by performing various techniques. In the computational part of the study, geometry optimizations, frequency analyses, frontier molecular orbital (FMO) calculations, molecular electrostatic potential (MEP) map calculations, FT-IR and NMR spectral analyses were performed on the compounds. Additionally, to reveal their potentials against SARS-CoV-2 main protease (SARS-CoV-2 M-pro), acetylcholine esterase (AChE), heat shock protein (HSP90 alpha) and nicotinamide N-methyl transferase (NNMT), molecular docking calculations were also performed on the synthesized compounds. Molecular dynamics (MD) simulations were carried out on the top-scoring ligand-receptor complexes to evaluate the stability of the complexes and the interactions between ligands and receptors in more detail. Results showed that synthesized compounds can interact with all these four receptors effectively and can be promising structures for further studies. (C) 2021 Elsevier B.V. All rights reserved.Pamukkale University Scientific Research Project Coordination Unit [2016FBE08]This study has been supported by Pamukkale University Sci-entific Research Project Coordination Unit by the project number of 2016FBE08. In the computational studies Kocaeli University and TUBITAK ULAKBIM, High Performance and Grid Computing Center (TRUBA) resources were used

Application of High Resolution Magnetic Resonance Imaging Methods for Spinal Cord Tissue Segmentation

This paper presents the primitive results of high resolution Magnetic Resonance (MR) Imaging experiments that are performed for spinal cord segmentation purposes. In the study, it is aimed to image the epidural space, the cerebrospinal fluid, the white matter and the gray matter tissues in the lower cervical and upper thoracic regions of the spine with a maximum voxel size of 1x1x1 mm(3). For this purpose, the MRI sequences providing T2 and T2* images and used for spinal cord segmentation in the literature are investigated and some of them are modified to fulfill the voxel requirement

Combined biofuel production from cotton stalk and seed with a biorefinery approach

CELIKTAS, Melih Soner/0000-0003-0597-5133; OGUT, TUBA CEREN/0000-0002-8775-2285; Bastabak, Benginur/0000-0002-4359-9880WOS: 000538051200015Due to usage of fossil fuels, the depletion of world crude oil reserves and increased deteriorating climate conditions have reached a high level. These circumstances have led researches to search for alternative and efficient fuels. the main biofuels considered are bioethanol and biodiesel. in this study, ethanol and biodiesel production from cotton stalk and seed were aimed using liquid hot water (LHW) along with consecutive processes, where separate saccharification and fermentation (SHF) process was carried out. the maximum ethanol concentrations of 0.348 g/L and 0.721 g/L were achieved at 24 h and 72 h, respectively. For biodiesel conversion, cottonseed oil was subjected to transesterification, where the main interest was to utilize the by-product, glycerol. Three different glycerol concentrations were investigated in terms of ethanol fermentation using Escherichia coli K1 active culture. the maximum ethanol concentration of 0.415 g/L was achieved at 20 mL glycerol concentration for 48 h. Overall, cotton stalk and seed have the potential to be utilized on an industrial scale

Relationship of radiometabolic biomarkers to KRAS mutation status and ALK rearrangements in cases of lung adenocarcinoma

Purpose: Rapid diagnosis of genetic mutations is important for targeted therapies such as EGFR tyrosine kinase inhibitors. KRAS mutation and ALK rearrangement are also important in determining treatment. The purpose of our study was to evaluate the diagnostic value of 18F-FDG PET to predict KRAS mutation and ALK rearrangement in order to determine the frequency of these genetic markers in our lung adenocarcinoma cases and contribute to forthcoming meta-analysis studies. Methods: A total of 218 patients with lung adenocarcinoma (EGFR analyzed) who were seen at our clinic between 2012 and 2014 were included in the study. The results of the 18 F-FDG-PET scans for each patient were retrospectively recorded with the associated medical documents. ALK rearrangements were analyzed in 166 of the 218 patients, while 50 of the 218 patients were analyzed for KRAS mutational status. SPSS 15.0 for Windows was used for statistical analysis. Results: FDG avidity was higher in cases with KRAS mutations and ALK rearrangements than those without, but the difference was not significant. ALK rearrangements were more common in younger, female, and nonsmoking patients with lung adenocarcinoma. Conclusions: The small numbers of KRAS mutations and ALK rearrangements are the limitation of this study for evaluation of diagnostic imaging. The frequency of these genetic alterations was as reported in the literature. We believe that our work will contribute to future meta-analysis