László Puskás

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Papers by László Puskás

Kalman filtering for disease-state estimation from microarray data

Bioinformatics/computer Applications in The Biosciences, 2006

Motivation: In this paper we propose using the Kalman filter as a pre-processing step in microarr... more Motivation: In this paper we propose using the Kalman filter as a pre-processing step in microarray-based molecular diagnosis. Incor- porating the expression covariance between genes is important in such classification problems, since this represents the functional relationships that govern tissue state. Failing to fulfil such require- ments may result in biologically implausible class prediction models. Here we show that employing

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Nanoformulated Novel Curcumin Analogue as a Potent Multi-Target Agent Against Glioblastoma

Current pharmaceutical design

Nutrigenomic approaches to study the effects of n-3 PUFA diet in the central nervous system

Nutrition and health, 2006

Deficiencies in essential, mainly omega-3 and omega-6 (n-3, n-6) long chain polyunsaturated fatty... more Deficiencies in essential, mainly omega-3 and omega-6 (n-3, n-6) long chain polyunsaturated fatty acids (LC-PUFA) result in visual and cognitive impairment and disturbances in mental functions in animals and could be the main reason for the increasing incidence of different mental disorders in humans. Traditional approaches cannot give us a detailed picture on how dietary lipids exert their effects, because they focus on only a few genes or biomarkers. Dietary lipids not only influence the biophysical state of the cell membranes but, via direct and indirect routes, they also act on multiple pathways including signalling and gene and protein activities. Therefore, to understand the molecular basis of the effects and roles of n-3 PUFA in the central nervous system global screening techniques such as DNA- or protein microarrays were used to assess the changes, in a global way, at the transcriptome and at the proteome level. With DNA microarrays we found that cholesterol and fish oil (h...

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Gene expression in thyroid tumors

Cancer treatment and research, 2004

RNA amplification results in reproducible microarray data with slight ratio bias

BioTechniques, 2002

Microarray expression analysis demands large amounts of RNA that are often not available. RNA amp... more Microarray expression analysis demands large amounts of RNA that are often not available. RNA amplification techniques have been developed to overcome this prcblem, but limited data are available regarding the reproducibility and maintenance of original transcript ratios. We optimized and validated two amplification techniques: a modified in vitro transcription for the linear amplification of 3 microg total RNA and a SMART PCR-based technique for the exponential amplification of 50 ng total RNA. To determine bias between transcript ratios, we compared the expression profiles in mouse testis versus spleen between the two amplification methods and a standard labeling protocol, using microarrays containing 4596 cDNAs spotted in duplicate. With each method, replicate hybridizations were highly reproducible. However, when comparing the amplification methods to standard labeling, correlation coefficients were lower. Twelve genes that exhibited inconsistent or contradictory expression rati...

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Heat-tolerant methanotrophic bacteria from the hot water effluent of a natural gas field

Applied and environmental microbiology, 1995

Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-to... more Methanotrophic bacteria were isolated from a natural environment potentially favorable to heat-tolerant methanotrophs. An improved colony plate assay was developed and used to identify putative methanotrophic colonies with high confidence. Fourteen new isolates were purified and partially characterized. These new isolates exhibit a DNA sequence homology of up to 97% with the conserved regions in the mmoX and mmoC genes of the soluble methane monooxygenase (MMO)-coding gene cluster of Methylococcus capsulatus Bath. The copper regulation of soluble MMO expression in the same isolates, however, differs from that of M. capsulatus Bath, as the new isolates can tolerate up to 0.8 microM copper without loss of MMO activity while a drastic reduction of MMO activity occurs already at 0.1 microM copper in M. capsulatus Bath. The isolates can be cultivated and utilized at elevated temperatures, and their copper- and heat-tolerant MMO activity makes these bacteria ideal candidates for future bi...

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MicroRNAs associated with ischemia-reperfusion injury and cardioprotection by ischemic pre- and postconditioning: protectomiRs

American journal of physiology. Heart and circulatory physiology, Jan 15, 2014

We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischem... more We aimed to characterize early changes in microRNA expression in acute cardioprotection by ischemic pre- and postconditioning in rat hearts. Hearts isolated from male Wistar rats were subjected to 1) time-matched nonischemic perfusion, 2) ischemia-reperfusion (30 min of coronary occlusion and 120 min of reperfusion), 3) preconditioning (3 × 5 min of coronary occlusion) followed by ischemia-reperfusion, or 4) ischemia-reperfusion with postconditioning (6 × 10 s of global ischemia-reperfusion at the onset of reperfusion). Infarct size was significantly reduced by both interventions. Of 350 different microRNAs assessed by microarray analysis, 147-160 microRNAs showed detectable expression levels. Compared with microRNA alterations induced by ischemia-reperfusion versus time-matched nonischemic controls, five microRNAs were significantly affected by both pre- and postconditioning (microRNA-125b*, microRNA-139-3p, microRNA-320, microRNA-532-3p, and microRNA-188), four microRNAs were sign...

Production of bulk amounts of universal RNA for DNA microarrays

BioTechniques

In DNA microarray technology, repeatability and reliability are very important to compare multipl... more In DNA microarray technology, repeatability and reliability are very important to compare multiple RNA samplesfrom different experiments. The application of common or universal RNA as a standard control equalizes the differences in hybridization parameters and array variations. For this purpose, high-quality reference RNA is necessary in bulk amounts. A novel approach was developed to get milligrams of sense or antisense RNA, starting from micrograms of pooled total RNA from different cell lines, tissues, or organisms. This method is inexpensive and allows further labeling procedures using poly(dT) or random oligomers as primers. In addition, amplified, sense reference RNA is suitable for standard labeling protocols, while the antisense reference RNA can be used with antisense RNA from the linear sample amplification method. Here we produced universal RNA for human, rat, and alfalfa and demonstrated the quality using specific cDNA microarrays.

[Comparative chemical proteomics: simultaneous identification of disease-specific protein targets and their small molecule-binding partners, suitable as drug candidates]

The simultaneous identification of disease-specific protein targets and their small molecule bind... more The simultaneous identification of disease-specific protein targets and their small molecule binding partners, suitable as drug candidates, could radically reduce the timeline and costs of drug discovery and development. Comparative chemical proteomics provides a novel approach to achieve this goal through rapid detection of overexpressed proteins in diseased samples by the application of small molecule microarrays. The interacting small molecules enables direct affinity-based isolation and identification of the proteins. In the present paper we report comparative chemical proteomics studies on melanocytes and melanoma cell-lines, which led to the identification of 3 overexpressed proteins (e.g. -tubulin) together with their small molecule binding partner.

Gene profiling identifies genes specific for well-differentiated epithelial thyroid tumors

Thyroid nodules are common. It would very helpful if genetic markers that can diagnose malignancy... more Thyroid nodules are common. It would very helpful if genetic markers that can diagnose malignancy from fine needle aspiration samples were available. Few such markers has been thus identified and none are specific. Large panels of potential markers can be screened by microarray technology. Studies done to date have concentrated on single tumor types and thus provide no help in identifying tumor subtype specific markers. To that end we have studied gene profiles of 5 types of benign and malignant thyroid nodular tissue (multinodular goiter, follicular adenoma, papillary and follicular carcinomas). We have identified 195 genes whose differential expression clustered into clinically relevant groups. Twenty-eight genes were selected for further confirmation using real time quantitative polymerase chain reaction. Despite the differences in the microarray panels used, we confirmed the differential regulation of 12 genes previously reported in thyroid cancer, although we found the expression of several genes to be regulated in other histological tumor subtypes than originally described. We found, PCSK2, TRIB1, RAP1 GA1 to be specifically overexpressed in follicular cancer and S100A4 and GK2 in papillary carcinoma. SERP1, RNASE 2 and STATA5 were suppressed in papillary thyroid cancer. We have thus identified new potential markers specific to malignant thyroid tumors. It is apparent that a range of nodular thyroid tissue using large tumor sample numbers is necessary to establish robust markers for malignancy and to categorize tumors on the basis of small tumor samples.

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Extracellular deposition of matrilin-2 controls the timing of the myogenic program during muscle regeneration

Development, 2014

Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling t... more Here, we identify a role for the matrilin-2 (Matn2) extracellular matrix protein in controlling the early stages of myogenic differentiation. We observed Matn2 deposition around proliferating, differentiating and fusing myoblasts in culture and during muscle regeneration in vivo. Silencing of Matn2 delayed the expression of the Cdk inhibitor p21 and of the myogenic genes Nfix, MyoD and Myog, explaining the retarded cell cycle exit and myoblast differentiation. Rescue of Matn2 expression restored differentiation and the expression of p21 and of the myogenic genes. TGF-b1 inhibited myogenic differentiation at least in part by repressing Matn2 expression, which inhibited the onset of a positive-feedback loop whereby Matn2 and Nfix activate the expression of one another and activate myoblast differentiation. In vivo, myoblast cell cycle arrest and muscle regeneration was delayed in Matn2 2/2 relative to wild-type mice. The expression levels of Trf3 and myogenic genes were robustly reduced in Matn2 2/2 fetal limbs and in differentiating primary myoblast cultures, establishing Matn2 as a key modulator of the regulatory cascade that initiates terminal myogenic differentiation. Our data thus identify Matn2 as a crucial component of a genetic switch that modulates the onset of tissue repair.

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Tissue-specific gene expression in rat hearts and aortas in a model of vascular nitrate tolerance

Journal of Cardiovascular Pharmacology, 2014

Nitroglycerin exerts a direct myocardial anti-ischemic effect even in the state of vascular nitra... more Nitroglycerin exerts a direct myocardial anti-ischemic effect even in the state of vascular nitrate tolerance. To examine the potentially diverse molecular responses in vascular and cardiac tissues, we investigated the gene expression profile of the heart and the aorta by DNA microarray in male Wistar rats that were previously made tolerant to the vascular effects of nitroglycerin. The blood pressure lowering effect of nitroglycerin (1-100 µg/kg) was markedly attenuated in rats pre-treated for 3 days with 3x100 mg/kg nitroglycerin. Nitric oxide content was significantly elevated in the heart but not in the aorta of nitrate-tolerant animals, which indicated tissue-specific differences in nitroglycerin bioconversion. Out of 7742 genes analyzed by DNA microarray, we found that while the expression of 25 genes changed significantly in the heart (increased: Tas2r119, Map6, Cd59, Kcnh2, Kcnh3, Senp6, Mcpt1, Tshb, Haus1, Vipr1, Lrn3, Lifr; decreased: Ihh, Fgfr1, Cryge, Krt9, Agrn, C4bpb, Fcer1a, Csf3, Hsd17b11, Hsd11b2, Ctnnbl1, Prpg1, Hsf1), only 14 genes were altered in the aorta (increased: Tas2r119, Ihh, Rrad, Npm1, Snai1; decreased: Tubb2b, Usp15, Sema6c, Wfdc2, Rps21, Ramp2, Galr1, Atxn1, Lhx1) in vascular nitrate tolerance. QRT-PCR analysis of genes related to oxidative/nitrative/nitrosative stress also showed differential expression pattern in the heart and aorta. This is the first pharmacogenomic analysis showing that nitroglycerin treatment leading to vascular nitrate tolerance differentially impacts gene expression in vascular and cardiac tissues, which indicates different tissue-specific downstream signaling pathways.

Compounds Blocking Methylglyoxal-induced Protein Modification and Brain Endothelial Injury

Archives of Medical Research, 2014

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Combination of unsaturated fatty acids and ionizing radiation on human glioma cells: cellular, biochemical and gene expression analysis

Lipids in Health and Disease, 2014

Background: Based on previous observations a potential resort in the therapy of the particularly ... more Background: Based on previous observations a potential resort in the therapy of the particularly radioresistant glioma would be its treatment with unsaturated fatty acids (UFAs) combined with irradiation. Methods: We evaluated the effect of different UFAs (arachidonic acid (AA), docosahexaenoic acid (DHA), gamma-linolenic acid (GLA), eicosapentaenoic acid (EPA) and oleic acid (OA)) on human U87 MG glioma cell line by classical biochemical end-point assays, impedance-based, real-time cellular and holographic microscopic analysis. We further analyzed AA, DHA, and GLA at morphological, gene and miRNA expression level.

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Facile synthesis of 1 H -imidazo[1,2- b ]pyrazoles via a sequential one-pot synthetic approach

Beilstein Journal of Organic Chemistry, 2014

5-Aminopyrazole-4-carbonitrile and ethyl 5-aminopyrazole-4-carboxylate, as potential trifunctiona... more 5-Aminopyrazole-4-carbonitrile and ethyl 5-aminopyrazole-4-carboxylate, as potential trifunctional building blocks are introduced in a facile, chemo-and regioselective multicomponent assembly of imidazo[1,2-b]pyrazoles via the Groebke-Blackburn-Bienaymé reaction (GBB reaction). Besides the synthetic elaboration of a green-compatible isocyanide-based access in three-component mode, we describe an operationally simple, one-pot two-step GBB protocol for the rapid construction of a 46 membered imidazo[1,2-b]pyrazole library with yields up to 83%.

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New molecular methods for classification, diagnosis and therapy prediction of hematological malignancies

Pathology & Oncology Research, 2002

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Development of chemically modified glass surfaces for nucleic acid, protein and small molecule microarrays

Molecular Diversity, 2003

Microarrays have become a widely used tool to investigate the living cell at different levels. DN... more Microarrays have become a widely used tool to investigate the living cell at different levels. DNA microarrays enable the expression analysis of thousand of genes simultaneously, while protein arrays investigate the properties and interactions of proteins with other proteins and with non-proteinaceous molecules. One crucial step in producing such microarrays is the permanent immobilization of samples on a solid surface. Our goal was to develop diverse linker systems capable of anchoring different biological samples, especially DNA and drug-like small molecules. We developed 6 different chemical surfaces having a 3-D-like linker system for biomolecule immobilization, and compared them to previously described immobilization strategies. The attachment chemistry utilizes the amino reactive properties of acrylic and epoxy functions. The capacity of the support was increased by creating a branching structure holding the reactive functions. The method of anchoring was investigated through a model reaction. From HPLC and mass spectrometry measurements we concluded that the covalent binding of DNA occurs through nucleobases. The tested systems offer the capability to permanently immobilize several biomolecular species in an array format.

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Pattern Triggered Immunity (PTI) in Tobacco: Isolation of Activated Genes Suggests Role of the Phenylpropanoid Pathway in Inhibition of Bacterial Pathogens

PLoS ONE, 2014

Background: Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless fo... more Background: Pattern Triggered Immunity (PTI) or Basal Resistance (BR) is a potent, symptomless form of plant resistance. Upon inoculation of a plant with non-pathogens or pathogenicity-mutant bacteria, the induced PTI will prevent bacterial proliferation. Developed PTI is also able to protect the plant from disease or HR (Hypersensitive Response) after a challenging infection with pathogenic bacteria. Our aim was to reveal those PTI-related genes of tobacco (Nicotiana tabacum) that could possibly play a role in the protection of the plant from disease.

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Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

Background: Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl... more Background: Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/ D3 cell line) treated with methylglyoxal.

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Anti-Inflammatory Effect of Recreational Exercise in TNBS-Induced Colitis in Rats: Role of NOS/HO/MPO System

Oxidative Medicine and Cellular Longevity, 2014

There are opposite views in the available literature: Whether physical exercise has a protective ... more There are opposite views in the available literature: Whether physical exercise has a protective effect or not on the onset of inflammatory bowel disease (IBD). Therefore, we investigated the effects of recreational physical exercise before the induction of colitis. After 6 weeks of voluntary physical activity (running wheel), male Wistar rats were treated with TNBS (10 mg). 72 hrs after trinitrobenzene sulphonic acid (TNBS) challenge we measured colonic gene (TNF-, IL-1 , CXCL1 and IL-10) and protein (TNF-) expressions of various inflammatory mediators and enzyme activities of heme oxygenase (HO), nitric oxide synthase (NOS), and myeloperoxidase (MPO) enzymes. Wheel running significantly increased the activities of HO, constitutive NOS (cNOS) isoform. Furthermore, 6 weeks of running significantly decreased TNBS-induced inflammatory markers, including extent of lesions, severity of mucosal damage, and gene expression of IL-1 , CXCL1, and MPO activity, while IL-10 gene expression and cNOS activity were increased. iNOS activity decreased and the activity of HO enzyme increased, but not significantly, compared to the sedentary TNBS-treated group. In conclusion, recreational physical exercise can play an anti-inflammatory role by downregulating the gene expression of proinflammatory mediators, inducing anti-inflammatory mediators, and modulating the activities of HO and NOS enzymes in a rat model of colitis.

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