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1.  Quantitative RT-PCR based platform for rapid quantification of the transcripts of highly homologous multigene families and their members during grain development 
BMC Plant Biology  2012;12:184.
Cereal storage proteins represent one of the most important sources of protein for food and feed and they are coded by multigene families. The expression of the storage protein genes exhibits a temporal fluctuation but also a response to environmental stimuli. Analysis of temporal gene expression combined with genetic variation in large multigene families with high homology among the alleles is very challenging.
We designed a rapid qRT-PCR system with the aim of characterising the variation in the expression of hordein genes families. All the known D-, C-, B-, and γ-hordein sequences coding full length open reading frames were collected from commonly available databases. Phylogenetic analysis was performed and the members of the different hordein families were classified into subfamilies. Primer sets were designed to discriminate the gene expression level of whole families, subfamilies or individual members. The specificity of the primer sets was validated before successfully applying them to a cDNA population derived from developing grains of field grown Hordeum vulgare cv. Barke. The results quantify the number of moles of transcript contributed to a particular gene family and its subgroups. More over the results indicate the genotypic specific gene expression.
Quantitative RT-PCR with SYBR Green labelling can be a useful technique to follow gene expression levels of large gene families with highly homologues members. We showed variation in the temporal expression of genes coding for barley storage proteins. The results imply that our rapid qRT-PCR system was sensitive enough to identify the presence of alleles and their expression profiles. It can be used to check the temporal fluctuations in hordein expressions or to find differences in their response to environmental stimuli. The method could be extended for cultivar recognition as some of the sequences from the database originated from cv. Golden Promise were not expressed in the studied barley cultivar Barke although showed primer specificity with their cloned DNA sequences.
PMCID: PMC3492166  PMID: 23043496
SYBR Green; High homology multigene families; Transcript abundance; Hordeins
2.  Time-Dependent Changes of Plasma Concentrations of Angiopoietins, Vascular Endothelial Growth Factor, and Soluble Forms of Their Receptors in Nonsmall Cell Lung Cancer Patients Following Surgical Resection 
ISRN Oncology  2012;2012:638352.
Even when patients with nonsmall cell lung cancer undergo surgical resection at an early stage, recurrent disease often impairs the clinical outcome. There are numerous causes potentially responsible for a relapse of the disease, one of them being extensive angiogenesis. The balance of at least two systems, VEGF VEGFR and Ang Tie, regulates vessel formation. The aim of this study was to determine the impact of surgery on the plasma levels of the main angiogenic factors during the first month after surgery in nonsmall cell lung cancer patients. The study group consisted of 37 patients with stage I nonsmall cell lung cancer. Plasma concentrations of Ang1, Ang2, sTie2, VEGF, and sVEGF R1 were evaluated by ELISA three times: before surgical resection and on postoperative days 7 and 30. The median of Ang2 and VEGF concentrations increased on postoperative day 7 and decreased on day 30. On the other hand, the concentration of sTie2 decreased on the 7th day after resection and did not change statistically later on. The concentrations of Ang1 and sVEGF R1 did not change after the surgery. Lung cancer resection results in proangiogenic plasma protein changes that may stimulate tumor recurrences and metastases after early resection.
PMCID: PMC3324894  PMID: 22550599
3.  Premitotic Assembly of Human CENPs -T and -W Switches Centromeric Chromatin to a Mitotic State 
PLoS Biology  2011;9(6):e1001082.
Centromeres are differentiated chromatin domains, present once per chromosome, that direct segregation of the genome in mitosis and meiosis by specifying assembly of the kinetochore. They are distinct genetic loci in that their identity in most organisms is determined not by the DNA sequences they are associated with, but through specific chromatin composition and context. The core nucleosomal protein CENP-A/cenH3 plays a primary role in centromere determination in all species and directs assembly of a large complex of associated proteins in vertebrates. While CENP-A itself is stably transmitted from one generation to the next, the nature of the template for centromere replication and its relationship to kinetochore function are as yet poorly understood. Here, we investigate the assembly and inheritance of a histone fold complex of the centromere, the CENP-T/W complex, which is integrated with centromeric chromatin in association with canonical histone H3 nucleosomes. We have investigated the cell cycle regulation, timing of assembly, generational persistence, and requirement for function of CENPs -T and -W in the cell cycle in human cells. The CENP-T/W complex assembles through a dynamic exchange mechanism in late S-phase and G2, is required for mitosis in each cell cycle and does not persist across cell generations, properties reciprocal to those measured for CENP-A. We propose that the CENP-A and H3-CENP-T/W nucleosome components of the centromere are specialized for centromeric and kinetochore activities, respectively. Segregation of the assembly mechanisms for the two allows the cell to switch between chromatin configurations that reciprocally support the replication of the centromere and its conversion to a mitotic state on postreplicative chromatin.
Author Summary
The centromere is a strange locus that derives its identity from the proteins that shape it rather than the DNA sequences it contains. It also functions in a remarkably singular way, providing a motor and command control center for the chromosome in conjunction with the kinetochore. Key to centromere identity is the chromatin that comprises it, which has a unique nucleosomal “bead on a string” including a special centromeric histone H3, called CENP-A. Found in alternating clusters of nucleosomes with “regular” histone H3, CENP-A is crucial for propagating centromere identity as well as for regulating kinetochore function. In this study, we have analysed the cell cycle dynamics of CENP-T and CENP-W, another two components of the constitutive centromere associated network. We show that, unlike CENP-A, CENP-T/W are not inherited stringently by daughter cells. Instead, these complexes - which are bound to the interstitial “regular” H3 nucleosome domains - assemble after DNA replication and are required for kinetochore formation. Thus, we propose that a stable CENP-A nucleosome population plays a role in centromere locus inheritance to daughter cells, while dynamic CENP-T/W and H3 nucleosomes provide a cycling function that triggers kinetochore assembly as cells enter mitosis in each new cell cycle.
PMCID: PMC3114758  PMID: 21695110

Results 1-3 (3)