Physical-chemical analysis of different types of flours available in the Romanian market.

The physical-chemical characteristics of different types of flours are essential for understanding their composition, nutritional value, and functional properties as well. The aim of this research was to identify the variability of the different wheat flours available in Romania. In this study 39 different wheat flours were selected and the following parameters were analyzed in the laboratory: moisture content, ash content, gluten content (wet and dry) and wet gluten spreading. The tested flours were classified into four different classes according to their ash content: 480 (ash content 0.48%) (N = 11), 550 (0.55%) (N = 9), 650 (0.65%) (N = 8), 1100 (1.1%) (N = 11). Mathematical and statistical methods were used to analyze the obtained results: descriptive statistics, box-plot, Spearman correlation and hierarchical cluster analysis. The results revealed that moisture content varied between 9.5 and 11.8%. In terms of ash content, the lowest and highest measured values were 0.427-2.04 g/100 g. The average wet gluten content of the studied flours varied between 30 and 32%, while the average dry gluten content was 12.8%. The findings indicate that the moisture content of all examined flour samples was within permissible levels for extended storage, aligning with established standards. Gluten is a key and essential parameter for bread making because influences the dough mixing and baking properties. The mineral content, represented by ash content, is influenced by cereal type and milling process, with wheat's ash content ranging between 1.5 and 2%. Flours with high wet gluten content (> 34%) can be used to improve the properties of lower quality flours. Further studies are necessary in order to determine the possible health effects of different cereal varieties.

Setting up a probe based, closed tube real-time PCR assay for focused detection of variable sequence alterations.

During diagnostic workflow when detecting sequence alterations, sometimes it is important to design an algorithm that includes screening and direct tests in combination. Normally the use of direct test, which is mainly sequencing, is limited. There is an increased need for effective screening tests, with "closed tube" during the whole process and therefore decreasing the risk of PCR product contamination. The aim of this study was to design such a closed tube, detection probe based screening assay to detect different kind of sequence alterations in the exon 11 of the human c-kit gene region. Inside this region there are variable possible deletions and single nucleotide changes. During assay setup, more probe chemistry formats were screened and tested. After some optimization steps the taqman probe format was selected.

Molecular characterization of 103 ovarian serous and mucinous tumors.

The pathogenesis of ovarian carcinomas is heterogeneous, with even the same entities showing great variance. In our study we investigated the mutations of the BRAF, KRAS, and p53 genes in serous and mucinous borderline tumors and in low grade and high grade serous and mucinous tumors. The mutations of BRAF and KRAS genes have been shown in 60% of borderline and low grade (well differentiated) serous and mucinous tumors, but very rarely in high grade (moderately and poorly differentiated) carcinomas. However mutations of p53 are very common in high grade tumors and this indicates a "dualistic" model of ovarian tumorigenesis. A total of 80 serous tumors, including serous borderline, low grade and high grade tumors, and 23 mucinous tumors, including borderline and invasive tumors were analysed for BRAF and KRAS mutations using real time PCR method followed by melting point analysis. P53 mutation was investigated by immunohistochemistry. We assumed mutation of the p53 gene when 100% of tumor cells showed strong nuclear positivity. We observed differences in genetic alterations in the development of the low grade tumors and between low and high grade tumors too. In some bilateral or stage II-III cases we observed differences between the mutation status of the left and right ovarian tumors and between the primary tumor and its implants. In one case in a tumor with micropapillary pattern showing high grade nuclear atypia we could detect mutations in both KRAS and p53 genes. The majority of our mucinous ovarian tumor cases showed a KRAS mutation. We have not found mutations of the BRAF and p53 genes in these cases. We have found as have others, that there is a dualistic pathway of ovarian carcinogenesis. In the majority of cases, low grade epithelial tumors develop in a stepwise manner due to genetic alterations of the members of MAP-kinase pathway; however mutation of the p53 gene is the key event in the development of high grade tumors.

High resolution melting curve analysis of DNA sequence alterations of various sizes.

In the diagnostic workflow we need to think in algorithms, containing more assays. One of the most important task in the management of cancer patient is to detect nucleic acid sequence changes in clinical specimens. Before using the most expensive method to analyze direct our targets, a screening assay is needed to reduce the number of samples. In the detection of gene-sequence alterations classical screening methods are available, as SSCP, DGGE or TGGE, (Finke Exp Clin Endocrinol Diabetes 104:92-97, 1996; Lessa and Applebaum Mol Ecol 2:119-129, 1993) however these are very time consuming processes. At this time in the molecular lab the real-time PCR equipments are very popular and with the function of melting curve analysis it can be a very convenient, simple and cost-efficient screening method.

Pathways of urothelial cancer progression suggested by Bayesian network analysis of allelotyping data.

Urothelial cancers of the bladder (UC) comprise biologically heterogeneous group of tumors and display complex genetic alterations. Several genetic changes have been analyzed in detail and some of them are associated with the development and progression of UCs. Only a few studies, however, are focused on identifying the order in which the aberrations may appear during UC tumorigenesis. We have analyzed 123 papillary UCs of the bladder by microsatellites for each of the chromosomal regions that have been suggested to be specifically involved in this type of tumor. We used Bayesian network modeling that enables to uncover multivariate probabilistic dependencies between variables. This methodology applied to LOH data allowed us to discover patterns of losses in UCs. Exploiting the mechanism of probabilistic reasoning in Bayesian networks we suggest primary and secondary events in tumor pathogenesis and reconstruct the possible flow of progression of allelic changes. Losses of chromosome 9p and 9q were found to be the primary events. Losses of 8p and 17p are important events leading to progression of tumor cell clones. The loss of 17p occurs when both abnormalities of chromosome 9 and 8p are already present. There are chromosomal losses related to 8p (1q, 18q, 10q) and some losses like 5q/5p were associated with 17p, leading to the hypothesis of different genetic pathways of UC progression. The abnormalities of chromosome regions 13q, 16q, 6q, 14q, 3p are suggested to be late events being accumulated during the progression of cancer. Although some genetic changes were associated only with the 8p pathway, most secondary genetic changes appear in both pathways. Supplementary material for this article can be found on the International Journal of Cancer website at http://www.interscience.wiley.com/jpages/0020-7136/suppmat/index.html.

[Fetal sex determination with real time PCR of fetal DNA in maternal plasma]. / Szabad magzati DNS kimutatása a magzati Y-kromoszóma igazolásával anyai vérplazmából.

INTRODUCTION: Non-invasive methods using maternal plasma and serum for molecular genetic diagnosis become an important field of interest in prenatal genetic diagnosis. Free fetal DNA in maternal plasma and serum has been shown to be useful for fetal gender determination, and seems to offer a new possibility to perform non-invasive prenatal genetic diagnosis. A possible application is fetal sex determination for couples at risk of X-linked diseases. The aim of this study was to control the reliability and reproducibility of the real-time PCR amplification of the SRY region. MATERIALS AND METHODS: Maternal serum before amniocentesis, and amnionic fluid samples were obtained from 56 pregnant women during the 11th to 22nd weeks of gestation. Real-time PCR analysis of the SRY region was performed in order to determine the fetal sex. Routine karyotyping of cultured amnionic cells was also performed on the samples. Six cases were excluded. RESULTS: In 26 of 50 pregnancies were found male fetuses by cytogenetic analysis. Real time PCR of maternal plasma has been positive for the SRY region in 27 cases. In 47 cases the cytogenetic gender and the real-time PCR result was correlating. In one case of 46,XY karyotype the PCR reaction for SRY region was negative, in two cases of SRY positivity the karyotype was 46,XX. In this study are presented the results of fetal sex determination in maternal plasma using real time PCR method. CONCLUSIONS: The real time PCR detection of fetal DNA in maternal plasma seems to be an easy non-invasive method to determine the fetal sex at this gestational age. Our experience is promising in terms of the specificity and sensitivity of the method.

[Genetic investigations in forensic medicine]. / DNS vizsgálatok az igazságügyi orvostanban.

Scientific research of the last decade including the introduction of new molecular biological methods and mapping of the human genome allowed the development of a revolutionary new molecular biological approach in forensic medicine. The traditional serological methods study proteins, the new DNA analysis goes further down to study DNA structures to analyze unique individual features. The two main areas of DNA application in forensic medicine are inheritance studies and personal identification in criminal cases using biological traces. Using this new, reliable and reproducible method we can answer questions they were almost impossible in the past. This article reviews how molecular techniques used to detect genetic polymorphism in forensic medicine.

Alteration of the LRP1B gene region is associated with high grade of urothelial cancer.

We have delineated regions of interest at chromosome 2q21.2, 2q36.3, and 2q37.1 by deletion mapping of 114 urothelial cancers (UC). Altogether, 17%, 18%, and 63% of the G1, G2, and G3 tumors displayed loss of heterozygosity at chromosome 2q, respectively, The region at 2q21.2 was narrowed down to the LRP1B gene (NT_005129.6). Hemi- and homozygous deletion at the LRP1B gene region was seen in 31 of 114 UCs. Only 8% of the UCs with G1 and none with G2 tumors showed loss of heterozygosity at the LRP1B gene, whereas 49% of the G3 UCs had allelic loss at this region. RT-PCR analysis of the LRP1B gene showed the lack of expression of several exons in 2 of 9 cases analyzed. Our analysis suggests that the LRP1B gene is a candidate tumor suppressor gene in UCs.