Development of a new DHPLC assay for genotyping UGT1A1(TA)n polymorphism associated with Gilbert’s syndrome

Introduction: Gilbert’s syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats in-stead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay. Materials and methods: Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes - (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplifi-ed by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis. Results: All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes. Conclusions: We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay

Neprototipni diskurzni označevalec zdaj

Prispevek predstavlja kvalitativno študijo primera diskurznega označevalca zdaj, ki je bil doslej proučevan v prozodično nevpeti strukturi oz. kot prototipni diskurzni označevalec. Strukturna, tj. prozodična, analiza kaže, da se diskurzni označevalec zdaj pojavlja tudi v neprototipni obliki. Na podlagi semantične analize, ki jo podpremo s testom parafraziranja in prevajanja, ugotavljamo, da neprototipni zdaj uvaja manjše tematske premike kot njegova prototipna različica in se pojavlja predvsem v vlogi poudarjanja, medtem ko je za prototipni zdaj značilna predvsem vloga strukturiranja diskurza. The present paper presents a qualitative case study of the Slovene discourse marker zdaj (now), which, thus far, has been explored in its prosodically prominent structure or as a prototypical discourse marker. The structural, i.e., the prosodical analysis, highlights that the discourse marker zdaj also manifests in its non-prototypical form. By means of semantic analysis, underpinned by p araphrase a nd t ranslation t ests, we s uggest t hat t he n on-prototypical form signals subtler frame shifts than its prototypical counterpart, and that it primarily functions as an emphasis device, whereas the prototypical zdaj predominately performs discourse structuring functions

Can Turn-Taking Highlight the Nature of Non-Verbal Behavior: A Case Study

The present research explores non-verbal behavior that accompanies the management of turns in naturally occurring conversations. To analyze turn management, we implemented the ISO 24617-2 multidimensional dialog act annotation scheme. The classification of the communicative intent of non-verbal behavior was performed with the annotation scheme for spontaneous authentic communication called the EVA annotation scheme. Both dialog acts and non-verbal communicative intent were observed according to their underlying nature and information exchange channel. Both concepts were divided into foreground and background expressions. We hypothesize that turn management dialog acts, being a background expression, co-occur with communication regulators, a class of non-verbal communicative intent, which are also of background nature. Our case analysis confirms this hypothesis. Furthermore, it reveals that another group of non-verbal communicative intent, the deictics, also often accompany turn management dialog acts. As deictics can be both foreground and background expressions, the premise that background non-verbal communicative intent is interlinked with background dialog acts is upheld. And when deictics were perceived as part of the foreground they co-occurred with foreground dialog acts. Therefore, dialog acts and non-verbal communicative intent share the same underlying nature, which implies a duality of the two concepts

Polymorphisms in Arsenic(+III Oxidation State) Methyltransferase (AS3MT) Predict Gene Expression of AS3MT as Well as Arsenic Metabolism

Background: Arsenic is mono- (MMA) and dimethylated (DMA) in humans and the methylation pattern demonstrates large inter-individual differences. The fraction of urinary MMA is a marker for susceptibility to arsenic-related diseases. Objectives: The impact of polymorphisms in five methyltransferase genes on arsenic metabolism was evaluated in two populations, one in South America, one in southeast Asia. The methyltransferase genes were arsenic(+III)methyltransferase (AS3MT), DNAmethyltransferase 1a and 3b (DNMT1a, DNMT3b), phosphatidylethanolamine Nmethyltransferase (PEMT) and betaine-homocysteine methyltransferase (BHMT). AS3MT expression was analyzed in peripheral blood. Methods: Subjects were women, exposed to arsenic in drinking water in the Argentinean Andes (N=172median urinary arsenic 200 [micro]g/L) and in rural Bangladesh (N=361100g/L, all in early pregnancy). Urinary arsenic metabolites were measured by HPLC-ICPMS. Polymorphisms (N=22) were genotyped with SequenomTM. AS3MT expression was measured with qPCR using TaqManr expression assays. Results: Six AS3MT polymorphisms were significantly associated with arsenic metabolite patterns in both populations (p-values ?0.01). The most frequent AS3MT haplotype in Bangladesh was associated with higher %MMA, and the most frequent in Argentina with lower %MMA and higher %DMA. Four polymorphisms in the DNMTs were associated with metabolite patterns in Bangladesh. Non-coding AS3MT polymorphisms affected gene expression of AS3MT in peripheral blood, demonstrating that one functional impact of AS3MT polymorphisms may be altering levels of gene expression. Conclusions: Polymorphisms in AS3MT significantly predicted As metabolism across these two very different populations, suggesting that AS3MT may have an impact on As metabolite patterns in populations worldwide

GSTM1 and GSTT1 double null genotypes determining cell fate and proliferation as potential risk factors of relapse in children with hematological malignancies after hematopoietic stem cell transplantation.

PURPOSE This study aimed to retrospectively evaluate the genetic association of null variants of glutathione S-transferases GSTM1 and GSTT1 with relapse incidence in children with hematological malignancies (HMs) undergoing busulfan (BU)- containing allogeneic hematopoietic stem cell transplantation (HSCT) and to assess the impact of these variants on BU-induced cytotoxicity on the immortalized lymphoblastoid cell lines (LCLs) and tumor THP1 GST gene-edited cell models. METHODS GSTM1- and GSTT1-null alleles were genotyped using germline DNA from whole blood prior to a conditioning BU-based regimen. Association of GSTM1- and GSTT1-null variants with relapse incidence was analyzed using multivariable competing risk analysis. BU-induced cell death studies were conducted in GSTs- null and non-null LCLs and CRISPR-Cas9 gene-edited THP1 leukemia cell lines. RESULTS Carrying GSTM1/GSTT1 double null genotype was found to be an independent risk factor for post-HSCT relapse in 86 children (adjusted HR: 6.52 [95% Cl, 2.76-15.42; p = 1.9 × 10-5]). BU-induced cell death preferentially in THP1GSTM1(non-null) and LCLsGSTM1(non-null) as shown by decreased viability, increased necrosis and levels of the oxidized form of glutathione compared to null cells, while GSTT1 non-null cells showed increased baseline proliferation. CONCLUSION The clinical association suggests that GSTM1/GSTT1 double null genotype could serve as genetic stratification biomarker for the high risk of post-HSCT relapse. Functional studies have indicated that GSTM1 status modulates BU-induced cell death. On the other hand, GSTT1 is proposed to be involved in baseline cell proliferation

Napotki za premagovanje neželenih učinkov sistemskega zdravljenja raka : kaj morate vedeti?

V knjižici opisujemo različne vrste sistemskega zdravljenja, kamor uvrščamo zdravljenje s citostatiki (to je s kemoterapijo), hormonskimi zdravili in tarčnimi zdravili. S temi zdravili pa ne vplivamo samo na rakave celice, ampak tudi na zdrave celice v telesu, zaradi česar med zdravljenjem lahko nastanejo neželeni učinki. Nekatere neželene učinke lahko preprečimo ali pa jih uspešno lajšamo, če se že pojavijo