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Hepatitis C virus (HCV) is a globally significant blood-borne agent causing liver diseases, and it has infected over 170 million people worldwide. HCV is a diverse group of RNA viruses currently divided into genotypes 1-7 as well as subtypes. HCV infection can be treated with antiviral drugs, but the HCV genotype has to be determined for optimal selection of treatment strategy. The aim of this study was to set up a sequencing-based HCV genotyping method suitable for the workflow of a diagnostic laboratory. The established method is robust and stable, and it utilizes a one-step reverse transcription and PCR amplification of the 5' untranslated region (5'UTR) and partial Core region of the HCV genome. Amplification products are sequenced using the standard Sanger method, and the genotype is determined by using a freely accessible web-based genotyping tool. The method was validated at the Helsinki University Hospital Laboratory using 238 previously genotyped serum samples. •A new one-step RT-PCR method for the amplification of the 5' untranslated region and partial Core region of hepatitis C virus was established.•HCV genotype is determined using Sanger sequencing and a freely accessible, easy-to-use web-based genotyping tool.•The method is robust, reproducible and suitable for diagnostic laboratory workflow, and it requires no costly instrumentation or specialized sequence analysis skills.

Background & Aims: The human hepatitis B virus (HBV) is a major cause of chronic hepatitis and hepatocellular carcinoma, but molecular mechanisms driving liver disease and carcinogenesis are largely unknown. We therefore studied cellular pathways altered by HBV infection., Methods: We performed gene expression profiling of primary human hepatocytes infected with HBV and proved the results in HBV-replicating cell lines and human liver tissue using real-time polymerase chain reaction and Western blotting. Activation of signal transducer and activator of transcription (STAT3) was examined in HBV-replicating human hepatocytes, HBV-replicating mice, and liver tissue from HBV-infected individuals using Western blotting, STAT3-luciferase reporter assay, and immunohistochemistry. The consequences of STAT3 activation on HBV infection and cell survival were studied by chemical inhibition of STAT3 phosphorylation and small interfering RNA-mediated knockdown of STAT3., Results: Gene expression profiling of HBV-infected primary human hepatocytes detected no interferon response, while genes encoding for acute phase and antiapoptotic proteins were up-regulated. This gene regulation was confirmed in liver tissue samples of patients with chronic HBV infection and in HBV-related hepatocellular carcinoma. Pathway analysis revealed activation of STAT3 to be the major regulator. Interleukin-6-dependent and -independent activation of STAT3 was detected in HBV-replicating hepatocytes in cell culture and in vivo. Prevention of STAT3 activation by inhibition of Janus tyrosine kinases as well as small interfering RNA-mediated knockdown of STAT3-induced apoptosis and reduced HBV replication and gene expression., Conclusions: HBV activates STAT3 signaling in hepatocytes to foster its own replication but also to prevent apoptosis of infected cells. This very likely supports HBV-related carcinogenesis.

Chronic myeloid leukemia (CML) is the paradigm for targeted cancer therapy. RT-qPCR is the gold standard for monitoring response to tyrosine kinase-inhibitor (TKI) therapy based on the reduction of blood or bone marrow BCR-ABL1 . Some patients with CML and very low or undetectable levels of BCR-ABL1 transcripts can stop TKI-therapy without CML recurrence. However, about 60 percent of patients discontinuing TKI-therapy have rapid leukaemia recurrence. This has increased the need for more sensitive and specific techniques to measure residual CML cells. The clinical challenge is to determine when it is safe to stop TKI-therapy. In this review we describe and critically evaluate the current state of CML clinical management, different technologies used to monitor measurable residual disease (MRD) focus on comparingRT-qPCR and new methods entering clinical practice. We discuss advantages and disadvantages of new methods.

The HER4 receptor tyrosine kinase and STAT5A cooperate to promote mammary luminal progenitor cell maturation and mammary epithelial cell differentiation. Coupled HER4 and STAT5A signaling is mediated, in part, through association of the HER4 intracellular domain (4ICD) with STAT5A at STAT5A target gene promoters where 4ICD functions as a STAT5A transcriptional coactivator. Despite an essential role for coupled 4ICD and STAT5A signaling in mammary gland development, the mechanistic basis of 4ICD and STAT5A cooperative signaling remains unexplored. Here we show for the first time that 4ICD and STAT5A directly interact through STAT5A recruitment and binding to HER4/4ICD residue Y984. Accordingly, altering the 4ICD Y984 to phenylalanine results in a dramatic reduction of STAT5A and 4ICD-Y984F interacting complexes coimmunoprecipitated with HER4 or STAT5A specific antibodies. We further show that disrupting the 4ICD and STAT5A interaction has an important physiological impact on mammary epithelial cell differentiation. HC11 mammary epithelial cells with stable expression of 4ICD undergo differentiation with significantly increased expression of the STAT5A target genes and differentiation markers β-casein and WAP. In contrast, HC11 cells stably expressing 4ICD-Y984F failed to undergo differentiation with basal expression levels of β-casein and WAP. Differentiation in this cell system was induced in the absence of exogenous prolactin indicating that 4ICD activity is sufficient to induce mammary epithelial cell differentiation. Finally, we show that suppression of STAT5A expression abolishes the ability of 4ICD to induce HC11 differentiation and activate β-casein or WAP expression. Taken together our results demonstrate for the first time that direct coupling of 4ICD and STAT5A is both necessary and sufficient to drive mammary epithelial differentiation. In conclusion, our findings that 4ICD and STAT5A directly interact to form a physiologically important transcriptional activation complex, provide a mechanistic basis for the in vivo observations that HER4/4ICD and STAT5A cooperate to promote mammary gland progenitor cell maturation and initiate lactation at parturition.

Background: microRNAs have recently been identified as powerful biomarkers of human disease. Reliable polymerase chain reaction (PCR)-based quantification of nucleic acids in clinical samples contaminated with polymerase inhibitor heparin requires deheparinization. However, the effects of deheparinization procedure on quantification of nucleic acids remain largely unknown. The aim of this study was to determine whether the deheparinization procedure completely eliminates the inhibition of amplification, while maintaining RNA integrity and technical variability of the measured microRNA levels., Methods: Heparinized plasma from 9 patients undergoing coronary artery bypass grafting (CABG) and the heparin-free plasma from 58 rats were spiked with a synthetic RNA oligonucleotide and total RNA was extracted. The RNA solutions were then treated with heparinase I to remove contaminating heparin prior to reverse transcription. Levels of synthetic spike-in RNA oligonucleotide, as well as endogenous hsa-miR-1-3p and hsa-miR-208a-3p, were measured using quantitative reverse transcription PCR (RT-qPCR). The amplification efficiency and presence of inhibitors in individual samples were directly determined using calibration curves., Results: In contrast to RNA samples from rat plasma, RNA samples derived from the CABG patient plasma contained inhibitors, which were completely eliminated by treatment with heparinase. The procedure caused a decrease in the amount of detected RNA; however, the technical variability of the measured targets did not change, allowing for the quantification of circulating endogenous hsa-miR-1-3p and hsa-miR-208a-3p in the plasma of CABG patients., Conclusions: The heparinase treatment procedure enables utilization of RT-qPCR for reliable microRNA quantification in heparinized plasma.

Watermelon mosaic potyvirus (WMV) is considered as an important virus infecting watermelon and causing adverse effects on crop productivity. To overcome this problem one of the main objectives of plant breeders is to make these strains less effective in the ability to infect plants by treatment with plant extracts. Due to the advantages of plant tissue culture, in vitro, in the process of the selection of different cultivars under biotic stress, this study was conducted to achieve this aim by evaluating the effect of three concentrations of Thuja extract on the multiplication of WMV in watermelon by measuring callus fresh weight and soluble proteins (mg g(-1) fresh weight) of healthy and infected hypocotyl explants. Also, WMV was isolated from naturally infected watermelon and characterized as potyvirus by serological and molecular analyses. The isolated virus gave a positive reaction with WMV antiserum compared with other antibodies of CMV, ZYMV and SqMV using DAS-ELISA. RT-PCR, with the specific primer for WMV-cp. gene, yielded 825 base pair DNA fragments. The results that belong to soluble protein analysis indicated that infected hypocotyl explants treated with 6 g L(-1) recorded the highest rate in the number of soluble protein bands compared with the rest of treatments. As a conclusion of these results, we can recommend to apply the Thuja extract at 6 g L(-1) as a optimum dosage to decrease the infection caused by watermelon mosaic potyvirus.

The rate of secretion of αs2-casein into bovine milk is approximately 25% of that of β-casein, yet mammary expression of their respective mRNA transcripts (csn1s2 and csn2) is not different. Our objective was to identify molecular mechanisms that explain the difference in translation efficiency between csn1s2 and csn2. Cell-free translational efficiency of csn2 was 5 times that of csn1s2. Transcripts of csn1s2 distributed into heavier polysomes than csn2 transcripts, indicating an attenuation of elongation and/or termination. Stimulatory and inhibitory effects of the 5' and 3' UTRs on translational efficiency were different with luciferase and casein sequences in the coding regions. Substituting the 5' and 3' UTRs from csn2 into csn1s2 did not improve csn1s2 translation, implicating the coding region itself in the translation difference. Deletion of a 28-codon fragment from the 3' terminus of the csn1s2 coding region, which displays codons with low correlations to cell fitness, increased translation to a par with csn2. We conclude that the usage of the last 28 codons of csn1s2 is the main regulatory element that attenuates its expression and is responsible for the differential translational expression of csn1s2 and csn2.

Cytochrome P450 (CYP) enzymes metabolize numerous endogenous substrates, such as retinoids, androgens, estrogens and vitamin D, that can modulate important cellular processes, including proliferation, differentiation and apoptosis. The aim of this study is to characterize the expression of CYP genes in CD34+ human cord blood hematopoietic stem and early progenitor cells (CBHSPCs) as a first step toward assessment of the potential biological functions of CYP enzymes in regulating the expansion or differentiation of these cells. CD34+ CBHSPCs were purified from umbilical cord blood via antibody affinity chromatography. Purity of CD34+ CBHSPCs was assessed using fluorescence-activated cell sorting. RNA was isolated from purified CD34+ CBHSPCs and total mononuclear cells (MNCs) for RNA-PCR analysis of CYP expression. Fourteen human CYPs were detected in the initial screening with qualitative RT-PCR in CD34+ CBHSPCs. Further quantitative RNA-PCR analysis of the detected CYP transcripts yielded evidence for preferential expression of CYP2R1 in CD34+ CBHSPCs relative to MNCs; and for greater expression of CYP1B1 in MNCs relative to CD34+ CBHSPCs. These findings provide the basis for further studies on possible functions of CYP2R1 and CYP1B1 in CBHSPCs׳ proliferation and/or differentiation and their potential utility as targets for drugs designed to modulate CD34+ CBHSPC expansion or differentiation.