Tumor expression of human chorionic gonadotropin beta mRNA and prognosis of prostate cancer treated by radical prostatectomy

The beta subunit of human chorionic gonadotropin (hCG beta) is encoded by six genes (CGB) classified as type I and type II. CGB mRNA is produced in large amounts by trophoblastic tissues and in small amounts by several cancerous tissues including prostate cancer and by a few benign tissues, including the prostate. Quantitative reverse-transcription polymerase chain reaction (RT-qPCR) was used to study the expression levels of all CGB mRNAs together (total CGB mRNA) and the two types of CGB mRNA separately in non-cancerous (n = 74) and cancerous prostatic tissue obtained by radical prostatectomy (n = 193). RNA was isolated from formalin-fixed paraffin-embedded (FFPE) samples and mRNA levels of CGB were correlated with disease-specific survival. Total CGB mRNA concentrations were significantly lower (p <.0001) in cancerous than non-cancerous prostatic tissue. Separate analysis of type I CGB and type II CGB mRNA showed that both type I CGB (p <.0001) and type II CGB mRNA (p = .007) are lower in cancerous tissue than in non-cancerous tissue. Low type II CGB mRNA level in cancerous tissue was associated with shorter cancer-specific survival (p = .001) of prostate cancer patients treated by radical prostatectomy.Peer reviewe

MAPK inhibitors induce serine peptidase inhibitor Kazal type 1 (SPINK1) secretion in BRAF V600E-mutant colorectal adenocarcinoma

The mitogen-activated protein kinase (MAPK) pathway plays a central role in colorectal cancers (CRC). In particular, BRAF V600E-mutant tumors, which represent around 10% of CRCs, are refractory to current therapies. Overexpression and secretion of serine peptidase inhibitor Kazal type 1 (SPINK1) are observed in around 50% of CRCs, and its serum level can be used as a biomarker for poor prognosis. Utilizing a recently developed extendable blocking probe assay, we analyzed the BRAF mutation status in a CRC patient cohort (N=571) using tissue-derived RNA as the starting material. From the same RNA samples, we measured the relative SPINK1 expression levels using a quantitative real-time PCR method. Expression of mutant BRAF V600E correlated with poor prognosis, as did low expression of SPINK1 mRNA. Further, BRAF V600E correlated negatively with SPINK1 levels. In order to investigate the effect of MAPK pathway-targeted therapies on SPINK1 secretion, we conducted invitro studies using both wild-type and V600E CRC cell lines. BRAF inhibitor vemurafenib, and subsequent MAPK pathway inhibitors trametinib and SCH772984, significantly increased SPINK1 secretion in V600E CRC cell lines Colo205 and HT-29 with a concomitant decrease in trypsin-1 and -2 secretion. Notably, no SPINK1 increase or trypsin-1 decrease was observed in BRAF wild-type CRC cell line Caco-2 in response to MAPK pathway inhibitors. In further mechanistic studies, we observed that only trametinib was able to diminish completely both MEK and ERK phosphorylation in the V600E CRC cells. Furthermore, the key regulator of integrated stress response, activating transcription factor 4 (ATF-4), was downregulated both at mRNA and at protein level in response to trametinib treatment. In conclusion, these data suggest that sustained inhibition of not only MAPK pathway activation, but also ATF-4 and trypsin, might be beneficial in the therapy of BRAF V600E-mutant CRC and that SPINK1 levels may serve as an indicator of therapy response.Peer reviewe

Extendable blocking probe in reverse transcription for analysis of RNA variants with superior selectivity

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Enrollment and completion rates of a nationwide guided digital parenting program for children with disruptive behavior before and during COVID-19

Our aim was to study enrollment and completion levels for the internet-based and telephone-assisted Finnish Strongest Families Smart Website (SFSW) parent training intervention, for parents of young children with disruptive behavior before and after the COVID-19 lockdown period. Population-based screening was carried out on 39,251 children during routine check- ups at 4 years of age. The parents of children scoring at least 5 on the Strengths and Difficulties Questionnaire were assessed against inclusion and exclusion criteria. Associations with enrollment or completion were analyzed using logistic regression models. The effects of COVID-19 restrictions on these were estimated using interrupted timeseries analysis. Of 39,251 families, 4894 screened positive and met the eligibility criteria. Of those, 3068 (62.6%) decided to enroll in the SFSW program and 2672 (87.1%) of those families completed it. The highest level of disruptive behavior (OR 1.33, 95% CI 1.12–1.57, p < 0.001) and overall severity of difficulties (OR 2.22, 95% CI 1.91–2.57, p < 0.001) were independently associated with enrollment. Higher parental education was associated with enrollment and completion. Higher paternal age was associated with enrollment, and parent depressive symptoms with non-completion. The SFSW enrollment did not significantly change following the COVID-19 restrictions, while the completion rate increased (COVID-19 completion OR 1.75, 95% CI 1.22–2.50, p = 0.002). Guided digital parenting interventions increase the sustainability of services, by addressing the child mental health treatment gap and ensuring service consistency during crisis situations.Peer reviewe

Technique for strand-specific gene-expression analysis and monitoring of primer-independent cDNA synthesis in reverse transcription

Primer-independent cDNA synthesis during reverse transcription hinders quantitative analysis of bidirectional mRNA synthesis in eukaryotes as well as in cells infected with RNA viruses. We report a simple RT-PCR-based assay for strand-specific gene-expression analysis. By modifying the cDNA sequence during reverse transcription, the opposite strands of target sequences can be simultaneously detected by postamplification melting curve analysis and primer-initiated transcripts are readily distinguished from nonspecifically primed cDNA. We have utilized this technique to optimize the specificity of reverse transcription on a panel of 15 target genes. Primer-independent reverse transcription occurred for all target sequences when reverse transcription was performed at 42°C and accounted for 11%–57% of the final PCR amplification products. By raising the reaction temperature to 55°C, the specificity of reverse transcription could be increased without significant loss of sensitivity. We have also demonstrated the utility of this technique for analysis of (+) and (-) RNA synthesis of influenza A virus in infected cells. Thus, this technique represents a powerful tool for analysis of bidirectional RNA synthesis

Prognostic impact of kallikrein-related peptidase transcript levels in prostate cancer

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