Transcription Factor STOX1A Promotes Mitotic Entry by Binding to the CCNB1 Promotor

Background: In this study we investigated the involvement of the transcription factor STOX1A in the regulation of the cell cycle. Methodology/Principal Findings: We found that several major cell cycle regulatory genes were differentially expressed upon STOX1A stimulation and knockdown in the neuroblastoma cell line SH-SY5Y. This includes STOX1A dependent differential regulation of cyclin B1 expression, a cyclin which is known to regulate mitotic entry during the cell cycle. The differential regulation of cyclin B1 expression by STOX1A is direct as shown with chromatin immunoprecipitation. Results furthermore suggest that mitotic entry is enhanced through the direct upregulation of cyclin B1 expression effectuated b

SFRS7-Mediated Splicing of Tau Exon 10 Is Directly Regulated by STOX1A in Glial Cells

Background: In this study, we performed a genome-wide search for effector genes bound by STOX1A, a winged helix transcription factor recently demonstrated to be involved in late onset Alzheimer’s disease and affecting the amyloid processing pathway. Methodology/Principal Findings: Our results show that out of 218 genes bound by STOX1A as identified by chromatinimmunoprecipitation followed by sequencing (ChIP-Seq), the serine/arginine-rich splicing factor 7 (SFRS7) was found to be induced, both at the mRNA and protein levels, by STOX1A after stable transfection in glial cells. The increase in SFRS7 was followed by an increase in the 4R/3R ratios of the microtubule-associated protein tau (MAPT) by differential exon 10 splicing. Secondly, STOX1A also induced expression of total tau both at the mRNA and protein levels. Upregulation of total tau expression (SFRS7-independent) and tau exon 10 splicing (SFRS7-dependent), as shown in this study to be both affected by STOX1A, is known to have implications in neurodegeneration

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

Evidence synthesis to inform model-based cost-effectiveness evaluations of diagnostic tests: a methodological systematic review of health technology assessments

Background: Evaluations of diagnostic tests are challenging because of the indirect nature of their impact on patient outcomes. Model-based health economic evaluations of tests allow different types of evidence from various sources to be incorporated and enable cost-effectiveness estimates to be made beyond the duration of available study data. To parameterize a health-economic model fully, all the ways a test impacts on patient health must be quantified, including but not limited to diagnostic test accuracy. Methods: We assessed all UK NIHR HTA reports published May 2009-July 2015. Reports were included if they evaluated a diagnostic test, included a model-based health economic evaluation and included a systematic review and meta-analysis of test accuracy. From each eligible report we extracted information on the following topics: 1) what evidence aside from test accuracy was searched for and synthesised, 2) which methods were used to synthesise test accuracy evidence and how did the results inform the economic model, 3) how/whether threshold effects were explored, 4) how the potential dependency between multiple tests in a pathway was accounted for, and 5) for evaluations of tests targeted at the primary care setting, how evidence from differing healthcare settings was incorporated. Results: The bivariate or HSROC model was implemented in 20/22 reports that met all inclusion criteria. Test accuracy data for health economic modelling was obtained from meta-analyses completely in four reports, partially in fourteen reports and not at all in four reports. Only 2/7 reports that used a quantitative test gave clear threshold recommendations. All 22 reports explored the effect of uncertainty in accuracy parameters but most of those that used multiple tests did not allow for dependence between test results. 7/22 tests were potentially suitable for primary care but the majority found limited evidence on test accuracy in primary care settings. Conclusions: The uptake of appropriate meta-analysis methods for synthesising evidence on diagnostic test accuracy in UK NIHR HTAs has improved in recent years. Future research should focus on other evidence requirements for cost-effectiveness assessment, threshold effects for quantitative tests and the impact of multiple diagnostic tests

Erratum to: Methods for evaluating medical tests and biomarkers

[This corrects the article DOI: 10.1186/s41512-016-0001-y.]

STOX1A induces changes in several major cell cycle regulatory genes.

<p>(A) The effect of stable STOX1A overexpression in the SH-SY5Y neuroblastoma cell line on four major mammalian cell cycle regulatory genes was determined with quantitative RT-PCR showing a mean 1,4 fold (mean ΔΔCt is −0,49) and mean 1,72 fold (mean ΔΔCt is −0,78) increased mRNA expression for CCNA2 and CCNB1, respectively, and a mean 1,22 fold (mean ΔΔCt is −0,29) decreased mRNA expression for CCNC in STOX1A stably transfected cell compared to their negative controls. (B) The effect of STOX1A knockdown in the SH-SY5Y neuroblastoma cell line on four major mammalian cell cycle regulatory genes was determined with quantitative RT-PCR showing a mean 11,8 fold (mean ΔΔCt is −3,56), mean 10.3 fold (mean ΔΔCt is −3,37) and mean 3,8 fold (mean ΔΔCt is −1,92) increased mRNA expression for CCNA2, CCNB1 and CCNE1 respectively, and a mean 1,7 fold (mean ΔΔCt is −0,77) increased mRNA expression for CCNC in STOX1A siRNA treated cells compared to their negative controls. (A,B) Bars are mean ± SEM. * indicate <i>P</i><0.05, ** indicate <i>P</i><0.01, *** indicate <i>P</i><0.001 (one sample t-test with theoretical mean 0). N = 4, each sample was measured in triplicate. (C) Expression of endogenous CCNB1 protein and the active form of the CDK1 protein was determined by western blot using total cell protein extracts obtained from STOX1A siRNA and control treated SH-SY5Y cells. CCNB1 proteins were detected by a specific antibody recognizing total CCNB1 protein. CDK1 proteins were detected using an antibody detecting total CDK1 protein levels and a specific antibody recognizing the active form of CDK1 phosphorylated at threonine 161. An antibody specific for actin was used as a loading control. Westernblot image is a representative of at least 3 independent experiments.</p

The effect of STOX1A on cell proliferation in the neuroblastoma cell-line SH-SY5Y.

<p>(A) The proliferation curve shows significantly decreased cell proliferation for STOX1A siRNA transfected SH-SY5Y cells compared to scrambled controls after 1, 2, and 3 days in culture. (A. left bar) In parallel we did not found significant differences in cell death between STOX1A siRNA and scrambled siRNA transfected cells at each of the indicated time points. (B) The proliferation curve shows significantly increased cell proliferation for STOX1A compared to MOCK stably transfected SH-SY5Y cells after 1, 2, and 3 days in culture. (B. left bar) In parallel we did not found significant differences in cell death between STOX1A and MOCK transfected cells at each of the indicated time points. Bars are mean ± SEM. <i>P</i>-values for each timepoint were calculated using two-tailed unpaired <i>t</i>-test. (C) Overexpression and knockdown of STOX1A was determined with quantitative RT-PCR showing a mean 21 fold (mean ΔΔCt 4.4) increase in mRNA expression for STOX1A in stably transfected SH-SH5Y cells compared to the controls (Left bar) and a mean 71 fold (mean ΔΔCt 6.15) decreased mRNA expression for STOX1A siRNA transfected SH-SY5Y cells compared to the scrambled controls (right bar). Bars are mean ± SEM. *** indicate <i>P</i><0.001 (one sample t-test with theoretical mean 0). N = 4, each sample was measured in triplicate. (D, Bottom left graph) Quantification of STOX1A-Halotag protein was performed using densitometry. The ratio number of obtained band intensities for STOX1A (at the expected band size of 150 Kd) divided by actin was compared to the ratio number of obtained band intensities for MOCK divided by actin for 3 independent experiments. A significant increase for the STOX1A ratio number was found compared to the MOCK ratio number. (D, Bottom right graph) Quantification of endogenous STOX1A protein was performed using densitometry. The ratio number of obtained band intensities for STOX1A siRNA (at the expected band size of 110 Kd) divided by actin was compared to the ratio number of obtained band intensities for scrambled siRNA divided by actin for 3 independent experiments. A significant increase for the STOX1A siRNA ratio number was found compared to the scrambled siRNA ratio number. <i>P</i>-values were calculated using two-tailed unpaired <i>t</i>-test, error bars represent ± SEM, * indicate <i>P</i><0.05, *** indicate <i>P</i><0.001. Westernblot images are a representative of at least 3 independent experiments.</p

Direct upregulation of CCNB1 effectuates enhanced mitotic entry.

<p>(A) Stably transfected STOX1A and MOCK SH-SY5Y cells were synchronized in the S-phase and total cell extracts were prepared from 0, 4 and 8 h after release from the 2× thymidine block. Western blotting was performed using antibodies to Phospho-Histone H3 (Ser 10) and CCNB1. An antibody specific for actin was used as a loading control. Westernblot image is a representative of at least 3 independent experiments. (B) Westernblot (see 5A) quantification of Phospho-H3 (ser 10) was performed using densitometry. The ratio number of obtained band intensities for STOX1A divided by actin was compared to the ratio number of obtained band intensities for MOCK divided by actin for 3 independent experiments. A significant increase for the STOX1A ratio number was found at all time points compared to the MOCK ratio number. (C) Westernblot (see 5A) quantification of CCNB1 was performed using densitometry. The ratio number of obtained band intensities for STOX1A divided by actin was compared to the ratio number of obtained band intensities for MOCK divided by actin for 3 independent experiments. A significant increase for the STOX1A ratio number was found at all time points compared to the MOCK ratio number. <i>P</i>-values were calculated using two-tailed unpaired <i>t</i>-test, error bars represent ± SEM, * indicate <i>P</i><0.05, ** indicate <i>P</i><0.01, *** indicate <i>P</i><0.001.</p