Background The consensus on how to choose a reference gene for serum or plasma miRNA expression qPCR studies has not been reached and none of the potential candidates have yet been convincingly validated. We proposed a new in silico approach of finding a suitable reference for human, circulating miRNAs and identified a new set of endogenous reference miRNA based on miRNA profiling experiments from Gene Expression Omnibus. We used 3 known normalization algorithms (NormFinder, BestKeeper, GeNorm) to calculate a new normalization score. We searched for a universal set of endogenous miRNAs and validated our findings on 2 new datasets using our approach. Results We discovered and validated a set of 13 miRNAs (miR-222, miR-92a, miR-27a, miR-17, miR-24, miR-320a, miR-25, miR-126, miR-19b, miR-199a-3p, miR-30b, miR-30c, miR-374a) that can be used to create a reliable reference combination of 3 miRNAs. We showed that on average the mean of 3 miRNAs (p = 0.0002) and 2 miRNAs (p = 0.0031) were a better reference than single miRNA. The arithmetic means of 3 miRNAs: miR-24, miR-222 and miR-27a was shown to be the most stable combination of 3 miRNAs in validation sets. Conclusions No single miRNA was suitable as a universal reference in serum miRNA qPCR profiling, but it was possible to designate a set of miRNAs, which consistently contributed to most stable combinations.
Objective: We aimed to compare glycemic control and variability parameters obtained from paired records of real-time continuous glucose monitoring (RT-CGM) and flash glucose monitoring (FGM). Methods: Ten Polish boys and 11 girls aged 15.3 ± 2.1 years with type 1 diabetes for 7.7 ± 4.5 years and glycated hemoglobin 7.35 ± 0.7% (57 ± 5 mmol/mol) were recruited between August 2017 and June 2018 and equipped with devices for RT-CGM (iPro2 system with Enlite electrodes) and FGM (FreeStyle Libre) for 1 week. Afterwards, Glyculator 2.0 software was used to calculate and compare key metrics of glycemic control listed in the International Consensus on Use of Continuous Glucose Monitoring, with distinction into all record/night-time/day-time blocks when appropriate. Results: Agreement between the two systems' measurements across patients ranged from poor (R 2 = .39) to nearly perfect (R 2 = .97). Significant differences between RT-CGM and FGM were observed in five important metrics: coefficient of variation (median difference: −4.12% [25%-75%: −7.50% to −2.96%], P = .0001), low blood glucose index (−0.88 [−1.88 to −0.18], P = .0004), % of time below 70 mg/dL (3.9 mmol/L) (−4.77 [−8.39 to −1.19], P = .0015) and 54 mg/dL (3 mmol/L) (−1.33 [−4.07 to 0.00], P = .0033) and primary time in range (TIR) 70-180 mg/dL (8.58 [1.31 to 12.66], P = .0006). Conclusions: RT-CGM and FGM differ in their estimates of clinically important indices of glycemic control. Therefore, such metrics cannot be directly compared between people using different systems. Our result necessitates system-specific guidelines and targets if TIR and glycemic variability are to be used as an endpoint in clinical trials.
Background Multi-gene expression assays are an attractive tool in revealing complex regulatory mechanisms in living organisms. Normalization is an indispensable step of data analysis in all those studies, since it removes unwanted, non-biological variability from data. In targeted qPCR assays it is typically performed with respect to prespecified reference genes, but the lack of robust strategy of their selection is reported in literature, especially in studies concerning circulating microRNAs (miRNA). Unfortunately, this problem impedes translation of scientific discoveries on miRNA biomarkers into widely available laboratory assays. Previous studies concluded that averaged expressions of multi-miRNA combinations are more stable references than single genes. However, due to the number of such combinations the computational load is considerable and may be hindering for objective reference selection in large datasets. Existing implementations of normalization algorithms (geNorm, NormFinder and BestKeeper) have poor performance and may require days to compute stability values for all potential reference as the evaluation is performed sequentially. Results We designed NormiRazor - an integrative tool which implements those methods in a parallel manner on a graphics processing unit (GPU) using CUDA platform. We tested our approach on publicly available miRNA expression datasets. As a result, the times of executions on 8 datasets containing from 50 to 400 miRNAs (subsets of GSE68314) decreased 18.7 ±0.6 (mean ±SD), 104.7 ±4.2 and 76.5 ±2.2 times for geNorm, BestKeeper and NormFinder with respect to previous Python implementation. To allow for easy access to normalization pipeline for biomedical researchers we implemented NormiRazor as an online platform where a user could normalize their datasets based on the automatically selected references. It is available at norm.btm.umed.pl, together with instruction manual and exemplary datasets. Conclusions NormiRazor allows for an easy, informed choice of reference genes for qPCR transcriptomic studies. As such it can improve comparability and repeatability of experiments and in longer perspective help translate newly discovered biomarkers into readily available assays.
Motivation:Multi-gene expression assays are an attractive tool in revealing complex regulatory mechanisms in living organisms. Normalization is an indispensable step of data analysis in all those studies, since it removes unwanted, non-biological variability from data. In targeted qPCR assays the normalization is typically performed with respect to prespecified reference genes, but the lack of robust strategy of their selection is reported in literature, especially in studies concerning circulating microRNAs (miRNA).Results: Previous studies concluded that averaged expressions of multi-miRNA combinations are more stable references than single genes. However, due to the number of such combinations the computational load is considerable and may be hindering for objective reference selection in large datasets. Existing implementations of normalization algorithms (geNorm, NormFinder and BestKeeper) have poor performance as every combination is evaluated sequentially. Thus, we designed an integrative tool which implemented those methods in a parallel manner on a graphics processing unit (GPU) using CUDA platform. We tested our approach on publicly available microRNA expression datasets. As a result the times of executions decreased 19-, 105-and 77-fold respectively for geNorm, BestKeeper and NormFinder.
Nie występujePraca wpłynęła do Redakcji: 21.04.2016 r. Po recenzji: 11.07.2016 r. Zaakceptowano do druku: 17.08.2016 r. StreszczenieWprowadzenie. Aktywność fizyczna i zdrowe żywienie to podstawa prawidłowego rozwoju i dobrego stanu zdrowia. Szczególnie ważne są w okresie dojrzewania, ponieważ zdrowy styl życia dzieci i młodzieży wpływa stymulująco na ich rozwój fizyczny i umysłowy, a także na dobre samopoczucie i sprawność w póź-niejszym wieku. Nieprawidłowości w sposobie odżywiania się i aktywności fizycznej przyczyniają się do powstania chorób: układu sercowo-naczyniowego, otyłości, cukrzycy i osteoporozy. W ostatnich latach zwięk-szyła się ich liczba u dzieci i młodzieży. Wskazuje to na potrzebę wielokierunkowych działań zmierzających do promocji zdrowia i edukacji na temat prawidłowego żywienia i aktywności fizycznej.Cel pracy. Poznanie zachowań zdrowotnych z zakresu sposobu żywienia i aktywności fizycznej wśród osób poniżej 18. r.ż.Materiał i metody. Na podstawie ankiety przeanalizowano sposób żywienia i aktywność fizyczną 114 uczniów w wieku do 18 lat. Udział w badaniu był dobrowolny i anonimowy.Wyniki. Wśród ankietowanych 42% wykonywało ćwiczenia fizycznie tylko 1-2 razy w tygodniu bądź wcale, a 28% przeznaczało jednorazowo na sport mniej niż 30 min. Prawie połowa dzieci (49%) jadła sło-dycze przynajmniej 5 razy w tygodniu, a 30% z nich codziennie. Trzydzieści osiem procent respondentów spożywało słone przekąski co najmniej 4 razy w tygodniu, 34% jadło żywność typu fast food co najmniej raz w tygodniu, 30% piło napoje wysokosłodzone minimum 5 razy w tygodniu. Aż 38% uczniów przyznało, że piło co najmniej jedną filiżankę kawy dziennie, 10% -napoje energetyczne, a 8% paliło papierosy. Jednocześnie 32% dzieci jadło mniej niż 2 porcje warzyw i owoców dziennie, 82% piło mniej niż 5 szklanek wody dziennie, a 47% dzieci nie piło mleka.Wnioski. Respondenci do 18. r.ż. wykazują niedostateczną aktywność fizyczną oraz nieprawidłowości w sposobie odżywiania się. Dotyczy to głównie zbyt małej ilości spożywanych warzyw i owoców oraz nadmiernego spożycia żywności typu fast food, słodyczy, słonych przekąsek oraz zastępowania wody i mleka napojami wysokosłodzonymi.Słowa kluczowe: dzieci, aktywność fizyczna, odżywianie, promowanie zdrowia
BackgroundFOXP1 is a pleiotropic protein that plays important roles in immune responses (B-cell development regulation and differentiation of monocyte), organ development (cardiac valves, lung, and esophagus), and neuronal development. Besides being the primary regulator of normal human tissue development, FOXP1 also plays a role in tumorigenesis. However, the potential value of FOXP1 expression in tumor prognosis remains controversial. FOXP1 expression was assessed in tumor cells (TCs) and stromal cells (SCs) of cutaneous melanomas with the aim of analyzing the associations between FOXP1 expression and clinicopathological characteristics. We believe this article to be the first report analyzing the correlations between FOXP1 expression and clinicopathological, as well as histological, characteristics in melanoma.Materials and methodsIn total, 96 formalin-fixed, paraffin-embedded primary cutaneous melanoma tissue specimens were subjected to immunohistochemical analysis for FOXP1, and the results were correlated with classical clinicopathological features and patient survival.ResultsFOXP1 overexpression in TCs was strongly associated with the presence of metastases in sentinel lymph nodes (p=0.0003, OR=11.66) and positive status of regional lymph nodes (p=0.0006, OR=22.15). In 96% (52 of 54) of patients presenting with low FOXP1 expression, no clinical or histopathological features of lymphatic dissemination were observed. However, thinner and nonulcerated tumors were reported to have increased numbers of FOXP1-positive SCs. In addition, a strong association was observed between FOXP1 upregulation in SCs and the absence of regional lymph node metastases. There was a significant correlation between FOXP1 upregulation in TCs and shorter cancer-specific overall survival (log-rank test, p=0.0040) and disease-free survival (log-rank test, p=0.0021). FOXP1 expression was confirmed in multivariate analysis as a factor that significantly unfavorably impacts prognosis in melanoma patients (HR=3.14, p=0.0299, adjusted for age, Breslow thickness, and sex).ConclusionThe findings from this study indicate that FOXP1 has a major role in melanoma progression, which makes it a candidate for molecular target-based cancer therapy.
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