Multiplex preamplification of specific cDNA targets prior to gene expression analysis by TaqMan Arrays

<p>Abstract</p> <p>Background</p> <p>An accurate gene expression quantification using TaqMan Arrays (TA) could be limited by the low RNA quantity obtained from some clinical samples. The novel cDNA preamplification system, the TaqMan PreAmp Master Mix kit (TPAMMK), enables a multiplex preamplification of cDNA targets and therefore, could provide a sufficient amount of specific amplicons for their posterior analysis on TA.</p> <p>Findings</p> <p>A multiplex preamplification of 47 genes was performed in 22 samples prior to their analysis by TA, and relative gene expression levels of non-preamplified (NPA) and preamplified (PA) samples were compared. Overall, the mean cycle threshold (CT) decrement in the PA genes was 3.85 (ranging from 2.07 to 5.01). A high correlation (r) between the gene expression measurements of NPA and PA samples was found (mean r = 0.970, ranging from 0.937 to 0.994; p < 0.001 in all selected cases). High correlation coefficients between NPA and PA samples were also obtained in the analysis of genes from degraded RNA samples and/or low abundance expressed genes.</p> <p>Conclusion</p> <p>We demonstrate that cDNA preamplification using the TPAMMK before TA analysis is a reliable approach to simultaneously measure gene expression of multiple targets in a single sample. Moreover, this procedure was validated in genes from degraded RNA samples and low abundance expressed genes. This combined methodology could have wide applications in clinical research, where scarce amounts of degraded RNA are usually obtained and several genes need to be quantified in each sample.</p

Impact on prostate cancer clinical presentation after non-screening policies at a tertiary-care medical center- a retrospective study

Background: In May 2012 the US Preventive Task Force issued a 'D' recommendation against routine PSA-based early detection of prostate cancer. This recommendation was implemented progressively in our health system. The aim of this study is to defne its impact on prostate cancer staging at a tertiary care institution. Methods: A retrospective analysis was performed from 2012 until 2015 at a single center. We analyzed the total number of biopsies performed per year and the positive biopsy rate. For those patients with positive biopsies we recorded diagnostic PSA, clinical stage, ISUP grade group, nodal involvement and metastatic status at diagnosis. Results: A total of 1686 biopsies were analyzed. The positive biopsy rate increased from 25% in 2012 to 40% in 2015 (p<0.05). No change in median PSA was noticed (p=0.627). The biopsies detected higher ISUP grades (p=0.000). In addition, newly diagnosed prostate cancer presented a higher clinical stage (p=0.005), higher metastatic rates (p=0.03) and a tendency to higher lymph node involvement although not statistically signifcant (p=0.09). Conclusion: After the 2012 recommendation, patients presented a higher probability of a prostate cancer diagnosis, with a more adverse ISUP group, clinical stage and metastatic disease. These results should be taken into consideration to implement a risk adapted strategy for prostate cancer screening. Keywords: Prostate cancer, Prostate specifc antigen, Prostate cancer screenin

Urinary cell microRNA-based prognostic classifier for nonmuscle invasive bladder cancer

Current prognostic tools for non-muscle invasive bladder cancer (NMIBC) do not have enough discriminative capacity to predict the risk of tumour progression. This study aimed to identify urinary cell microRNAs that may be useful as non-invasive predictive biomarkers of tumour progression in NMIBC patients. To this end, 210 urine samples from NMIBC patients were included in the study. RNA was extracted from urinary cells and expression of 8 microRNAs, previously described by our group, was analysed by quantitative PCR. A tumour progression predicting model was developed by Cox regression analysis and validated by bootstrapping. Regression analysis identified miR-140-5p and miR-92a-3p as independent predictors of tumour progression. The risk score derived from the model containing these two microRNAs was able to discriminate between two groups with a highly significant different probability of tumour progression (HR, 5.204; p<0.001) which was maintained when patients were stratified according to tumour risk. The algorithm was also able to identify two groups with different cancer-specific survival (HR, 3.879; p=0.021). Although the data needs to be externally validated, miRNA analysis in urine appears to be a valuable prognostic tool in NMIBC patients

Análisis multidisciplinar de la aplicación de técnicas de evaluación continua formativa

El presente trabajo presenta las actividades realizas para la mejora del proceso de aprendizaje en sesiones de prácticas mediante distintas estrategias de evaluación continua formativa por parte de un grupo de innovación educativa de la Universidad Politécnica de Valencia. Se ha estudiado el uso de cuestionarios temporizados realizados antes y al finalizar la práctica mediante el uso de una plataforma de e-learning así como el uso de rúbricas para la valoración del trabajo del alumno y las memorias de prácticas. La eficacia de las distintas estrategias ha sido establecida a partir de datos cualitativos y cuantitativos recogidos mediante observación durante su aplicación, calificaciones obtenidas y realizando un sondeo de opinión. La aplicación de estas metodologías ha sido llevada a cabo en 10 asignaturas de diferentes áreas de conocimiento (Teoría de la Señal y Comunicaciones, Química, Ingeniería Química y Economía) impartidas en diferentes carreras técnicas, por lo que se obtiene una visión multidisciplinar del efecto de las innovaciones

Prognostic implication of TERT promoter mutation and circulating tumor cells in muscle-invasive bladder cancer

Purpose: Current clinical prognostic factors are not accurate enough to identify and monitor those muscle-invasive bladder cancer (MIBC) patients at high risk of progression after radical cystectomy (RC). Here, we determined genetic alterations in the tumor and circulating tumor cell (CTC) enumeration to find biomarkers useful for the management of MIBC after RC. Methods: Thirty-nine MIBC patients undergoing RC were included. Tumoral tissue DNA was analyzed by next generation sequencing. CTCs were isolated from blood collected before RC and one, four and 12 months later. Results: Sixteen (41%) patients progressed in a median time of 8.5 months and 11 (69%) of these patients harbored the TERT c.-124C > T mutation. All progressive patients harboring the TERT c.-124C > T mutation presented a significant increase in CTC number 12 months after RC compared to those without the mutation. Additionally, CTC number at 12 months was identified as an independent prognostic biomarker for tumor progression and cancer specific survival (CSS). Ten (63%) progressive patients showed an increment of CTC number with a median anticipation period of four months compared with imaging techniques. Conclusions: The TERT c.-124C > T mutation could be considered a biomarker of aggressivity. CTC enumeration is a useful tool for identifying MIBC patients at high risk of progression and CSS after RC and for detecting tumor progression earlier than imaging techniques

Differential gene expression profile between progressive and de novo muscle invasive bladder cancer and its prognostic implication

This study aimed to ascertain gene expression profle diferences between progressive muscle-invasive bladder cancer (MIBC) and de novo MIBC, and to identify prognostic biomarkers to improve patients' treatment. Retrospective multicenter study in which 212 MIBC patients who underwent radical cystectomy between 2000 and 2019 were included. Gene expression profles were determined in 26 samples using Illumina microarrays. The expression levels of 94 genes were studied by quantitative PCR in an independent set of 186 MIBC patients. In a median follow-up of 16 months, 46.7% patients developed tumor progression after cystectomy. In our series, progressive MIBC patients show a worse tumor progression (p= 0.024) and cancer-specifc survival (CSS) (p= 0.049) than the de novo group. A total of 480 genes were found to be diferently expressed between both groups. Diferential expression of 24 out of the 94 selected genes was found in an independent cohort. RBPMC2 and DSC3 were found as independent prognostic biomarkers of tumor progression and CALD1 and LCOR were identifed as prognostic biomarkers of CSS between both groups. In conclusion, progressive and de novo MIBC patients show diferent clinical outcome and gene expression profles. Gene expression patterns may contribute to predict high-risk of progression to distant metastasis or CSS

Ability of a urine gene expression classifier to reduce the number of follow up cystoscopies in bladder cancer patients

This study aimed to improve our previous urine gene expression classifiers focusing on the detection of non-high-risk non-muscle-invasive bladder cancer (NMIBC), and develop a new classifier able to decrease the frequency of cystoscopies during bladder cancer (BC) patients' surveillance. A total of 597 urines from BC patients, controls and patients in follow-up for BC (PFBC) were included. The study has 3 phases. In the urinary biomarker discovery phase, 84 urines from BC and control patients were retrospectively included and analyzed by Ribonucleic Acid (RNA) sequencing. In the classifier development phase, a total of 132 selected genes from previous phase were evaluated by nCounter in 214 prospectively collected urines from PFBC (98 with tumor). A diagnostic classifier was generated by logistic regression. Finally, in the classifier validation phase, a multicentric and international cohort of 248 urines (134 BC and 114 nonrecurrent PFBC) was used to validate classifier performance. A total of 521 genes were found differentially expressed between non-high-risk NMIBC samples and all other groups (P < 0.05). An 8-gene diagnostic classifier with an area under curve (AUC) of 0.893 was developed. Validation of this classifier in a cohort of PFBC achieved an overall sensitivity (SN) and a negative predictive value (NPV) of 96% and 97%, respectively (AUC = 0.823). Notably, this accuracy was maintained in non-high-risk NMIBC group (SN = 94%; NPV = 98%). In conclusion, this 8-gene expression classifier has high SN and NPV in a real clinical scenario. The use of this classifier can reduce the number of follow-up cystoscopies in PFBC, although assessing its final place in clinical setting is necessary

Students' perception on learning methods in engineering disciplines

This article is (c) Emerald Group Publishing and permission has been granted for this version to appear here (please insert the web address here). Emerald does not grant permission for this article to be further copied/distributed or hosted elsewhere without the express permission from Emerald Group Publishing Limited.[EN] Purpose - This study explores the preferences for learning methods among the students of seven engineering disciplines in a Spanish technical university. The purpose of this paper is to investigate the students' views and from them contribute to the knowledge of the effectiveness of learning methodologies. Design/methodology/approach - An online anonymous questionnaire survey was adopted to collect students' perceptions. Seven learning methods were compared in seven engineering degrees. The authors sampled 1660 students, and 426 completed responses were analysed. In addition to a descriptive analysis of the results, a multiple correspondence analysis (MCA) was performed using R data processing software. Findings - It was found that project-based learning and problem-based learning were perceived as the more effective ones. MCA identified response patterns between the preference and the efficiency of learning methods showing that students can be classified into two groups according to their preferred level of activeness in learning. Research limitations/implications - The study focusses on a single technical university and not all engineering degrees could be sampled. However, five different engineering fields were studied and no significant differences among them were found. Practical implications - The results add up to the known literature showing that students have different learning needs and consequently they perceive some methods as more effective. Instructors can use this information to strengthen their learning activities. Results also suggest that students can be classified into two groups in relation to their level of activeness in learning. This can also help to enhance general student motivation if two paths with different levels of activeness are planned. Originality/value - No previous studies have compared several learning methods in different engineering fields. Thus, this study contributes to fill this gap and contributes to the body of evidence around learning methodologies from the perspective of students.Vidal Rodriguez, B.; Fenollosa Ribera, ML.; Ribal, J.; Sanchis Kilders, P.; García-Rupérez, J.; Bes-Piá, M.; Blasco-Tamarit, E.... (2022). Students' perception on learning methods in engineering disciplines. Journal of Applied Research in Higher Education. 14(3):946-957. https://doi.org/10.1108/JARHE-01-2021-0041S94695714

Economic feasibility of freeze-drying to obtain powdered fruit

[EN] Fruit is a highly valuable food whose consumption should be encouraged. In addition, it would be desirable to design processes and products to channel the surplus and take advantage of the post-harvest losses that limit its fresh marketing. Freeze-drying is a known industrial process that permits the obtaining of high quality products, despite having always been labeled as very expensive. In this study, the economic feasibility of freeze-drying to obtain powdered fruit has been proven, as it yields a product more than twice as cheap as when obtained by spray-drying, recognized for its low cost.The authors thank the Ministerio de Economía y Competitividad for the financial support given through the Project AGL 2012-39103.Camacho Vidal, MM.; Casanova, MÁ.; Fenollosa Ribera, ML.; Ribal Sanchis, FJ.; Martínez-Lahuerta, JJ.; Martínez Navarrete, N. (2018). Economic feasibility of freeze-drying to obtain powdered fruit. En IDS 2018. 21st International Drying Symposium Proceedings. Editorial Universitat Politècnica de València. 827-834. https://doi.org/10.4995/IDS2018.2018.7479OCS82783

Predictors, pathological characteristics and outcomes of bladder recurrences following nephroureterectomy

Objetivos: Analizar las variables predictoras de recidiva vesical (RV) tras nefroureterectomía(NU) por tumor de tracto urinario superior (TTUS), así como sus características patológicas,evolución y repercusión en supervivencia. Material y métodos: Estudio retrospectivo de 117 pacientes sometidos a NU laparoscópica por TTUS entre 2007-2012 en nuestro centro. Los posibles factores predictores de RV se analizaron mediante regresión de Cox y para el estudio de supervivencia se utilizaron las curvas de Kaplan-Meier.Resultados: Fueron 85 hombres (73%) y 32 mujeres (27%) con una edad media de 70 años. Tras un seguimiento medio de 26 meses, 23 presentaron RV (19,6%). En el análisis multivariante, el género (p = 0,003; HR mujer 3,8) y la localización del TTUS en uréter distal (p = 0,002; HR 4,8) fueron predictores independientes de RV. La mediana de tiempo hasta la RV fue de 8 meses. Quince pacientes presentaron una RV no músculo-invasiva (65,2%) y 8 músculo-invasiva (34,8%). Todas las RV excepto 2, aparecieron durante los primeros 2 años. Cinco casos con RV no músculo-invasiva presentaron nueva RV. Seispacientes con RV músculo-invasiva murieron sin poderse definir si fue por tumor vesical o de vías. La aparición de RV no mostró repercusión en la supervivencia de los pacientes con TTUS. Conclusiones: El género (mujer) y la localización del TTUS (uréter distal) son factores predic-tores de RV tras NU. Pacientes con estas características podrían beneficiarse de tratamientoadyuvante intravesical y de un seguimiento más estricto. La aparición de RV no tiene impacto en la supervivencia de los pacientes con TTUS. OBJECTIVES: To analyze the predictors for bladder recurrence (BR) after nephroureterectomy (NU) for upper urinary tract tumors (UUTT), as well as its pathological characteristics, outcomes and impact on survival. MATERIAL AND METHODS: Retrospective study of 117 patients who underwent laparoscopic nephroureterectomy by UUTT between 2007-2012 at our center. The potential predictors for BR were analyzed using Cox regression; Kaplan-Meier curves were employed to study survival. RESULTS: The sample was composed of 85 men (73%) and 32 women (27%), with a mean age of 70 years. After a mean follow-up of 26 months, 23 patients presented BR (19.6%). In the multivariate analysis, sex (p=.003; HR [female], 3.8) and the location of the UUTT in the distal ureter (p=.002; HR, 4.8) were independent predictors for BR. The median time to BR was 8 months. Fifteen patients presented a nonmuscle-invasive BR (65.2%), and 8 presented a muscle-invasive BR (34.8%). All BRs, except for 2, appeared during the first 2 years. Five cases with nonmuscle-invasive BR presented a new BR. Six patients with muscle-invasive BR died before it could be determined whether cause of death was the BR or an UUTT relapse. The onset of BR showed no repercussion on the survival of patients with UUTT. CONCLUSIONS: Sex (female) and the location of the UUTT (distal ureter) are predictors for BR after NU. Patients with these characteristics might benefit from adjuvant intravesical treatment and closer monitoring. The onset for RV has no impact on the survival of patients with UUTT