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BACKGROUND: Healthcare-associated infections and antibiotic resistance worsen globally. Antibiotic stewardship programs (ASP) aim to optimise infection treatment and curb resistance, yet implementation hurdles persist. This study examined ASP challenges in ICUs. METHODS: This study employed a qualitative methodological design to evaluate the implementation process of an antibiotic stewardship program (ASP) in eight intensive care units (ICUs) across Argentina. Thirty-four semi-structured interviews with healthcare workers (HCWs) were conducted. Interviews were analysed guided by Normalisation Process Theory, examining coherence, cognitive participation, collective action, and reflexive monitoring constructs. RESULTS: Key challenges included insufficient human resources, lack of institutional support, and resistance to change, particularly among staff not initially involved in the study. Despite these challenges, the program saw partial success in improving ICU practices, particularly in antibiotic use and communication across departments. The main strategy implemented in this quality improvement collaborative was the use of improvement cycles, which served as the central component for driving change. However, participation in improvement cycles was inconsistent, and sustainability post-intervention remains uncertain due to workload pressures and the need for continuous education. Concerns about workload and communication barriers persisted. Many participants did not perceive training as a separate component, which led to low engagement. Resistance to change became evident during modifications to clinical guidelines. The intervention had a positive impact on various processes, including communication and record keeping. CONCLUSION: This study underscores the persistent challenges in implementing ASPs in healthcare, emphasising the need for enhanced collaboration, workforce capacity building, and evidence-based practices to overcome barriers and optimize antimicrobial use to improve patient outcomes.

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Gene expression through RT-qPCR can be performed by the relative quantification method, which requires the expression normalization through reference genes. Therefore, it is essential to validate, experimentally, the candidate reference genes. Thus, although there are several studies that are performed to identify the most stable reference genes, most them validate genes for very specific conditions, not exploring the whole potential of the research since not all possible combinations of treatments and/or conditions of the study are explored. For this reason, new experiments must be conducted by researchers that have interest in analyzing gene expression of treatments and/or conditions present, but not explored, in these studies. Here, we present the RGeasy tool, which aims to facilitate the selection of reference genes, allowing the user to choose genes for a greater number of combinations of treatments/conditions, compared to the ones present in the original articles, through just a few clicks. RGeasy was validated with RT-qPCR data from gene expression studies performed in two coffee species, Coffea arabica and Coffea canephora, and it can be used for any animal, plant or microorganism species. In addition to displaying a rank of the most stable reference genes for each condition or treatment, the user also has access to the primer pairs for the selected reference genes.

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Viral infection disrupts the normal regulation of the host gene's expression. In order to normalise the expression of dysregulated host genes upon virus infection, analysis of stable reference housekeeping genes using quantitative real-time-PCR (qRT-PCR) is necessary. In the present study, healthy and African swine fever virus (ASFV) infected porcine tissues were assessed for the expression stability of five widely used housekeeping genes (HPRT1, B2M, 18 S rRNA, PGK1 and H3F3A) as reference genes using standard algorithm. Total RNA from each tissue sample (lymph node, spleen, kidney, heart and liver) from healthy and ASFV-infected pigs was extracted and subsequently cDNA was synthesized, and subjected to qRT-PCR. Stability analysis of reference genes expression was performed using the Comparative delta CT, geNorm, BestKeeper and NormFinder algorithm available at RefFinder for the different groups. Direct Cycle threshold (CT) values of samples were used as an input for the web-based tool RefFinder. HPRT1 in spleen, 18 S rRNA in liver and kidney and H3F3A in heart and lymph nodes were found to be stable in the individual healthy tissue group (group A). The majority of the ASFV-infected organs (liver, kidney, heart, lymph node) exhibited H3F3A as stable reference gene with the exception of the ASFV-infected spleen, where HPRT1 was found to be the stable gene (group B). HPRT1 was found to be stable in all combinations of all CT values of both healthy and ASFV-infected porcine tissues (group C). Of five different reference genes investigated for their stability in qPCR analysis, the present study revealed that the 18 S rRNA, H3F3A and HPRT1 genes were optimal reference genes in healthy and ASFV-infected different porcine tissue samples. The study revealed the stable reference genes found in healthy as well as ASF-infected pigs and these reference genes identified through this study will form the baseline data which will be very useful in future investigations on gene expression in ASFV-infected pigs.

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AIMS: This study aimed to assess the use of cross-assembled phage (crAssphage) as an endogenous control employing a multivariate normalization analysis and its application as a severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) data normalizer. METHODS AND RESULTS: A total of 188 twelve-hour composite raw sewage samples were obtained from eight wastewater treatment plants (WWTP) during a 1-year monitoring period. Employing the N1 and N2 target regions, SARS-CoV-2 RNA was detected in 94% (177) and 90% (170) of the samples, respectively, with a global median of 5 log10 genomic copies per liter (GC l-1). CrAssphage was detected in 100% of the samples, ranging from 8.29 to 10.43 log10 GC l-1, with a median of 9.46 ± 0.40 log10 GC l-1, presenting both spatial and temporal variabilities. CONCLUSIONS: Although SARS-CoV-2 data normalization employing crAssphage revealed a correlation with clinical cases occurring during the study period, crAssphage normalization by the flow per capita per day of each WWTP increased this correlation, corroborating the importance of normalizing wastewater surveillance data in disease trend monitoring.

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Banana (Musa spp.) is the most widely consumed fruit globally. Fusarium wilt, caused by Fusarium oxysporum f. sp. cubense (Foc), is a highly threatening disease to banana production. Resistance genes to Foc exist in wild Musa genotypes such as Musa acuminata subsp. burmannicoides var. Calcutta 4. Whilst real-time PCR (RT-qPCR) is appropriate for accurate analysis of gene expression in pathways involved in host defence responses, reference genes with stable expression under specific biotic stress conditions and host tissue types are necessary for normalization of sample variation. In this context, the stability in potential host reference genes ACT1, APT, EF1α, GAPDH, αTUB, RAN, UBIQ1, UBIQ2, ßTUB1, ßTUB3, L2 and ACTA1 was evaluated in total RNA samples from root tissues in Calcutta 4 (resistant) and Musa sp. cultivar Prata-anã (susceptible) extracted during interaction with Foc subtropical race 4 (STR4). Expression stability was calculated using the algorithms geNorm, NormFinder and BestKeeper. ßTUB3 and L2 were identified as the most stable in Calcutta 4, with ACTA1 and GAPDH the most stable in Prata-anã. These reference genes for analysis of gene expression modulation in the Musa-Foc STR4 pathosystem are fundamental for advancing understanding of host defence responses to this important pathogen.

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RNA-seq faces persistent challenges due to the ongoing, expanding array of data processing workflows, none of which have yet achieved standardization to date. It is imperative to determine which method most effectively preserves biological facts. Here, we used Shannon entropy as a tool for depicting the biological status of a system. Thus, we assessed the measurement of Shannon entropy by several RNA-seq workflow approaches, such as DESeq2 and edgeR, but also by combining nine normalization methods with log2 fold change on paired samples of TCGA RNA-seq representing datasets of 515 patients and spanning 12 different cancer types with 5-year overall survival rates ranging from 20% to 98%. Our analysis revealed that TPM, RLE, and TMM normalization, coupled with a threshold of log2 fold change ≥1, for identifying differentially expressed genes, yielded the best results. We propose that Shannon entropy can serve as an objective metric for refining the optimization of RNA-seq workflows and mRNA sequencing technologies.

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BACKGROUND: Normalization is a critical step in the analysis of single-cell RNA-sequencing (scRNA-seq) datasets. Its main goal is to make gene counts comparable within and between cells. To do so, normalization methods must account for technical and biological variability. Numerous normalization methods have been developed addressing different sources of dispersion and making specific assumptions about the count data. MAIN BODY: The selection of a normalization method has a direct impact on downstream analysis, for example differential gene expression and cluster identification. Thus, the objective of this review is to guide the reader in making an informed decision on the most appropriate normalization method to use. To this aim, we first give an overview of the different single cell sequencing platforms and methods commonly used including isolation and library preparation protocols. Next, we discuss the inherent sources of variability of scRNA-seq datasets. We describe the categories of normalization methods and include examples of each. We also delineate imputation and batch-effect correction methods. Furthermore, we describe data-driven metrics commonly used to evaluate the performance of normalization methods. We also discuss common scRNA-seq methods and toolkits used for integrated data analysis. CONCLUSIONS: According to the correction performed, normalization methods can be broadly classified as within and between-sample algorithms. Moreover, with respect to the mathematical model used, normalization methods can further be classified into: global scaling methods, generalized linear models, mixed methods, and machine learning-based methods. Each of these methods depict pros and cons and make different statistical assumptions. However, there is no better performing normalization method. Instead, metrics such as silhouette width, K-nearest neighbor batch-effect test, or Highly Variable Genes are recommended to assess the performance of normalization methods.

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Este artigo parte do exame da relação entre o poder de normalização, o dispositivo diagnóstico e a patologização da transexualidade para expor a função da norma em diferentes prismas na discussão sobre o normal e o patológico no contexto da experiência trans. São pautadas as dificuldades decorrentes dos pressupostos normativos que servem como diretrizes para políticas públicas e privadas de acesso ao processo transexualizador. Mediante uma discussão epistêmica, a experiência trans e sua patologização são analisadas em relação à função diagnóstica na psiquiatria e na psicanálise, observando o rigor de suas especificidades. Por fim, a orientação lacaniana é apresentada como alternativa à psiquiatrização e patologização da experiência trans, tendo em vista o seu distinto uso diagnóstico, o privilégio da singularidade em sua prática e o caráter subversivo de sua epistemologia.

Cet article commence par examiner la relation entre le pouvoir de normalisation, le dispositif de diagnostic et la pathologisation de la transsexualité pour exposer la fonction de la norme sous différents angles dans la discussion sur le normal et le pathologique dans le contexte de l'expérience trans. Les difficultés découlant des hypothèses normatives qui servent de lignes directrices aux politiques publiques et privées sur l'accès au processus de transsexualisation sont mises en question. À travers une discussion épistémique, l'expérience trans et sa pathologisation sont analysées en relation avec la fonction diagnostique en psychiatrie et en psychanalyse, en observant la rigueur de leurs spécificités. Enfin, l'orientation lacanienne est présentée comme une alternative à la psychiatrisation et à la pathologisation de l'expérience trans, compte tenu de son usage diagnostique distinct, du privilège de la singularité dans sa pratique et du caractère subversif de son épistémologie

This article begins by examining the relationship between the power of normalization, the diagnostic device and the pathologization of transsexuality to expose the function of the norm from different perspectives in the discussion about the normal and the pathological in the context of the trans experience. Difficulties arising from normative assumptions that serve as guidelines for public and private policies on access to the transsexualization process are examined. Through an epistemic discussion, the trans experience and its pathologization are analyzed in relation to the diagnostic function in psychiatry and psychoanalysis, observing the rigor of their specificities. Finally, the Lacanian orientation is presented as an alternative to the psychiatrization and pathologization of the trans experience, given its distinct diagnostic use, the privilege of singularity in its practice and the subversive character of its epistemology

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Early diagnosis of potentially malignant disorders, such as oral epithelial dysplasia, is the most reliable way to prevent oral cancer. Computational algorithms have been used as an auxiliary tool to aid specialists in this process. Usually, experiments are performed on private data, making it difficult to reproduce the results. There are several public datasets of histological images, but studies focused on oral dysplasia images use inaccessible datasets. This prevents the improvement of algorithms aimed at this lesion. This study introduces an annotated public dataset of oral epithelial dysplasia tissue images. The dataset includes 456 images acquired from 30 mouse tongues. The images were categorized among the lesion grades, with nuclear structures manually marked by a trained specialist and validated by a pathologist. Also, experiments were carried out in order to illustrate the potential of the proposed dataset in classification and segmentation processes commonly explored in the literature. Convolutional neural network (CNN) models for semantic and instance segmentation were employed on the images, which were pre-processed with stain normalization methods. Then, the segmented and non-segmented images were classified with CNN architectures and machine learning algorithms. The data obtained through these processes is available in the dataset. The segmentation stage showed the F1-score value of 0.83, obtained with the U-Net model using the ResNet-50 as a backbone. At the classification stage, the most expressive result was achieved with the Random Forest method, with an accuracy value of 94.22%. The results show that the segmentation contributed to the classification results, but studies are needed for the improvement of these stages of automated diagnosis. The original, gold standard, normalized, and segmented images are publicly available and may be used for the improvement of clinical applications of CAD methods on oral epithelial dysplasia tissue images.

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Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recent development for epigenome mapping, but its unique methodology can hamper proper quantitative analyses. As traditional normalization approaches have been shown to be inaccurate, we sought to determine endogenous normalization factors based on the human genome regions of constant nonspecific signal. This constancy was determined by applying Shannon's information entropy, and the set of normalizer regions, which we named the 'Greenlist', was extensively validated using publicly available datasets. We demonstrate here that the greenlist normalization outperforms the current top standards, and remains consistent across different experimental setups, cell lines and antibodies; the approach can even be applied to different species or to CUT&Tag. Requiring no additional experimental steps and no added cost, this approach can be universally applied to CUT&RUN experiments to greatly minimize the interference of technical variation over the biological epigenome changes of interest.

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Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recent development for epigenome mapping, but its unique methodology can hamper proper quantitative analyses. As traditional normalization approaches have been shown to be inaccurate, we sought to determine endogenous normalization factors based on the human genome regions of constant nonspecific signal. This constancy was determined by applying Shannon’s information entropy, and the set of normalizer regions, which we named the ‘Greenlist’, was extensively validated using publicly available datasets. We demonstrate here that the greenlist normalization outperforms the current top standards, and remains consistent across different experimental setups, cell lines and antibodies; the approach can even be applied to different species or to CUT&Tag. Requiring no additional experimental steps and no added cost, this approach can be universally applied to CUT&RUN experiments to greatly minimize the interference of technical variation over the biological epigenome changes of interest.

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Se abordan las formas de violencia que afectan los contextos urbanos, conocidas como "limpieza social", en el departamento de Caldas entre 1980 y 2006. Estas se analizarán junto con el crecimiento de los grupos armados ilegales, con especial énfasis en el paramilitarismo. El objetivo es evidenciar las estrategias discursivas que se aplican por parte de sectores poderosos para estigmatizar ciertas prácticas y actores sociales, principalmente a través de los medios de comunicación. Se apeló al Análisis Crítico del Discurso, aplicado a los enunciados referentes al fenómeno en cuestión, y se siguieron las tesis de Foucault en lo referente a los dispositivos de normalización y la discriminación entre lo normal y lo anormal en la dinámica de las relaciones de poder También se asume la perspectiva moral que se halla en la base de la "limpieza social" y se postulan las razones de su persistencia. Los resultados muestran un proceso que va de la dispersión a la sistematicidad en relación con el exterminio social y sus manifestaciones discursivas. Las conclusiones dan cuenta de la persistencia del fenómeno en contextos contemporáneos.

The forms of violence that affect urban contexts, generally known as "social cleansing", are addressed in the department of Caldas between 1980 and 2006. These will be analyzed along with the growth of the illegal armed groups, with special emphasis on paramilitarism. The objective is to highlight the discursive strategies that are applied by powerful groups to stigmatize certain practices and social actors, mainly through the media. Critical discourse analysis was used, applied to the statements referring the mentioned phenomenon, and Foucault's thesis regarding normalization devices and discrimination between normal and abnormal in the dynamics of power relations were followed. The moral perspective that is at the base of "social cleansing" is also assumed and the reasons for its persistence are postulated. The results show a process that goes from dispersion to systematicity in relation to social extermination and its discursive manifestations. The conclusions show the persistence of the phenomenon in contemporary contexts.

São abordadas as formas de violência que afetam os contextos urbanos, conhecidas como "limpeza social", no departamento de Caldas entre 1980 e 2006. Elas serão analisadas juntamente com o crescimento de grupos armados ilegais, com ênfase especial no paramilitarismo. O objetivo é destacar as estratégias discursivas aplicadas por setores poderosos para estigmatizar determinadas práticas e atores sociais, principalmente por meio da mídia. A Análise Crítica do Discurso foi aplicada às declarações referentes ao fenômeno em questão, e a tese de Foucault foi seguida em relação aos dispositivos de normalização e à discriminação entre o normal e o anormal na dinâmica das relações de poder. Ela também assume a perspectiva moral que está na base da "limpeza social" e postula as razões de sua persistência. Os resultados mostram um proceso que vai da dispersão à sistematicidade em relação ao extermínio social e suas manifestações discursivas. As conclusões mostram a persistência do fenômeno em contextos contemporâneos.

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Toxoplasmosis causes serious harm to the fetus, as tachyzoite dissemination, during pregnancy in women developing the primo-infection. The microRNAs (miRNAs) are small non-coding RNAs, which have regulatory roles in cells by silencing messenger RNA. Circulating miRNA are promising biomarkers for diagnosis and prognosis of numerous diseases. The miRNAs levels are estimated by quantitative real-time PCR (qPCR), however, the relative quantification of each miRNA expression requires proper normalization methods using endogenous miRNAs as control. This study analyzed the expression of three endogenous miRNAs (miR-484, miR -423-3p and miR-26b-5p) for use as normalizers in future studies of target miRNAs for gestational toxoplasmosis (GT). A total of 32 plasma samples were used in all assays divided in 21 from women with GT and 11 from healthy women. The stability of each endogenous miRNA was evaluated by the algorithm methods RefFinder that included GeNorm, Normfinder, BestKeeper and comparative delta-CT programs. The miR-484 was the most stably gene, and equivalently expressed in GT and NC groups. These results contribute to future studies of target miRNAs in clinical samples of women with gestational toxoplasmosis.

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The properties presented by Candida viswanathii's lipases turn this specie into a promising producer of potentially applicable lipases in several industrial sectors, such as: food, textiles, in the oleochemical and paper industries, and also in different pharmaceutical applications. However, studies for elucidating growth and developmental processes at the molecular level in this species are still incipient. Performing such kinds of studies often rely on the use of the RT-qPCR, which is a highly sensitivity technique, but whose parameters must be carefully planned for achieving reliable data. Among the crucial parameters required for achieving reliable results through this technique, the use of appropriated and validated reference genes is one the most important, constituting a bottleneck, mainly in species where molecular studies are scarce. Thus, the aim of this study was to determine the best reference genes for RT-qPCR gene expression studies in C. viswanathii grown in culture media containing four different carbon sources (Olive oil, Triolein, Tributyrin, and Glucose). Eleven candidate reference genes (ACT, GPH1, AGL9, RPB2, SAP1, PGK1, TAF10, UBC13, TFC1, UBP6, and FBA1) were analyzed for their expression patterns and stability. Analysis of gene expression stability was performed using the RefFinder tool, which integrates the geNorm, NormFinder, BestKeeper and Delta-Ct algorithms, and validation of the results was performed through analyzing the expression of a lipase gene, CvLIP4. Analyzing the four treatments together, CvACT and CvRPB2 constituted the best reference gene pair. When treatments are analyzed individually, CvRPB2/CvACT, CvFBA1/CvAGL9, CvPGK1/CvAGL9 and CvACT/CvRPB2 were the best reference gene pairs for the culture media containing olive oil, triolein, tributyrin, and glucose as carbon sources, respectively. These results are essential and form the basis for the development of relative gene expression studies in C. viswanathii, since adequate reference genes are crucial for the reliability of RT-qPCR data.

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Antecedentes: la presencia de hipertrofia ventricular izquierda (HVI) es un marcador pronóstico y de severidad en condiciones de sobrecarga de presión. Se determina por masa ventricular (MV) aumentada en el ecocardiograma, debiéndose ajustar la MV por tamaño corporal en pediatría (normalización). Existen múltiples métodos de normalización, pero se desconoce si estos métodos son comparables. Objetivo: comparar distintos métodos de normalización de MV en sujetos con y sin sobrecarga de presión y evaluar el impacto del estado nutricional en el diagnóstico de HVI. Métodos: estudio de corte transversal en niños entre 5-18 años, divididos en 3 grupos: A) sin sobrecarga de presión (SSP), B) HTA (confirmado o sospecha), o C) Con cardiopatía obstructiva izquierda (CCOI, gradiente ≥25 mmHg). Se analizó antropometría, presión arterial y parámetros ecocardiográficos convencionales. Se determinó HVI por 4 mé-todos de normalización de MV: 1) Superficie corporal (SC) según sexo, 2) Talla2,7>51g/m2,7 3) Z-Score de Talla ≥ Z+2 y 4) Masa corporal magra ≥ Z+2. Se evaluó la concordancia de los métodos por grupo y según el diagnóstico nutricional. Resultados: se analizaron 1162 sujetos, 673(57,9%) hombres, edad 10,2 ± 3,2 años, 852(73,3%) SSP, 192(16,5%) con HTA y 118(10,2%) con CCOI. Un 38,6% presentaba malnutrición por exceso. Se observó diferencia entre los métodos para HVI en pacientes SSP y con CCOI(p=0,01), y en obesos con CCOI. La concordancia entre los métodos fue variable (rango de Kappa 0,38­0,71). Conclusiones: existe variabilidad y discrepancia entre los distintos métodos de normalización utilizados para definir HVI, influenciados por el estado nutricional.

Background: The presence of left ventricular hypertrophy (LVH) is both prognostic and severity marker in pressure overload conditions. It is determined by increased ventricular mass (MV) in the echocardiogram. MV must be adjusted for body size in pediatrics (normali-zation). There are multiple normalization methods, but it is unknown whether these methods are comparable. Objective: To compare different methods of MV normalization in subjects with and without pressure overload and to evaluate the impact of nutritional status on LVH diagnosis. Methods: Cross-sectional study in children aged 5-18 years, divided into 3 groups: A) without pressure overload (SSP), B) Systemic hypertension (confirmed or suspected), or C) Left obstructive heart disease (CCOI, gradient ≥ 25mmHg). Anthropometrics, blood pressure, and conventional echocardiographic parameters were analyzed. LVH was determined by 4 MV normalization methods: 1) Body surface area (BSA) adjusted by gender, 2) height2.7, 3) Z-Score height ≥ Z+2, and 4) lean body mass ≥ Z+2. The concordance of the methods was evaluated by group and according to the nutritional diagnosis. Results: 1162 subjects were analyzed, 673(57.9%) men, age 10.2 ± 3.2 years, 852(73.3%) SSP, 192(16.5%) with hypertension and 118(10.2%) with CCOI. 38.6% presented overnutrition. A difference was demonstrated between the methods for LVH diagnosis in patients with SSP and with CCOI (p=0.01), and in obese patients with CCOI. Agreement between methods was variable (Kappa range 0.38­0.71). Conclusions: There is variability and discrepancy between the different normalization methods used to define LVH, influenced by nutritional status.

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El propósito de este artículo es exponer la metodología didáctica utilizada para la creación de catálogos de autoridad de autor en los cursos de catalogación de la Escuela de Bibliotecología y Ciencias de la Información de la Universidad de Costa Rica. Se muestran los principales materiales didácticos creados para los cursos, así como la importancia de la utilización de la metodología lúdica. Lo anterior se desarrolló para la enseñanza de la creación de puntos de acceso de personas, familias y entidades corporativas aplicando las RDA, además de la creación de los catálogos de autoridades como medio de normalización de estos puntos de acceso. La metodología utilizada se basó en una investigación cuantitativa de alcance descriptivo en la cual se realizó un cuestionario en línea con el objetivo de analizar la percepción de las personas estudiantes del curso BI-2003 Catalogación II, respecto a la utilización de las herramientas didácticas para la elaboración de los catálogos de autoridad de autor y de la metodología lúdica como método de aprendizaje. Uno de los principales hallazgos de esta investigación es que las herramientas didácticas creadas para la elaboración de catálogos de autoridades, así como la estrategia lúdica, favorecen la comprensión y aplicación de ejercicios prácticos, en los cuales tienen la posibilidad de trabajar con la normativa vigente.

The purpose of this article is to present the didactic methodology used for the creation of author's authority catalogs in the cataloging courses of the School of Librarianship and Information Sciences of the University of Costa Rica. The main didactic materials created for the courses are shown, as well as the importance of using the ludic methodology. The foregoing was developed to teach the creation of access points for individuals, families and corporate entities applying the RDA, in addition to the creation of authority catalogs as a means of standardizing these access points. The methodology used was based on quantitative research of descriptive scope in which an online questionnaire was carried out in order to analyze the perception of the students of the BI-2003 Cataloging II course, regarding the use of didactic tools for the preparation of author's authority catalogs and ludic methodology as a learning method. As part of the conclusions, it was established that one of the main findings of this research is that the didactic tools created for the elaboration of authority catalogs, as well as the ludic strategy, favor the understanding and application of practical exercises, in which they have the possibility of working with current regulations.

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Quantitative real-time PCR (RT-qPCR) is used extensively in gene expression studies. For adequate comparisons, the identification and use of reliable reference genes are crucial. Nevertheless, the availability of such genes in strawberry species is limited and has yet to be described for the Chilean strawberry, Fragaria chiloensis. In this study, the expression dynamics of a set of 10 candidate reference genes were analyzed in various F. chiloensis vegetative tissues (root, runners, stem, leaf, and flower), and fruits at different ripening stages or subjected to different hormonal treatments (ABA, auxin). The expression stability of candidate genes was examined by a series of algorithms, such as geNorm, NormFinder, BestKeeper, and ΔCt, for comparisons and rankings. Finally, by using RefFinder, a comprehensive and comparative ranking of the four methods was achieved. The results highlight that the expression stability of candidate reference genes fluctuates depending on tissue type, fruit stage, and hormonal treatment. As reference genes, the use of FcCHP2 and FcACTIN1 is recommended for F. chiloensis vegetative tissues; FcDBP and FcCHP1 for fruit ripening stages; FcGAPDH and FcDBP for fruit subjected to ABA and NDGA treatments; FcCHP1 and FcCHP2 for fruit under AUXIN and TIBA treatments; and FcDBP and FcCHP2 when all fruit stages and hormonal treatments are compared. If just one reference gene is employed as a normalizer, FcDBP should be chosen as it is the most stable internal control in most conditions. Therefore, the present study delivers a set of reliable reference genes for RT-qPCR expression analysis in F. chiloensis tissues and fruits subjected to several hormonal treatments. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-022-01227-y.

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Abstract Introduction: Galvanic vestibular evoked myogenic potentials evaluate vestibular nerve responses using electric stimulation by records collected from the sternocleidomastoid muscle. A normal vestibular evoked myogenic potential response consists of the first positive, P1, and negative, N1, peaks. The response can be affected by factors such as age and gender and is also consequential in the diagnosis of pathologies. Objectives: The present study was performed to obtain normative data on healthy adults, to help in diagnosis by establishing clinical norms as well as to investigate changing test parameters with age in galvanic vestibular evoked myogenic potentials. Methods: A total of 100 healthy participants were included in the study. Galvanic vestibular evoked myogenic potential (current 3 mA, duration 1ms) was performed randomly on both ears of each participant. The participants between the ages of 18-65 (mean age 39.7 ± 13.9) were divided into 5 groups according to their ages. Normative data of galvanic vestibular evoked myogenic potentials parameters were calculated in groups and in total, and age-related changes were examined. Results: The galvanic vestibular evoked myogenic potential waveform was elicited from all participants (200 ears). The latency of P1 and N1 was 7.82 ± 3.29ms and 22.06 ± 3.95 ms, respectively. The P1-N1 amplitude value was 66.64 ± 24.5 μV. The percentage of vestibular asymmetry was 16.29 ±11.99%. The latencies of P1 and N1 and P1-N1 amplitude values demonstrated significant differences among different age groups (p < 0.01). Conclusions: The results of this study show that as age increased, latencies were prolonged, and amplitudes gradually decreased. The normative data aids in the diagnosis of retrolabyrinthine lesions and the increase in the clinical use of galvanic vestibular evoked myogenic potentials.

Resumo Introdução: Os potenciais evocados miogênicos vestibulares galvânicos avaliam as respostas do nervo vestibular com estimulação elétrica por meio de registros coletados do músculo esternocleidomastóideo. Uma resposta normal de potenciais evocados miogênicos vestibulares consiste nos primeiros picos positivo, P1, e negativo, N1. A resposta pode ser afetada por fatores como idade e sexo e também tem importância no diagnóstico de doenças. Objetivos: Obter dados normativos em adultos saudáveis, para ajudar no diagnóstico através do estabelecimento de normas clínicas, e investigar a alteração dos parâmetros de teste com a idade em potenciais evocados miogênicos vestibulares galvânicos. Método: Foram incluídos no estudo 100 participantes saudáveis. O potencial evocado miogênico vestibular galvânico (corrente 3mA, duração 1ms) foi realizado de forma aleatória nas duas orelhas de cada participante. Os participantes entre 18 e 65 anos (média de 39,7 ±13,9) foram divididos em 5 grupos de acordo com a idade. Os dados normativos dos parâmetros dos potenciais evocados miogênicos vestibulares galvânicos foram calculados nos grupos e no total e as alterações relacionadas à idade foram examinadas. Resultados: A forma de onda do potencial evocado miogênico vestibular galvânico foi obtida de todos os participantes (200 orelhas). A latência de P1 e N1 foi de 7,82±3,29ms e 22,06 ±3,95 ms, respectivamente. O valor da amplitude P1-N1 foi de 66,64 ±24,5 μV. O percentual de assimetria vestibular foi de 16,29± 11,99%. Os valores das latências de P1 e N1 e da amplitude P1-N1 mostraram diferenças significantes entre os diferentes grupos etários (p < 0,01). Conclusão: Os resultados deste estudo mostram que à medida que a idade aumentou as latências foram prolongadas e as amplitudes diminuíram gradualmente. Os dados normativos auxiliam no diagnóstico de lesões retrolabirínticas e na disseminação do uso clínico dos potenciais evocados miogênicos vestibulares galvânicos.

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The aim of this work was to evaluate if rivers could be used for SARS-CoV-2 surveillance. Five sampling points from three rivers (AR-1 and AR-2 in Arenales River, MR-1 and MR-2 in Mojotoro River, and CR in La Caldera River) from Salta (Argentina), two of them receiving discharges from wastewater plants (WWTP), were monitored from July to December 2020. Fifteen water samples from each point (75 in total) were collected and characterized physico-chemically and microbiologically and SARS-CoV-2 was quantified by RT-qPCR. Also, two targets linked to human contributions, human polyomavirus (HPyV) and RNase P, were quantified and used to normalize SARS-CoV-2 concentration, which was compared to reported COVID-19 cases. Statistical analyses allowed us to verify the correlation between SARS-CoV-2 and the concentration of fecal indicator bacteria (FIB), as well as to find similarities and differences between sampling points. La Caldera River showed the best water quality; FIBs were within acceptable limits for recreational activities. Mojotoro River's water quality was not affected by the northern WWTP of the city. Instead, Arenales River presented the poorest water quality; at AR-2 was negatively affected by the discharges of the southern WWTP, which contributed to significant increase of fecal contamination. SARS-CoV-2 was found in about half of samples in low concentrations in La Caldera and Mojotoro Rivers, while it was high and persistent in Arenales River. No human tracers were detected in CR, only HPyV was found in MR-1, MR-2 and AR-1, and both were quantified in AR-2. The experimental and normalized viral concentrations strongly correlated with reported COVID-19 cases; thus, Arenales River at AR-2 reflected the epidemiological situation of the city. This is the first study showing the dynamic of SARS-CoV-2 concentration in an urban river highly impacted by wastewater and proved that can be used for SARS-CoV-2 surveillance to support health authorities.

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BACKGROUND: Hypoxia pathways are deregulated in clear renal cell carcinoma (ccRCC) because of the loss of the von Hippel-Lindau tumor suppressor function. Quantitative PCR is a powerful tool for quantifying differential expression between normal and cancer cells. Reliable gene expression analysis requires the use of genes encoding housekeeping genes. Therefore, in this study, eight reference candidate genes were evaluated to determine their stability in 786-0 cells under normoxic and hypoxic conditions. METHODS AND RESULTS: Four different tools were used to rank the most stable genes-geNorm, NormFinder, BestKeeper, and Comparative Ct (ΔCt), and a general ranking was performed using RankAggreg. According to the four algorithms, the TFRC reference gene was identified as the most stable. There was no agreement among the results from the algorithms for the 2nd and 3rd positions. A general classification was then established using the RankAggreg tool. Finally, the three most suitable reference genes for use in 786-0 cells under normoxic and hypoxic conditions were TFRC, RPLP0, and SDHA. CONCLUSIONS: To the best of our knowledge, this is the first study to identify reliable genes that can be used for gene expression analysis in ccRCC in a hypoxic environment.

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