Unique challenges and best practices for single cell transcriptomic analysis in toxicology.

The application and analysis of single-cell transcriptomics in toxicology presents unique challenges. These include identifying cell sub-populations sensitive to perturbation; interpreting dynamic shifts in cell type proportions in response to chemical exposures; and performing differential expression analysis in dose-response studies spanning multiple treatment conditions. This review examines these challenges while presenting best practices for critical single cell analysis tasks. This covers areas such as cell type identification; analysis of differential cell type abundance; differential gene expression; and cellular trajectories. Towards enhancing the use of single-cell transcriptomics in toxicology, this review aims to address key challenges in this field and offer practical analytical solutions. Overall, applying appropriate bioinformatic techniques to single-cell transcriptomic data can yield valuable insights into cellular responses to toxic exposures.

Interpretable predictive models of genome-wide aryl hydrocarbon receptor-DNA binding reveal tissue-specific binding determinants.

The aryl hydrocarbon receptor (AhR) is an inducible transcription factor whose ligands include the potent environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Ligand-activated AhR binds to DNA at dioxin response elements (DREs) containing the core motif 5'-GCGTG-3'. However, AhR binding is highly tissue specific. Most DREs in accessible chromatin are not bound by TCDD-activated AhR, and DREs accessible in multiple tissues can be bound in some and unbound in others. As such, AhR functions similarly to many nuclear receptors. Given that AhR possesses a strong core motif, it is suited for a motif-centered analysis of its binding. We developed interpretable machine learning models predicting the AhR binding status of DREs in MCF-7, GM17212, and HepG2 cells, as well as primary human hepatocytes. Cross-tissue models predicting transcription factor (TF)-DNA binding generally perform poorly. However, reasons for the low performance remain unexplored. By interpreting the results of individual within-tissue models and by examining the features leading to low cross-tissue performance, we identified sequence and chromatin context patterns correlated with AhR binding. We conclude that AhR binding is driven by a complex interplay of tissue-agnostic DRE flanking DNA sequence and tissue-specific local chromatin context. Additionally, we demonstrate that interpretable machine learning models can provide novel and experimentally testable mechanistic insights into DNA binding by inducible TFs.

Generative modeling of single-cell gene expression for dose-dependent chemical perturbations.

Single-cell sequencing reveals the heterogeneity of cellular response to chemical perturbations. However, testing all relevant combinations of cell types, chemicals, and doses is a daunting task. A deep generative learning formalism called variational autoencoders (VAEs) has been effective in predicting single-cell gene expression perturbations for single doses. Here, we introduce single-cell variational inference of dose-response (scVIDR), a VAE-based model that predicts both single-dose and multiple-dose cellular responses better than existing models. We show that scVIDR can predict dose-dependent gene expression across mouse hepatocytes, human blood cells, and cancer cell lines. We biologically interpret the latent space of scVIDR using a regression model and use scVIDR to order individual cells based on their sensitivity to chemical perturbation by assigning each cell a "pseudo-dose" value. We envision that scVIDR can help reduce the need for repeated animal testing across tissues, chemicals, and doses.

Prediction of mammalian tissue-specific CLOCK-BMAL1 binding to E-box DNA motifs.

The Brain and Muscle ARNTL-Like 1 protein (BMAL1) forms a heterodimer with either Circadian Locomotor Output Cycles Kaput (CLOCK) or Neuronal PAS domain protein 2 (NPAS2) to act as a master regulator of the mammalian circadian clock gene network. The dimer binds to E-box gene regulatory elements on DNA, activating downstream transcription of clock genes. Identification of transcription factor binding sites and genomic features that correlate to DNA binding by BMAL1 is a challenging problem, given that CLOCK-BMAL1 or NPAS2-BMAL1 bind to several distinct binding motifs (CANNTG) on DNA. Using three different types of tissue-specific machine learning models with features based on (1) DNA sequence, (2) DNA sequence plus DNA shape, and (3) DNA sequence and shape plus histone modifications, we developed an interpretable predictive model of genome-wide BMAL1 binding to E-box motifs and dissected the mechanisms underlying BMAL1-DNA binding. Our results indicated that histone modifications, the local shape of the DNA, and the flanking sequence of the E-box motif are sufficient predictive features for BMAL1-DNA binding. Our models also provide mechanistic insights into tissue specificity of DNA binding by BMAL1.

[Design and validation of a handbook and photographic atlas on complementary food for use as a food education tool]. / Diseño y validación de un manual y atlas fotográfico sobre alimentación complementaria para uso como herramienta de educación alimentaria.

Introduction: The use of visual tools to calculate the adequate portion of the infant's food with an adequate education would allow to provide correct, simple and clear information to those responsible for their feeding. The objective was to design a manual and photographic atlas on complementary feeding (CF) for children between 6-12 months of age, to be used as a tool in food and nutrition education. Methods: Methods: The instrument was divided into two sections: a manual that includes advice on CF, and another corresponding to the food atlas, adapted to children aged 6 to 12 months. Content validity was performed by expert judgment, and the degree of agreement was measured using the Interclass Correlation Coefficient (ICC). Results: The designed tool includes 47 foods, 323 photographs and 6 chapters, where key concepts were developed, feeding in the sick child, measurements and capacities of the utensils used, images accompanied with recommendations for food and nutritional education about consistency, frequency, quantity, progression in the incorporation of food, safety, recipes and menu ideas according to age. The atlas was divided into food groups, subdivided into raw and cooked foods, and in different presentations: puree, pieces and whole. The content validation showed a high percentage of general acceptance (85.86%) and reported a 95% ICC of 0.884 (0.712-0.973). Conclusion: The designed tool is very useful for carrying out nutrition counseling and education interventions for children of the age of starting their CF.

Introducción: El uso de herramientas visuales para calcular la porción adecuada de los alimentos del lactante junto con una adecuada educación permitiría proporcionar información correcta, simple y clara a los responsables de su alimentación. El objetivo fue diseñar un manual y atlas fotográfico sobre alimentación complementaria (AC) para niños entre 6-12 meses de edad, para ser utilizado como herramienta en educación alimentaria y nutricional. Métodos: El instrumento se dividió en dos secciones: un manual que incluye consejería sobre AC, y otra correspondiente al atlas de alimentos, adaptados a niños de 6 a 12 meses. Se realizó la validez de contenido por juicio de expertos, y se midió el grado acuerdo mediante el Coeficiente de Correlación Interclase (CCI). Resultados: La herramienta diseñada incluye 47 alimentos, 323 fotografías y 6 capítulos, donde se desarrollaron conceptos claves, la alimentación en el niño enfermo, medidas y capacidades de los utensilios utilizados, imágenes acompañadas con recomendaciones de educación alimentaria y nutricional acerca de la consistencia, frecuencia, cantidad, progresión en la incorporación de los alimentos, inocuidad, recetas e ideas de menú de acuerdo a la edad. El atlas se dividió por grupos de alimentos, subdividido en alimentos crudos y cocidos, y en diferentes presentaciones: puré, trozos y entero. La validación de contenido mostro un alto porcentaje de aceptación general (85,86%) y reportó un CCI 95% de 0,884 (0,712-0,973). Conclusión: La herramienta diseñada resulta de gran utilidad para la realización de intervenciones de consejería y educación alimentaria para niños en edad de iniciar su AC.

Validación de un instrumento para identificar conocimientos y prácticas maternas de alimentación complementaria en una población vulnerable / Validation of an instrument to identify knowledge and maternal practices of complementary feeding in a vulnerable population

FUNDAMENTOS: Una alimentación adecuada durante los primeros años de vida resulta imprescindible para alcanzar un crecimiento óptimo. Esto depende fundamentalmente de los conocimientos y prácticas maternas, por lo que es esencial contar con instrumentos validados que permitan su identificación. El objetivo fue diseñar y validar un cuestionario para identificar conocimientos y prácticas maternas de alimentación complementaria en una población vulnerable. MÉTODOS: Estudio de validación. El cuestionario fue diseñado a partir de una revisión de la literatura, validado mediante juicio de 6 expertos y un pilotaje cognitivo en 50 madres de niños de 6 a 23 meses de edad de la localidad de Taco Pozo, Chaco, Argentina. RESULTADOS: El instrumento inicial contó con 40 ítems, distribuidos en 4 bloques. El test demostró una excelente claridad y comprensión (97%), y una aceptación superior al 90% para la coherencia, claridad y relevancia evaluados por los jueces, con un Coeficiente de Correlación Intraclase de 0,823; 0,888 y 0,508, respectivamente. Se demostró la unidimensionalidad de la herramienta (Kaiser-Meyer-Olkin = 0,414 y prueba de esfericidad de Bartlett = 0,007). CONCLUSIONES: La validación del cuestionario resultó satisfactoria para la determinación de los conocimientos y prácticas maternas de alimentación complementaria, útil para la planificación de futuras intervenciones educativas

BACKGROUND: Adequate food during the first years of life is essential to achieve optimal growth. This depends fundamentally on maternal knowledge and practices, so it is essential to have validated instruments that allow their identification. The objective was to design and validate a questionnaire to identify knowledge or maternal practices of complementary feeding. METHODS: Validation study. The questionnaire was designed based on a review of the literature, and validated by trial of 6 experts and a cognitive pilot in 50 mothers of children 6 to 23 months of age from the town of Taco Pozo, Chaco, Argentina. RESULTS: The initial instrument had 40 items, distributed in 4 blocks. The test showed excellent clarity and understanding (97%), and an acceptance greater than 90% for the coherence, clarity and relevance evaluated by the judges, with an Intraclass Correlation Coefficient of 0.823; 0.888 and 0.508, respectively. The unidimensionality of the tool was demonstrated (Kaiser-Meyer-Olkin = 0.414 and Bartlett's test of sphericity = 0.007). CONCLUSIONS: The validation of the questionnaire was satisfactory for the determination of the knowledge and maternal practices of complementary feeding, useful for the planning of future educational interventions