RESUMEN
The majority of aneuploid fetuses are spontaneously miscarried. Nevertheless, some aneuploid individuals survive despite the strong genetic insult. Here, we investigate if the survival probability of aneuploid fetuses is affected by the genome-wide burden of slightly deleterious variants. We analyzed two cohorts of live-born Down syndrome individuals (388 genotyped samples and 16 fibroblast transcriptomes) and observed a deficit of slightly deleterious variants on Chromosome 21 and decreased transcriptome-wide variation in the expression level of highly constrained genes. We interpret these results as signatures of embryonic selection, and propose a genetic handicap model whereby an individual bearing an extremely severe deleterious variant (such as aneuploidy) could escape embryonic lethality if the genome-wide burden of slightly deleterious variants is sufficiently low. This approach can be used to study the composition and effect of the numerous slightly deleterious variants in humans and model organisms.
Asunto(s)
Aneuploidia , Cromosomas Humanos Par 21/genética , Síndrome de Down , Genotipo , Transcriptoma , Aborto Espontáneo , Síndrome de Down/embriología , Síndrome de Down/genética , Femenino , Humanos , EmbarazoRESUMEN
X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.
Asunto(s)
Cromosomas Humanos X/genética , Fibroblastos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de la Célula Individual/métodos , Inactivación del Cromosoma X , Alelos , Células Cultivadas , Femenino , Fibroblastos/citología , Humanos , TranscriptomaRESUMEN
Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.
Asunto(s)
Alelos , Expresión Génica , Impresión Genómica , Análisis de Secuencia de ARN/métodos , Análisis de la Célula Individual/métodos , Línea Celular , Fibroblastos/citología , Fibroblastos/metabolismo , Estudios de Asociación Genética , Técnicas de Genotipaje , Heterocigoto , Humanos , Modelos Teóricos , Polimorfismo de Nucleótido Simple , TranscriptomaRESUMEN
OBJECTIVE: F-FDG-PET/MRI is a novel hybrid techinque that has been recently introduced in oncological imaging, showing promising results. The aim of this study is to assess the value of whole-body F-FDG-PET/MRI for predicting the pathological stage of locally advanced rectal cancer after preoperative chemoradiotherapy. DESIGN: This was a prospective observational study. SETTINGS: The study was conducted at a tertiary care hospital. PATIENTS: Thirty-six patients with locally advanced rectal cancer (25 male, median age 68.5 years) were prospectively assessed with PET/MRI and thoracoabdominal CT before and after preoperative chemoradiotherapy. Twenty-seven patients underwent low anterior or abdominoperineal resection. Nine patients with a complete clinical response underwent organ-preserving treatment (8 local excision and 1 watch-and-wait approach) with >1-year follow-up. MAIN OUTCOME MEASURES: One radiologist evaluated pelvic MRI and CT. A second radiologist and a nuclear medicine physician jointly assessed PET/MRI. The imaging was compared with histology or follow-up (ypT0 vs T ≥1 and ypN0 vs ypN+ categories). Metastases were confirmed with biopsy or a follow-up CT scan at least at 1 year after preoperative chemoradiotherapy. The sensitivity, specificity, and accuracy values of the imaging techniques were calculated using standard formulas. RESULTS: The accuracy for ypT staging was 89% and 92%, and the accuracy for ypN was 86% and 92% for MRI and PET/MRI. Compared with CT, PET/MRI correctly diagnosed 4 of 5 metastases, but it did not detect a lung metastatic nodule. In 11% of the patients, the PET/MRI changed the treatment strategy. LIMITATIONS: This study is limited by its small sample size. CONCLUSIONS: Although the whole-body PET/MRI was more accurate than the pelvic MRI alone for the prediction of tumor and node response to preoperative chemoradiotherapy, the technique performed worse than CT in detecting small lung metastasis. See Video Abstract at http://links.lww.com/DCR/B108. TOMOGRAFÍA POR EMISIÓN DE POSITRONES DE 18F- FLUORODEOXIGLUCOSA (FDG) / RESONANCIA MAGNÉTICA (TEP/RM) PARA ESTADIFICACIÓN TUMORAL TNM DE CÁNCER DEL RECTO DESPUÉS DE LA QUIMIORRADIOTERAPIA PREOPERATORIA - EXPERIENCIA INICIAL: Evaluar el valor de la tomografía por emisión de positrones de 18F-fluorodeoxiglucosa / resonancia magnética (TEP/RM) para predecir el estadio patológico del cáncer de recto localmente avanzado después de la quimiorradioterapia preoperatoria.Este fue un estudio prospectivo observacional.El estudio se realizó en un hospital de atención terciaria.Treinta y seis pacientes con cáncer rectal localmente avanzado (25 hombres, edad media de 68.5 años) fueron evaluados prospectivamente con TEP/RM y tomografía computarizada (TC) toraco-abdominal antes y después de la quimiorradioterapia preoperatoria. Veintisiete pacientes se sometieron a resección anterior baja o abdominoperineal. Nueve pacientes con una respuesta clínica completa se sometieron a un tratamiento de preservación de órganos (8 escisión local y 1 un enfoque de observar y esperar) con un seguimiento de> 1 año.Un radiólogo evaluó la RM pélvica y la TC. Un segundo radiólogo y un médico de medicina nuclear evaluaron conjuntamente TEP / RM. La imagen se comparó con la histología o el seguimiento (ypT0 vs T ≥1 y ypN0 vs ypN + categorías). Las metástasis se confirmaron con biopsia o una TC de seguimiento al menos 1 año después de la quimiorradioterapia preoperatoria. Los valores de sensibilidad, especificidad y precisión de las técnicas de imagen se calcularon utilizando fórmulas estándar.La precisión para la estadificación ypT fue del 89% y 92%, y la precisión para ypN fue del 86% y 92% para RM y TEP/RM respectivamente. En comparación con la TC, la TEP / RM diagnosticó correctamente 4 de 5 metástasis, pero no detectó un nódulo metastásico pulmonar. En el 11% de los pacientes, la TEP / RM cambió la estrategia de tratamiento.Este estudio está limitado por su pequeño tamaño de muestra.Si bien la TEP / RM de todo el cuerpo fue más precisa que la RM pélvica sola para la predicción de la respuesta tumoral y ganglionar a la quimiorradioterapia preoperatoria, la técnica funcionó peor que la TC para detectar metástasis pulmonares pequeños. Consulte Video Resumen en http://links.lww.com/DCR/B108.
Asunto(s)
Quimioradioterapia , Imagen Multimodal , Neoplasias del Recto/diagnóstico por imagen , Neoplasias del Recto/terapia , Adulto , Anciano , Anciano de 80 o más Años , Colectomía , Terapia Combinada , Femenino , Fluorodesoxiglucosa F18 , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Tomografía de Emisión de Positrones , Cuidados Preoperatorios , Proctectomía , Estudios Prospectivos , Radiofármacos , Neoplasias del Recto/patología , Tomografía Computarizada por Rayos XRESUMEN
Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.
Asunto(s)
Síndrome de Down/genética , Regulación de la Expresión Génica/genética , Genoma/genética , Transcriptoma/genética , Animales , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Cromosomas Humanos Par 21/genética , Cromosomas de los Mamíferos/genética , Momento de Replicación del ADN , Síndrome de Down/patología , Femenino , Feto/citología , Fibroblastos , Histonas/química , Histonas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Lisina/metabolismo , Masculino , Metilación , Ratones , Gemelos Monocigóticos/genéticaRESUMEN
The study of gene expression in mammalian single cells via genomic technologies now provides the possibility to investigate the patterns of allelic gene expression. We used single-cell RNA sequencing to detect the allele-specific mRNA level in 203 single human primary fibroblasts over 133,633 unique heterozygous single-nucleotide variants (hetSNVs). We observed that at the snapshot of analyses, each cell contained mostly transcripts from one allele from the majority of genes; indeed, 76.4% of the hetSNVs displayed stochastic monoallelic expression in single cells. Remarkably, adjacent hetSNVs exhibited a haplotype-consistent allelic ratio; in contrast, distant sites located in two different genes were independent of the haplotype structure. Moreover, the allele-specific expression in single cells correlated with the abundance of the cellular transcript. We observed that genes expressing both alleles in the majority of the single cells at a given time point were rare and enriched with highly expressed genes. The relative abundance of each allele in a cell was controlled by some regulatory mechanisms given that we observed related single-cell allelic profiles according to genes. Overall, these results have direct implications in cellular phenotypic variability.
Asunto(s)
Alelos , Fibroblastos/citología , Genoma Humano , Análisis de Secuencia de ARN , ADN Complementario/genética , ADN Complementario/metabolismo , Haplotipos , Heterocigoto , Humanos , Fenotipo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Análisis de la Célula IndividualRESUMEN
MOTIVATION: Large genomic datasets combining genotype and sequence data, such as for expression quantitative trait loci (eQTL) detection, require perfect matching between both data types. RESULTS: We described here MBV (Match BAM to VCF); a method to quickly solve sample mislabeling and detect cross-sample contamination and PCR amplification bias. AVAILABILITY AND IMPLEMENTATION: MBV is implemented in C ++ as an independent component of the QTLtools software package, the binary and source codes are freely available at https://qtltools.github.io/qtltools/ . CONTACT: olivier.delaneau@unige.ch or emmanouil.dermitzakis@unige.ch. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Técnicas de Genotipaje/métodos , Sitios de Carácter Cuantitativo , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Sesgo , Genómica/métodos , Genómica/normas , Técnicas de Genotipaje/normas , Humanos , Análisis de Secuencia de ADN/normasRESUMEN
Thrombosis is a leading cause of morbidity and mortality. Fibrinogen, the soluble substrate for fibrin-based clotting, has a central role in haemostasis and thrombosis and its plasma concentration correlates with cardiovascular disease event risk and a prothrombotic state in experimental models. We aimed to identify chemical entities capable of changing fibrinogen production and test their impact on experimental thrombosis. A total of 1,280 bioactive compounds were screened for their ability to alter fibrinogen production by hepatocyte-derived cancer cells and a selected panel was tested in zebrafish larvae. Anthralin and all-trans retinoic acid (RA) were identified as fibrinogen-lowering and fibrinogen-increasing moieties, respectively. In zebrafish larvae, anthralin prolonged laser-induced venous- occlusion times and reduced thrombocyte accumulation at injury sites. RA had opposite effects. Treatment with RA, a nuclear receptor ligand, increased fibrinogen mRNA levels. Using an antisense morpholino oligonucleotide to deplete zebrafish fibrinogen, we correlated a shortening of laser-induced venous thrombosis times with RA treatment and fibrinogen protein levels. Anthralin had little effect on fibrinogen mRNA in zebrafish larvae, despite leading to lower detectable fibrinogen. Therefore, we made a proteomic scan of anthralin-treated cells and larvae. A reduced representation of proteins linked to the canonical secretory pathway was detected, suggesting that anthralin affects protein secretion. In summary, we found that chemical modulation of fibrinogen levels correlates with measured effects on experimental venous thrombosis and could be investigated as a therapeutic avenue for thrombosis prevention.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Fibrinógeno/metabolismo , Fibrinolíticos/farmacología , Trombosis de la Vena/tratamiento farmacológico , Proteínas de Pez Cebra/metabolismo , Animales , Animales Modificados Genéticamente , Antralina/farmacología , Modelos Animales de Enfermedad , Fibrinógeno/genética , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Humanos , Integrina alfa2/genética , Integrina alfa2/metabolismo , Morfolinos/farmacología , Mutación , Oligonucleótidos Antisentido/farmacología , Proteómica , Bibliotecas de Moléculas Pequeñas , Tretinoina/farmacología , Trombosis de la Vena/genética , Trombosis de la Vena/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.
Asunto(s)
Variaciones en el Número de Copia de ADN , Dosificación de Gen , Modelos Genéticos , Transcriptoma/genética , Trisomía/genética , Alelos , Cromosomas Humanos Par 13/genética , Cromosomas Humanos Par 18/genética , Cromosomas Humanos Par 8/genética , Síndrome de Down/genética , Femenino , Fibroblastos , Perfilación de la Expresión Génica , Humanos , Masculino , Mosaicismo , Fenotipo , RNA-Seq , Análisis de la Célula IndividualRESUMEN
The identification of genetic variants affecting gene expression, namely expression quantitative trait loci (eQTLs), has contributed to the understanding of mechanisms underlying human traits and diseases. The majority of these variants map in non-coding regulatory regions of the genome and their identification remains challenging. Here, we use natural genetic variation and CAGE transcriptomes from 154 EBV-transformed lymphoblastoid cell lines, derived from unrelated individuals, to map 5376 and 110 regulatory variants associated with promoter usage (puQTLs) and enhancer activity (eaQTLs), respectively. We characterize five categories of genes associated with puQTLs, distinguishing single from multi-promoter genes. Among multi-promoter genes, we find puQTL effects either specific to a single promoter or to multiple promoters with variable effect orientations. Regulatory variants associated with opposite effects on different mRNA isoforms suggest compensatory mechanisms occurring between alternative promoters. Our analyses identify differential promoter usage and modulation of enhancer activity as molecular mechanisms underlying eQTLs related to regulatory elements.
Asunto(s)
Elementos de Facilitación Genéticos/genética , Variación Genética , Regiones Promotoras Genéticas , Línea Celular Transformada , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Herpesvirus Humano 4/patogenicidad , Humanos , Sitios de Carácter Cuantitativo , TranscriptomaRESUMEN
Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10(-8)) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.
Asunto(s)
Carcinoma Basocelular/genética , Transducción de Señal/efectos de la radiación , Neoplasias Cutáneas/genética , Anilidas/uso terapéutico , Antineoplásicos/uso terapéutico , Carcinogénesis/genética , Carcinoma Basocelular/tratamiento farmacológico , Carcinoma Basocelular/patología , Análisis Mutacional de ADN , Progresión de la Enfermedad , Exoma , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Células HEK293 , Humanos , Mutación , Piridinas/uso terapéutico , Neoplasias Cutáneas/tratamiento farmacológico , Neoplasias Cutáneas/patología , TranscriptomaRESUMEN
The HSA21 encoded Single-minded 2 (SIM2) transcription factor has key neurological functions and is a good candidate to be involved in the cognitive impairment of Down syndrome. We aimed to explore the functional capacity of SIM2 by mapping its DNA binding sites in mouse embryonic stem cells. ChIP-sequencing revealed 1229 high-confidence SIM2-binding sites. Analysis of the SIM2 target genes confirmed the importance of SIM2 in developmental and neuronal processes and indicated that SIM2 may be a master transcription regulator. Indeed, SIM2 DNA binding sites share sequence specificity and overlapping domains of occupancy with master transcription factors such as SOX2, OCT4 (Pou5f1), NANOG or KLF4. The association between SIM2 and these pioneer factors is supported by co-immunoprecipitation of SIM2 with SOX2, OCT4, NANOG or KLF4. Furthermore, the binding of SIM2 marks a particular sub-category of enhancers known as super-enhancers. These regions are characterized by typical DNA modifications and Mediator co-occupancy (MED1 and MED12). Altogether, we provide evidence that SIM2 binds a specific set of enhancer elements thus explaining how SIM2 can regulate its gene network in neuronal features.
Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , ADN/metabolismo , Elementos de Facilitación Genéticos , Células Madre Embrionarias de Ratones/metabolismo , Factores de Transcripción/genética , Animales , Sitios de Unión , Células Cultivadas , Inmunoprecipitación de Cromatina/métodos , ADN/genética , Factor 4 Similar a Kruppel , Ratones , Análisis de Secuencia de ARN , Factores de Transcripción/metabolismoRESUMEN
Children with Down syndrome (DS) and acute lymphoblastic leukaemia (ALL) have poorer survival and more relapses than non-DS children with ALL, highlighting an urgent need for deeper mechanistic understanding of DS-ALL. Here, using full-exome or cancer genes-targeted sequencing of 42 ALL samples from 39 DS patients, we uncover driver mutations in RAS, (KRAS and NRAS) recurring to a similar extent (15/42) as JAK2 (12/42) mutations or P2RY8-CRLF2 fusions (14/42). RAS mutations are almost completely mutually exclusive with JAK2 mutations (P=0.016), driving a combined total of two-thirds of analysed cases. Clonal architecture analysis reveals that both RAS and JAK2 drove sub-clonal expansions primarily initiated by CRLF2 rearrangements, and/or mutations in chromatin remodellers and lymphocyte differentiation factors. Remarkably, in 2/3 relapsed cases, there is a switch from a primary JAK2- or PTPN11-mutated sub-clone to a RAS-mutated sub-clone in relapse. These results provide important new insights informing the patient stratification strategies for targeted therapeutic approaches for DS-ALL.
Asunto(s)
Síndrome de Down/genética , Janus Quinasa 2/genética , Mutación , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Proteínas ras/metabolismo , Niño , Cromatina/química , Análisis Mutacional de ADN , Síndrome de Down/metabolismo , Femenino , Eliminación de Gen , Regulación Leucémica de la Expresión Génica , Humanos , Linfocitos/citología , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Receptores de Citocinas/genéticaRESUMEN
Alteration of the number of copies of double minutes (DMs) with oncogenic EGFR mutations in response to tyrosine kinase inhibitors is a novel adaptive mechanism of glioblastoma. Here we provide evidence that such mutations in DMs, called here amplification-linked extrachromosomal mutations (ALEMs), originate extrachromosomally and could therefore be completely eliminated from the cancer cells. By exome sequencing of seven glioblastoma patients we reveal ALEMs in EGFR, PDGFRA and other genes. These mutations together with DMs are lost by cancer cells in culture. We confirm the extrachromosomal origin of such mutations by showing that wild-type and mutated DMs may coexist in the same tumour. Analysis of 4,198 tumours suggests the presence of ALEMs across different tumour types with the highest prevalence in glioblastomas and low-grade gliomas. The extrachromosomal nature of ALEMs explains the observed drastic changes in the amounts of mutated oncogenes (like EGFR or PDGFRA) in glioblastoma in response to environmental changes.