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Brissett, N.

Publications and source records attributed to Brissett, N..

3 recordsLinked to original sources
bioRxiv2024

Sexually dimorphic effects of prenatal alcohol exposure on the murine skeleton

BackgroundPrenatal alcohol exposure (PAE) can result in lifelong disabilities known as foetal alcohol spectrum disorder (FASD) and is associated with childhood growth deficiencies and increased bone fracture risk. However, the effects of PAE on the adult skeleton remain unclear and any potential sexual dimorphism is undetermined. Therefore, we utilised a murine model to examine sex differences with PAE on in vitro bone formation, and in the juvenile and adult skeleton. MethodsPregnant C57BL/6J female mice received 5% ethanol in their drinking water during gestation. Primary calvarial osteoblasts were isolated from neonatal offspring and mineralised bone nodule formation and gene expression assessed. Skeletal phenotyping of 4- and 12-week-old male and female offspring was conducted by micro-computed tomography ({micro}CT), 3-point bending, growth plate analyses, and histology. ResultsOsteoblasts from male and female PAE mice displayed reduced bone formation, compared to control ([≤]30%). Bglap and Ahsg were upregulated with PAE in both sexes compared to control, whereas Vegfa, Bmp6, Tgfbr1 and Flt1 were downregulated in PAE male osteoblasts only. In 12-week-old mice, {micro}CT analysis revealed a sex and exposure interaction across several trabecular bone parameters. PAE was detrimental to the trabecular compartment in male mice compared to control, yet PAE females were unaffected. Both male and female mice had significant reductions in cortical parameters with PAE. Whilst male mice were negatively affected along the tibia length, females were only distally affected. Posterior cortical porosity was increased in PAE females only. Mechanical testing revealed PAE males had significantly reduced bone stiffness compared to controls; maximum load and yield was reduced in both sexes. PAE had no effect on total body weight or tibial bone length in either sex. However, total growth plate width in male PAE mice compared to control was reduced, whilst female PAE mice were unaffected. 4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals. ConclusionsEvidence herein suggests for the first time that PAE exerts divergent sex effects on the skeleton, possibly influenced by underlying sex specific transcriptional mechanisms of osteoblasts. Establishing these sex differences will support future policies and clinical management of FASD. Plain English summaryPrenatal alcohol exposure (PAE) can lead to a set of lifelong cognitive, behavioural, and physical disabilities known as foetal alcohol spectrum disorder (FASD). FASD is a significant burden on healthcare, justice and education systems, which is set to worsen with rising alcohol consumption rates. FASD children have an increased risk of long bone fracture and adolescents are smaller in stature. However, sex differences and the long-term effects of PAE on the skeleton have not been investigated and was the aim of this study. Using a mouse model of PAE, we examined the function and gene expression of bone-forming cells (osteoblasts). We then analysed the skeletons of male and female mice at 12-weeks-old (adult) and 4-weeks-old (juvenile). PAE reduced osteoblast bone formation in both sexes, compared to control. Differential gene expression was predominantly observed in PAE males and largely involved genes related to blood vessel formation. High resolution x-ray imaging (micro-CT) revealed PAE had a detrimental effect on the inner trabecular bone component in 12-week-old male mice only. Analysis of the outer cortical bone revealed that whilst both male and female PAE mice were negatively affected, anatomical variations were observed. Mechanical testing also revealed differences in bone strength in PAE mice, compared to control. Interestingly, 4-week-old mice did not possess these sex differences observed in our PAE model at 12 weeks of age. Our data suggest PAE has detrimental and yet sex-dependent effects on the skeleton. Establishing these sex differences will support future policies and clinical management of FASD. HighlightsO_LIPrimary calvarial osteoblasts isolated from male and female PAE mice displayed reduced mineralised bone nodule formation and differential gene expression compared to control. C_LIO_LIPAE had a detrimental effect on trabecular bone parameters in 12-week-old male mice only. C_LIO_LIPAE leads to spatial variation in cortical bone parameters and geometry, with male mice negatively affected along the tibia length and female mice only affected at the distal end. C_LIO_LIMechanical testing revealed PAE male mice had significantly reduced bone stiffness compared to controls; PAE in both sexes reduced maximum load and yield. C_LIO_LI4-week-old mice did not display the altered skeletal phenotype with PAE observed in 12-week-old animals. C_LI

cell biology↗
bioRxiv2022

Targeted and untargeted nanopore sequencing approaches to profile the gut microbiota of mice infants exposed to ethanol in utero

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as foetal alcohol spectrum disorders. Here we assessed the effectiveness of targeted and untargeted (metagenomic) nanopore sequencing approaches to profile the gut microbiota of infant mice exposed to ethanol in utero. DNA extracts from the gut content of 12 infant mice exposed to ethanol in utero were analysed using one untargeted and two targeted (full-length 16S rRNA gene and the 16S-ITS-23S region of the ribosomal RNA operon) nanopore sequencing approaches. The targeting of the full-length 16S rRNA gene provided the most comprehensive analysis of the mouse gut microbiota. The differences in diversity between approaches were accounted by the sequencing target (p-value < 0.001). Faecalibaculum rodentium and Duncaniella sp. were the two most prevalent taxa detected using targeted sequencing approaches, while bacterial taxa were more evenly represented when using the metagenomic approach. Full-length 16S rRNA gene nanopore sequencing provides the most discriminatory microbiota compositional analysis of mice faecal samples. However, using nanopore sequencing approaches targeting the metagenome or different taxonomically-informative DNA region appears to introduce significant target-related biases. ImportanceCurrent nanopore approaches have not been standardized which may confound the biological interpretations of hight-throughput sequencing datasets. Additionally, nanopore sequencing still present a high error-rate compared to other more mature sequencing technologies, such as Illumina sequencing. These technological handicaps create the need to study and optimize nanopore sequencing approaches to answer biological questions, such as interrogations of the microbial composition and abundance of clinical and environmental samples. In this work, three nanopore sequencing approaches were designed and attempted to optimize fungal and bacterial profiling sequencing methodologies. Two targeted methods based on the bacterial 16S rRNA gene, and 16S-ITS-23S rrn operon region, and one untargeted shotgun/metagenomic approach were tested. Despite potential experimental and/or bioinformatical biases were found, the 16S rRNA gene-targeted nanopore sequencing was the most comprehensive approach to study the microbial composition of the infant mice gut microbiotas.

microbiology↗
bioRxiv2022

Effect of ethanol exposure in utero on infant mice gut microbiotas assessed by nanopore and illumina sequencing

The gut microbiome plays a vital role in host homeostasis and understanding of its biology is essential for a better comprehension of the etiology of disorders such as Foetal Alcohol Spectrum Disorder. Foetal Alcohol Spectrum Disorder represents a cluster of abnormalities including growth deficiencies and neurological impairments, which are not easily diagnosed nor treated. Here the effect of ethanol exposure in utero on the gut microbial profiles of 16 infant mice (nine exposed in utero and seven non-exposed) was assessed by targeted nanopore sequencing and Illumina sequencing approaches. The nanopore sequencing was implemented using MinION system targeting PCR-amplified amplicons made from the full-length 16S rRNA gene. The Illumina sequencing was performed using Miseq system targeting the V3-V4 region of the 16S rRNA gene. Ethanol exposure did not affect the microbial profiles. Several low prevalent taxa, like Akkermansia muciniphila, were detected but further studies must be performed to detail the effect of ethanol exposure to these taxa since no clear pattern was detected throughout this study. ImportanceDetailed knowledge about the interactions between gut microbes and the developing nervous system is still scarce. Foetal Alcohol Spectrum Disorder represents a clinically relevant set of conditions with cumbersome diagnostic and treatment. In this work the microbial profiles of infant mice gut exposed to ethanol in utero were analysed through third-generation Illumina and optimized next-generation nanopore sequencing technologies. The fungal (albeit not detected) and bacterial microbial profiles here obtained through nanopore and Illumina sequencing represent a technological and biological advancement towards a better comprehension of the microbial landscape in Foetal Alcohol Spectrum Disorder at early post-natal periods.

microbiology↗