An efficient method for high molecular weight bacterial DNA extraction suitable for shotgun metagenomics from skin swabs.

The human skin microbiome represents a variety of complex microbial ecosystems that play a key role in host health. Molecular methods to study these communities have been developed but have been largely limited to low-throughput quantification and short amplicon-based sequencing, providing limited functional information about the communities present. Shotgun metagenomic sequencing has emerged as a preferred method for microbiome studies as it provides more comprehensive information about the species/strains present in a niche and the genes they encode. However, the relatively low bacterial biomass of skin, in comparison to other areas such as the gut microbiome, makes obtaining sufficient DNA for shotgun metagenomic sequencing challenging. Here we describe an optimised high-throughput method for extraction of high molecular weight DNA suitable for shotgun metagenomic sequencing. We validated the performance of the extraction method, and analysis pipeline on skin swabs collected from both adults and babies. The pipeline effectively characterised the bacterial skin microbiota with a cost and throughput suitable for larger longitudinal sets of samples. Application of this method will allow greater insights into community compositions and functional capabilities of the skin microbiome.

A protocol paper for the MOTION Study-A longitudinal study in a cohort aged 60 years and older to obtain mechanistic knowledge of the role of the gut microbiome during normal healthy ageing in order to develop strategies that will improve lifelong health and wellbeing.

BACKGROUND: Advances in medicine and public health mean that people are living longer; however, a significant proportion of that increased lifespan is spent in a prolonged state of declining health and wellbeing which places increasing pressure on medical, health and social services. There is a social and economic need to develop strategies to prevent or delay age-related disease and maintain lifelong health. Several studies have suggested links between the gut microbiome and age-related disease, which if confirmed would present a modifiable target for intervention development. The MOTION study aims to determine whether and how changes in the gut microbiome are associated with physical and mental capacity. A comprehensive longitudinal multiparameter study such as this has not been previously undertaken. METHODS: MOTION is a longitudinal prospective cohort study with a focus on gut health and cognitive function. 360 healthy individuals aged 60 years and older, living in East Anglia, UK will be recruited to the study, stratified into one of three risk groups (cohorts) for developing dementia based on their cognitive function. Participants will attend study appointments every six months over four years, providing stool and blood samples and a health questionnaire. Participants will also undergo physical measurements and cognitive tests at alternating appointments, and undergo Optical Coherence Tomography scans at 3 timepoints. Two subgroups of participants in the study will provide colonic tissue biopsies (n = ≥30 from each cohort), and brain imaging (n = 30) at two timepoints. DISCUSSION: This study will provide new insights into the gut-(microbiota)-brain axis and the relationship between age-associated changes in gut microbe populations and cognitive health. Such insights could help develop new microbe-based strategies to improve lifelong health and wellbeing. TRIAL REGISTRATION: This study is registered in the ClinicalTrials.gov Database with ID: NCT04199195 Registered: May 14, 2019.

The Pregnancy and EARly Life study (PEARL) - a longitudinal study to understand how gut microbes contribute to maintaining health during pregnancy and early life.

BACKGROUND: The early life period represents the first step in establishing a beneficial microbial ecosystem, which in turn affects both short and longer-term health. Changes during pregnancy influence the neonatal microbiome; through transmission of maternal microbes during childbirth, and beyond, through nutritional programming. However, in-depth exploration of longitudinal maternal-infant cohorts, with sampling of multiple body sites, complemented by clinical and nutritional metadata, and use of cutting-edge experimental systems are limited. The PEARL study will increase our knowledge of; how microbes (including viruses/phages, bacteria, fungi and archaea) change in composition and functional capacity during pregnancy; transmission pathways from mother to infant; the impact of various factors on microbial communities across pregnancy and early life (e.g. diet), and how these microbes interact with other microbes and modulate host processes, including links to disease onset. METHODS: PEARL is a longitudinal observational prospective study of 250 pregnant women and their newborns, with stool and blood samples, questionnaires and routine clinical data collected during pregnancy, labour, birth and up to 24 months post birth. Metagenomic sequencing of samples will be used to define microbiome profiles, and allow for genus, species and strain-level taxonomic identification and corresponding functional analysis. A subset of samples will be analysed for host (immune/metabolite) molecules to identify factors that alter the host gut environment. Culturing will be used to identify new strains of health-promoting bacteria, and potential pathogens. Various in vitro and in vivo experiments will probe underlying mechanisms governing microbe-microbe and microbe-host interactions. DISCUSSION: Longitudinal studies, like PEARL, are critical if we are to define biomarkers, determine mechanisms underlying microbiome profiles in health and disease, and develop new diet- and microbe-based therapies to be tested in future studies and clinical trials. TRIAL REGISTRATION: This study is registered in the ClinicalTrials.gov Database with ID: NCT03916874 .

Neonates' responses to repeated exposure to a still face.

AIM: The main aims of the study were to examine whether human neonates' responses to communication disturbance modelled by the still-face paradigm were stable and whether their responses were affected by their previous experience with the still-face paradigm. METHODS: The still face procedure, as a laboratory model of interpersonal stress, was administered repeatedly, twice, to 84 neonates (0 to 4 day olds), with a delay of an average of 1.25 day. RESULTS: Frame-by-frame analysis of the frequency and duration of gaze, distressed face, crying, sleeping and sucking behaviours showed that the procedure was stressful to them both times, that is, the still face effect was stable after repeated administration and newborns consistently responded to such nonverbal violation of communication. They averted their gaze, showed distress and cried more during the still-face phase in both the first and the second administration. They also showed a carry-over effect in that they continued to avert their gaze and displayed increased distress and crying in the first reunion period, but their gaze behaviour changed with experience, in the second administration. While in the first administration the babies continued averting their gaze even after the stressful still-face phase was over, this carry-over effect disappeared in the second administration, and the babies significantly increased their gaze following the still-face phase. CONCLUSION: After excluding explanations of fatigue, habituation and random effects, a self-other regulatory model is discussed as a possible explanation for this pattern.