To Örebro University

BACKGROUND: 16S rRNA gene sequencing is widely used for bacterial community profiling in both clinical and research contexts. The expanding availability of library preparation protocols and bioinformatic pipelines increases analytical flexibility but may also introduce method-dependent biases that affect inferred microbial composition and relative abundance estimates. The relative impact of library preparation protocol, amplicon region, and bioinformatic pipeline on species-level taxonomic inference and compositional agreement remains insufficiently characterised. We therefore compared the Illumina 16S Metagenomic Sequencing Library Preparation protocol (V3-V4) and the Zymo Quick-16S Plus NGS Library Prep Kit (V1-V2 and V3-V4) in combination with two bioinformatic pipelines, nf-core/ampliseq and TRANA. Performance was assessed using defined microbial community standards and human faecal and colonic biopsy samples.

RESULTS: Pipeline choice was the dominant driver of variation in inferred community composition, exceeding the effects of amplicon regions and library preparation protocols. Genus-level profiles were broadly concordant across methods. Species-level resolution and agreement with expected community composition differed systematically between pipelines, with TRANA demonstrating lower Bray-Curtis dissimilarities to expected compositions than nf-core/ampliseq. Amplicon region had a secondary, pipeline-dependent effect, while protocol differences were minor. In clinical samples, inter-individual biological variation exceeded technical variation.

CONCLUSIONS: Bioinformatic processing substantially influenced species-level inference in short-read 16S sequencing, highlighting the importance of pipeline selection for microbiome study design and cross-study comparability.

Intestinal luminal microbial metabolites affect tryptophan and serotonin metabolism, and cross or modify the blood-brain barrier (BBB). Understanding those mechanisms further necessitates integrated gut-brain axis model systems. Using an ex vivo-in vitro approach, H2O2-stressed or non-stressed human dermal fibroblasts - representing the BBB - are cultured with serosal fluids of healthy or irritable bowel syndrome human colonic biopsies collected from Ussing chamber experiments, after participant's colon was exposed to butyrate in vivo, fecal fiber fermentation or control supernatant ex vivo. Culturing fibroblasts with serosal fluids does not compromise viability or have cytotoxic effects. Serosal fluids alone do not alter expression of tryptophan-related large amino acid membrane transporter genes and proteins, nor their activity (i.e., tryptophan uptake). However, adding serosal fluids to fibroblasts prior to oxidative stress indicate a protective role. This new model allows investigation of direct effects of serosal content on BBB-representing fibroblasts and is highly promising for more personalized applications.

Oral intake of probiotics has been shown to positively impact depression, anxiety, stress and cognition. Recently, an effort was made to more objectively assess their impact on brain structure and function. However, there has been no exhaustive systematic assessment of outcomes of these studies, nor the techniques utilised. Therefore, we performed a systematic review on randomised, placebo-controlled trials assessing the effects of oral probiotic interventions on brain health by imaging or electrophysiology techniques in human adults. Of 2307 articles screened, 26 articles comprising 19 studies, totalling 762 healthy subjects or patients with various diseases, were ultimately included. The quality of most studies was high. Overall, probiotic intake appears to modify resting state connectivity and activity, decrease involvement of several brain regions during negative emotional stimulation, and improve sleep quality. Several studies found correlations between brain outcomes and clinical symptom ratings, supporting the relevance of brain imaging and electrophysiology techniques in this field.

Stress induction tests such as carbon dioxide (CO2) inhalation challenges, are often used in research of anxiety and panic disorders. Physiological parameters of the autonomic response, e.g., heart rate variability (HRV) and skin conductance (SC), are often measured alongside questionnaires for evaluation. Previous studies have shown varied results on CO2 inhalation-induced physiological reactivity and further knowledge is of interest. The aim of this study was to explore the effects of a 35 % CO2 inhalation test on HRV frequencies and SC, in healthy subjects; and to set those into relation with subjective psychological ratings. In this single-blinded, repeated measures study, healthy subjects underwent a 35 % CO2 inhalation challenge, whereof the first and third double vital capacity inhalations were with normal air and the second with 35 % CO2. HRV (low and high frequency) and SC (as electrodermal activity (EDA)) were measured throughout. CO2 inhalation resulted in a significant increase of HRV's high and very high frequencies compared to the first air inhalation (median difference + 0.0001633; + 0.0000348) and of HRV's very high and very low frequencies compared to the last air inhalation (+ 0.0000321; + 0.0000154). Mean and maximum SC increased significantly during the CO2 inhalation compared to both air inhalations (mean difference to first air inhalation + 1.151; + 1.964; to last air inhalation + 0.5751; +1.484), but also between the separate air inhalations (+ 0.5754; + 0.4799). The HRV results indicate increased parasympathetic activity, while the SC results indicate increased sympathetic activity during CO2 inhalation. SC only minorly correlated with provoked panic symptoms (EDA minimum to VAS minimum r = -0.559, and not between any other EDA and VAS/PSL measure). While those results seem contradictory, this study confirms that a 35 % CO2 inhalation challenge in young healthy adults, provokes a physiological as well as psychological reaction.

Increasing evidence suggests that modulations of the gut-brain axis with probiotics impact healthy ageing. This double-blinded, randomised, placebo-controlled study compared effects of micro-encapsulated and non-encapsulated Lacticaseibacillus rhamnosus HN001 in 87 community-dwelling elderly (60-80 years). Resting state functional connectivity differed significantly in regions involved in visual processing and perception between the two probiotic groups (p < 0.0001). Brain morphometry was not altered. Significant time*group effects (p < 0.05) were observed for processing speed, non-significant effects for short-term memory and anxiety symptoms, while other cognitive domains, depression, perceived stress, and sleep quality were unaffected. Distribution of available and stored peripheral serotonin was significantly affected (p < 0.05), while levels of γ-aminobutyric acid and glutamate in striatum and circulating brain-derived neurotrophic factor did not show significant time*group effects. Micro-encapsulated probiotics target the gut differently, which impacts the effects on brain health assessed by (functional) magnetic resonance imaging in older adults. The trial is registered at ClinicalTrials.gov under ID: NCT05801042.

Psychiatric disorders are categorized on the basis of presence and absence of diagnostic criteria using classification systems such as the international classification of diseases (ICD) and the diagnostic and statistical manual for mental disorders (DSM). The research domain criteria (RDoC) initiative provides an alternative dimensional framework for conceptualizing mental disorders. In the present paper, we studied neural and behavioral effects of central stimulant (CS) medication in adults with attention deficit hyperactivity disorder (ADHD) and healthy controls using categorical and dimensional stratifications. AX-Continuous Performance Task (AX-CPT) was utilized for the later purpose, and participants were classified as "reactive" or "proactive" based on their baseline proactive behavioral index (PBI). Out of the 65 individuals who participated (33 healthy controls and 32 patients with ADHD), 53 were included in the final analysis that consisted of 31 healthy controls and 22 ADHD patients. For the dimensional stratification, a median split of PBI scores divided participants into "reactive" and "proactive" groups irrespective of whether they had ADHD or not. Participants performed AX-CPT in conjunction with functional magnetic resonance imaging (fMRI) before and after CS medication. We found no significant within or between group CS effect when participants were categorically assigned as healthy controls and ADHD patients. For the dimensional stratification, however, CS selectively increased activation in frontoparietal cognitive areas and induced a shift towards proactive control mode in the reactive group, without significantly affecting the proactive group. In conclusion, the neural and behavioral effects of CS were more clear-cut when participants were stratified into dimensional groups rather than diagnostic categories.

The appropriateness of the fecal microbiota to adequately reflect the gut microbiota composition from more difficult to access luminal content at different colonic locations has been debated. Here, in a healthy population, luminal samples were collected from terminal ileum to rectum using an unique sampling technique without the need of prior bowel cleansing/preparation. Rectal swabs were collected immediately prior colonoscopy by an experienced physician, and fecal samples were collected at home by the participants themselves. Microbiota composition was evaluated as relative abundance, α-diversity and Bray-Curtis dissimilarities. Our data suggest that fecal samples and rectal swabs present noninvasive, easily accessible, low-cost sampling tools that are accurate proxies to characterize luminal large intestinal microbiota composition.

BACKGROUND: An increasing number of cross-sectional studies suggests that diet may impact memory and cognition in healthy older adults. However, randomized, controlled trials investigating the effects of whole-diet interventions on memory and cognition in healthy older adults are rather rare and conflicting results are often reported.

OBJECTIVE: Therefore, a systematic review was conducted to compile the current evidence regarding the potential effects of whole-diet interventions on 1) memory and, 2) other cognitive outcomes in older adults.

METHODS: Studies that reported on randomized, controlled trials with dietary interventions in healthy older adults (60 yrs. and older) were included. Studies utilizing supplements, single food items or trials in specific patient groups (ie neurodegenerative diagnoses) were excluded.

RESULTS: For the 23 included articles, the main outcomes examined fell into one or more of the following categories: cognitive task-based outcomes related to memory, other cognitive task-based outcomes, and additional outcomes related to cognitive function or disease risk. Three of the studies that investigated dietary interventions alone and two multi-domain study showed positive effects on memory function, whereas five multi-domain interventions and one intervention that focused on diet alone showed positive effects on other cognitive outcomes.

CONCLUSIONS: The effect of randomized, controlled whole-diet interventions on memory and cognitive function in healthy older adults is modest and inconclusive, highlighting the need for more well-designed, sufficiently powered studies. Furthermore, the potential mechanisms by which diet impacts cognition in healthy aging need to be elucidated.

REGISTRY AND REGISTRY NUMBER FOR SYSTEMATIC REVIEWS OR META-ANALYSES: This systematic review is registered in PROSPERO under ID CRD42022329759.

BACKGROUND: An increased intestinal permeability is a common feature in patients with diarrhoea-predominant irritable bowel syndrome (IBS-D). Probiotics have shown to improve IBS symptoms and might also affect intestinal barrier function.

AIM: The aim of this study was to investigate the effects of a 6-week intervention with Limosilactobacillus reuteri ATCC PTA 6475 alone (single strain) or in combination with Limosilactobacillus reuteri DSM 17938 (dual strain) on gut barrier function, immune markers, and symptoms in IBS-D patients (ClinicalTrials.gov registration number: NCT03986476).

METHODS: 65 IBS-D patients were randomised into three groups (placebo, single strain, dual strain). Small and large intestinal permeability were assessed using a multi-sugar urinary recovery test. Blood, saliva, faecal samples, and several symptom scales were collected before, and after three and six weeks of intervention.

RESULTS: Small and large intestinal permeability as well as other markers of gut barrier function were not significantly affected by the probiotic interventions. Serum IL-6 levels showed a tendency to be reduced in the single strain group (descriptive p = 0.052). In addition, high-sensitivity C-reactive protein was significantly reduced in the dual strain group (p = 0.041). The participants in both treatment groups reported less gastrointestinal symptoms after three weeks, but this reached significance only in the dual strain group (total score: p = 0.032, pain subscore: p = 0.028). After six weeks, none of the assessed symptoms were significantly different from the placebo.

CONCLUSION: The probiotic compounds investigated in this study did not seem to affect IBS-D patients' gut barrier function, but showed potential anti-inflammatory and symptom-improving properties, which need to be confirmed in larger study cohorts.