Background. Blastocystis, the most prevalent microbial eukaryote in humans, has a global distribution. Studies have linked its presence with distinct gut microbiome and metabolome profiles compared to those where the organism is absent. However, the interplay of antibiotic administration, Blastocystis and the surrounding gut microbiome remains understudied. This case study aimed to explore antibiotic consumption and the presence of Blastocystis with subsequent changes in the gut microbiome and metabolome of an individual diagnosed with irritable bowel syndrome (IBS). Methods. Stool samples from an IBS patient, collected at 12 time points, were tested for the presence of Blastocystis using real-time PCR targeting the SSUrRNA gene, followed by sequencing of positive samples. Illumina sequencing determined the gut microbiome composition, while one-dimensional proton NMR spectroscopy was used to analyse the metabolome composition. Statistical analyses were conducted to identify relationships between antibiotic consumption, bacterial diversity, metabolome composition and Blastocystis presence. Results. Antibiotics significantly impacted the gut microbiome, with diversity declining early in the antibiotic course, then recovering later and post-course. Blastocystis was detected early, late and post-course but was not detectable mid-course, coinciding with the decline in bacterial diversity. Significant differences were observed between Blastocystis-positive and Blastocystis-negative samples, with bacterial composition significantly changing between samples collected before, early and after the antibiotic course compared to those collected mid-course. Metabolite groups, including short-chain fatty acids, amino acids and succinate, exhibited changes throughout the antibiotic course, indicating that gut metabolite composition is affected by antibiotic consumption. Discussion/Conclusion. While antibiotics did not significantly impact Blastocystis colonization, they did cause a mid-course decline in microbial diversity and Blastocystis presence. The study also revealed significant alterations in important metabolites such as short-chain fatty acids and amino acids throughout the antibiotic course, with an altered metabolome observed post-course. This case study underscores the complex interactions between antibiotics, gut microbiota and metabolites, highlighting the resilience of Blastocystis in the gut ecosystem.

Talin-1 is the core mechanosensitive adapter protein linking integrins to the cytoskeleton. The TLN1 gene is comprised of 57 exons that encode the 2,541 amino acid TLN1 protein. TLN1 was previously considered to be expressed as a single isoform. However, through differential pre-mRNA splicing analysis, we discovered a cancer-enriched, non-annotated 51-nucleotide exon in TLN1 between exons 17 and 18, which we refer to as exon 17b. TLN1 is comprised of an N-terminal FERM domain, linked to 13 force-dependent switch domains, R1-R13. Inclusion of exon 17b introduces an in-frame insertion of 17 amino acids immediately after Gln665 in the region between R1 and R2 which lowers the force required to open the R1-R2 switches potentially altering downstream mechanotransduction. Biochemical analysis of this isoform revealed enhanced vinculin binding, and cells expressing this variant show altered adhesion dynamics and motility. Finally, we showed that the TGF-β/SMAD3 signaling pathway regulates this isoform switch. Future studies will need to consider the balance of these two TLN1 isoforms.

Systemic capillary leak syndrome (SCLS) is a rare life-threatening disorder due to profound vascular leak. The trigger and the cause of the disease are currently unknown and there is no specific treatment. Here, we identified a rare heterozygous splice-site variant in the TLN1 gene in a familial SCLS case, suggestive of autosomal dominant inheritance with incomplete penetrance. Talin1 has a key role in cell adhesion by activating and linking integrins to the actin cytoskeleton. This variant causes in-frame skipping of exon 54 and is predicted to affect talin’s C-terminal actin-binding site (ABS3). Modeling the SCLS- TLN1 variant in TLN1 -heterozygous endothelial cells (ECs) disturbed the endothelial barrier function. Similarly, mimicking the predicted actin-binding disruption in TLN1 -heterozygous ECs resulted in disorganized endothelial adherens junctions. Mechanistically, we established that the SCLS- TLN1 variant, through the disruption of talin’s ABS3, sequestrates talin’s interacting partner, vinculin, at cell–extracellular matrix adhesions, leading to destabilization of the endothelial barrier. We propose that pathogenic variants in TLN1 underlie SCLS, providing insight into the molecular mechanism of the disease that can be explored for future therapeutic interventions.

Blastocystis is the most prevalent intestinal protist in humans, yet its role in gut health remains poorly understood. Increasing evidence suggests subtype-specific interactions with the gut microbiome and metabolome may underlie its variable associations with health and disease. In this pilot study, we performed an integrated analysis of the microbiota and metabolite profiles of nine Blastocystis subtypes (ST1-ST9) grown in vitro using xenic cultures. Using 16S rRNA amplicon sequencing and proton nuclear magnetic resonance (¹H-NMR) metabolomics, we characterised the microbial communities and extracellular metabolites across subtypes. ST3 exhibited the most distinct microbiome and metabolomic profile, characterised by a significant enrichment of short-chain fatty acids (SCFAs) and amino-acid derivatives. Benzoate, a known antimicrobial, was uniquely downregulated in ST3. Linear discriminant analysis identified several bacterial genera, such as Methanobrevibacter and Enterobacter, as biomarkers for ST3. Correlations between key metabolites and microbial taxa suggest potential syntrophic interactions. These findings suggest that individual Blastocystis subtypes establish distinct microenvironments in vitro, with implications for their ecological roles in vivo. Our study provides a foundational framework for understanding subtype-specific biology and offers a platform for improving culture conditions and investigating host-microbe interactions.

Cryptosporidium spp. and Giardia duodenalis are globally important intestinal protozoa causing diarrheal disease in humans and animals, with significant zoonotic potential. This study aimed to investigate the prevalence, molecular diversity, and potential transmission dynamics of these parasites in humans, livestock, and environmental samples from a rural community in Türkiye using a One Health approach, and to assess their associations with gut microbiome composition. Faecal samples were collected from 124 humans, 305 livestock (cattle, sheep, and goats), and 40 environmental samples (water and mud). Parasites were detected using qPCR and nested PCR, with positive samples genotyped by sequencing. Microbiome profiling was performed using 16S rRNA gene amplicon sequencing. Giardia duodenalis was detected in 12.1% of humans, 17.5% of livestock, and 2.5% of environmental samples, with assemblages A, B, and E identified, including the first detection of assemblage E in a human in Türkiye. Cryptosporidium spp. were found in 8.9% of humans, 19.3% of livestock, and 55% of environmental samples, with C. parvum as the dominant zoonotic species. Microbiome analysis revealed no significant differences in overall diversity. This study provides the first One Health assessment of Cryptosporidium spp. and Giardia duodenalis in Türkiye, emphasizing zoonotic transmission risks linked to livestock and the environment.

Biochar is widely promoted as a ruminant feed additive for microbiome modulation, primarily being touted as a method for reducing enteric methane emission reduction, but evidence on biochar’s broader gastrointestinal effects remains limited. We conducted a seven-month, on-farm longitudinal study of UK Sussex beef cattle in Kent, monitoring two herds (n=25 each). One herd received ad libitum hardwood (Ash and Chestnut) biochar, while the other herd served as a contemporaneous control. Faecal DNA was collected to provide insight into the lower gastrointestinal tract microbiome. Metagenome DNA was profiled by 16S rRNA (V3–V4) amplicon sequencing, and alpha/beta-diversity metrics were analysed, including multivariable association testing (MaAsLin3). Redundancy analysis indicated biochar supplementation explained notably little variance, and biochar supplementation did not induce a significant change in alpha diversity metrics. Overall, biochar did not drive temporal changes in diversity and was instead associated with increased abundance of specific taxa. These differences may reflect herd-specific effects rather than biochar per se. As such, these findings highlight the need for further, large scale biochar research to elucidate its impact on the complete gastrointestinal tract of ruminants.