Systemic infection and inflammation impair mental function through a combination of altered attention and cognition. Here, we comprehensively review the relevant literature and report personal clinical observations to discuss the relationship between infection, peripheral inflammation, and cerebral and cognitive dysfunction in patients with myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). Cognitive dysfunction in ME/CFS could result from low-grade persistent inflammation associated with raised pro-inflammatory cytokines. This may be caused by both infectious and non-infectious stimuli and lead to altered regional cerebral blood flow accompanied by disturbed neuronal function. Immune dysregulation that manifests as a subtle immunodeficiency or the autoimmunity targeting of one or more neuronal receptors may also be a contributing factor. Efforts to reduce low-grade systemic inflammation and viral reactivation and to improve mitochondrial energy generation in ME/CFS have the potential to improve cognitive dysfunction in this highly disabling condition.

The gut microbiome plays a vital role in physiological functions including metabolism, immune regulation, and gut-brain communication. Alterations in gut microbe makeup and function, termed microbial dysbiosis, are associated with various metabolic, inflammatory, and neurological disorders. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) patients often display gut microbial dysbiosis and increased intestinal barrier permeability ("leaky gut"). This "leaky gut" allows for microbial products and toxins, such as lipopolysaccharides (LPS), to enter the bloodstream, triggering systemic inflammation and immune dysregulation. ME/CFS patients exhibit altered immune responses, including production of antibodies reactive with gut microbial antigens, although the significance of these antibodies in promoting pathogenic or protective immune responses remains unclear. This chapter outlines methodologies for quantifying antibody reactivity to intestinal microbes and identifying stool-bound IgG in ME/CFS patients and healthy same household controls, to further investigate the role of anti-microbial IgG in ME/CFS pathogenesis.

The relationship between the intestinal microbiota and human health during ageing is an area of increasing interest due to increasing health challenges experienced by ageing populations. This paper develops a mathematical model describing the age-related biological changes associated with alterations to the microbiota, vitamin D levels, immunosenescence and inflammageing to determine the likely impact of manipulating the intestinal microbiota with dietary components. Age-dependent parameters are incorporated into a previously developed model to determine the evolution of intestinal bacterial populations, vitamin D receptor:1,25 dihydroxyvitamin D levels, epithelial barrier integrity and immune response with increasing age. Results suggest an age-related decline in both innate and adaptive immunity, weakening of the intestinal barrier, elevation in systemic inflammation and reduced serum vitamin D, resulting in individuals over 60 years old becoming vitamin D deficient (<50 nmol/L). Numerical simulations indicate that administration of probiotics and/or vitamin D supplements upregulates the VDR complex at all ages, which helps restore epithelial barrier function, particularly in older adults in whom the intestinal barrier has been compromised. The greatest benefit is derived from co-supplementation with probiotics and age-dependent doses of vitamin D. Finally, the value of gathering additional experimental data motivated by the modelling insights is discussed.

Candida parapsilosis is a common human commensal and opportunistic fungal pathogen that is also found in non-human primates (NHPs). Here, we report the first draft sequence of C. parapsilosis NCYC 4418, a fecal isolate from an adult cynomolgus macaque.

Genome-wide association studies have identified numerous susceptibility loci in complex diseases, such as chronic immune-mediated inflammatory disorders (IMIDs), yet their impact on pathomechanisms remains poorly understood. Low effect sizes, polygenicity, and predominance within non-coding genomic regions remain major challenges to the functional interpretation of IMID-associated single-nucleotide polymorphisms (SNPs). To address this, we present a novel systems genomics approach which models the cumulative impact of non-coding SNPs on downstream cellular signalling and gene regulatory networks. Applying this to the prototypical chronic IMIDs of Crohn's disease (CD) and ulcerative colitis (UC), both forms of inflammatory bowel disease (IBD), we individually analysed 2,636 patient genomes. Signals from non-coding SNPs were found to propagate towards well-established and novel CD- and UC-associated pathogenic pathways through the signalling and gene regulatory layers. The SNP-propagated gene regulatory networks stratified CD and UC patients into distinct clusters corresponding to cell type-specific gene dysregulation and potential therapeutic response. This approach bridges the gap between genotype and phenotype, laying the foundations for accelerating precision medicine in complex diseases.

Whole transcriptome amplification (WTA2) and sequence-independent single primer amplification (SISPA) are two widely used methods for combined metagenomic sequencing of RNA and DNA viruses. However, information on the reproducibility and bias of these methods on diverse viruses in faecal samples is currently lacking. A mock community (MC) of diverse viruses was developed and used to spike faecal samples at different concentrations. Virus-like particles (VLPs) were extracted, nucleic acid isolated, reverse-transcribed, and PCR amplified using either WTA2 or SISPA and sequenced for metagenomic analysis. A bioinformatics pipeline measured the recovery of MC viruses in replicates of faecal samples from three human donors, analysing the consistency of viral abundance measures and taxonomy. Viruses had different recovery levels with VLP extraction introducing variability between replicates, while WTA2 and SISPA produced comparable results. In comparing WTA2- and SISPA-generated libraries, WTA2 gave more uniform coverage depth profiles and improved assembly quality and virus identification. SISPA produced more consistent abundance, with a 50% difference between replicates occurring in ~20% and ~10% of sequences for WTA2 and SISPA, respectively. In conclusion, a bioinformatics pipeline has been developed to assess the methodological variability and bias of WTA2 and SISPA, demonstrating higher sensitivity with WTA2 and higher consistency with SISPA.

Gastrointestinal bacteria interact with the host and each other through various mechanisms, including the production of extracellular vesicles (EVs). However, the composition and potential roles of EVs released by gut archaea are poorly understood. Here, we study EVs produced by four strains of human gut-derived methanogenic archaea: Methanobrevibacter smithii ALI, M. smithii GRAZ-2, M. intestini, and Methanosphaera stadtmanae. The size (~130 nm) and morphology of these EVs are comparable to those of bacterial EVs. Proteomic and metabolomic analyses reveal that the archaeal EVs are enriched in putative adhesins or adhesin-like proteins, free glutamic and aspartic acid, and choline glycerophosphate. The archaeal EVs are taken up by macrophages in vitro and elicit species-specific responses in immune and epithelial cell lines, including production of chemokines such as CXCL9, CXCL11, and CX3CL1. The EVs produced by M. intestini strongly induce pro-inflammatory cytokine IL-8 in epithelial cells. Future work should examine whether archaeal EVs play roles in the interactions of archaea with other gut microbes and with the host.

Bacterial extracellular vesicles (BEVs) produced by members of the intestinal microbiota can not only contribute to digestion but also mediate microbe–host cell communication via the transfer of functional biomolecules to mammalian host cells. An unresolved question is which host factors and conditions influence BEV cargo and how they impact host cell function. To address this question, we analysed and compared the proteomes of BEVs released by the major human gastrointestinal tract (GIT) symbiont Bacteroides thetaiotaomicron (Bt) in vivo in fed versus fasted animals using nano-liquid chromatography with tandem mass spectrometry (LC-MSMS). Among the proteins whose abundance was negatively affected by fasting, nine of ten proteins of the serine protease family, including the regulatory protein dipeptidyl peptidase-4 (DPP-4), were significantly decreased in BEVs produced in the GITs of fasted animals. Strikingly, in extracellular vesicles produced by the intestinal epithelia of the same fasted mice, the proteins with the most increased abundance were serine protease inhibitors (serpins). Together, these findings suggest a dynamic interaction between GI bacteria and the host. Additionally, they indicate a regulatory role for the host in determining the balance between bacterial serine proteases and host serpins exported in bacterial and host extracellular vesicles.

Vitamin B12 (cobalamin) and related cobamides are essential cofactors for many gut bacteria, yet their acquisition requires complex uptake systems due to limited availability. In the human gut commensal Bacteroides thetaiotaomicron, cobamide uptake is mediated by multiple operons encoding outer membrane proteins, transporters and uncharacterised lipoproteins, some of which are incorporated into bacterial extracellular vesicles (BEVs). Here, we advance the functional and structural understanding of this cobamide acquisition system by examining previously uncharacterised features. Bioinformatic and promoter–reporter analyses revealed four uptake operons, including novel genes we designate btuK, btuJ, btuL and btuX, with evidence for internal promoters and riboswitch regulation. Recombinant expression and binding assays identified ten cobamide-binding proteins, including three novel lipoproteins (BtuK1, BtuJ1 and BtuJ2). Biophysical measurements demonstrated affinities in the nano- to picomolar range, with BtuJ proteins displaying exceptionally tight binding. High-resolution crystal structures of BtuJ1 and BtuJ2 revealed an augmented β-jelly-roll fold, with conserved tyrosine residues forming a ‘halo’ around the corrin, suggesting a conserved binding mechanism within the IPR027828 protein family. Comparative proteomics of cells and BEVs under cobamide starvation showed selective enrichment of BtuJ and BtuL in BEVs. Functional assays demonstrated that BEV-mediated cobamide uptake depends specifically on BtuJ1 and BtuJ2, whereas BtuL promotes early-phase BEV release. These findings establish the BtuJ proteins as critical BEV-associated cobamide-binding components, provide structural insights into their tight binding, and suggest a model where BEVs act analogously to siderophores, capturing cobamides for delivery to cells. This work highlights the central role of BEVs in microbial nutrient competition.

Bacterial extracellular vesicles (BEVs) are nano-size vesicles containing a cargo of bioactive molecules that can play key roles in microbe-microbe and microbe-host interactions. In tracking their biodistribution in vivo, BEVs can cross several physical host barriers including the intestinal epithelium, vascular endothelium, and blood-brain-barrier (BBB) to ultimately accumulate in tissues such as the liver, lungs, spleen, and the brain. This tissue-specific dissemination has been exploited for the delivery of biomolecules such as vaccines for mucosal delivery. Although numerous strategies for labeling and tracking BEVs have been described, most have constraints that impact on interpreting in vivo bioimaging patterns. Here, we describe a general method for labeling BEVs using lipophilic fluorescent membrane stains which can be adopted by non-expert users. We also describe how the procedure can be used to overcome potential limitations. Furthermore, we outline methods of quantitative ex vivo tissue imaging that can be used to evaluate BEV organ trafficking.

The advent of viral metagenomics, or viromics, has improved our knowledge and understanding of global viral diversity. High-throughput sequencing technologies enable explorations of the ecological roles, contributions to host metabolism, and the influence of viruses in various environments, including the human intestinal microbiome. However, bacterial metagenomic studies frequently have the advantage. The adoption of advanced technologies like long-read sequencing has the potential to be transformative in refining viromics and metagenomics. Here, we examined the effectiveness of long-read and hybrid sequencing by comparing Illumina short-read and Oxford Nanopore Technology (ONT) long-read sequencing technologies and different assembly strategies on recovering viral genomes from human faecal samples. Our findings showed that if a single sequencing technology is to be chosen for virome analysis, Illumina is preferable due to its superior ability to recover fully resolved viral genomes and minimise erroneous genomes. While ONT assemblies were effective in recovering viral diversity, the challenges related to input requirements and the necessity for amplification made it less ideal as a standalone solution. However, using a combined, hybrid approach enabled a more authentic representation of viral diversity to be obtained within samples.

Bacterial extracellular vesicles (BEVs) contribute to stress responses, quorum sensing, biofilm formation and interspecies and interkingdom communication. However, the factors that regulate their release and heterogeneity are not well understood. We set out to investigate these factors in the common gut commensal Bacteroides thetaiotaomicron by studying BEV release throughout their growth cycle. Utilising a range of methods, we demonstrate that vesicles released at different stages of growth have significantly different composition, with early vesicles enriched in specifically released outer membrane vesicles (OMVs) containing a larger proportion of lipoproteins, while late phase BEVs primarily contain lytic vesicles with enrichment of cytoplasmic proteins. Furthermore, we demonstrate that lipoproteins containing a negatively charged signal peptide are preferentially incorporated in OMVs. We use this observation to predict all Bacteroides thetaiotaomicron OMV enriched lipoproteins and analyse their function. Overall, our findings highlight the need to understand media composition and BEV release dynamics prior to functional characterisation and define the theoretical functional capacity of Bacteroides thetaiotaomicron OMVs.

Acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) remain significant challenges in haematological oncology. This review examines the pathophysiology, classification, and risk stratification of these aggressive malignancies, emphasising their impact on treatment strategies and prognosis. We discuss current standard-of-care treatments, including chemother-apy regimens and targeted therapies, while addressing the associated adverse effects and hyper-sensitivity reactions. Delving into the metabolic characteristics and vulnerabilities of leukaemia cells, the review highlights the key differences between lymphoid and myeloid leukaemia and how metabolic insights can be utilised for therapeutic purposes, with special focus on asparagi-nase therapy and its potential for improvement in both ALL and AML treatment. The review conveys the importance of personalised medicine approaches based on individual metabolic pro-files and the challenges posed by metabolic heterogeneity and plasticity in leukaemia cells. Inte-grating molecular and metabolic profiling can inform and advance treatment strategies for acute leukaemia, potentially improving patient outcomes and quality of life.

Microorganisms thriving in the human gastrointestinal microbiome have developed numerous mechanisms for microbiome-host and microbe-microbe interaction, including the formation of extracellular vesicles (EVs). However, the interactive mechanisms of members of the human archaeome remained largely unexplored. Here, we report the capability of two Methanobrevibacter strains M. smithii, Cand. M. intestini and Methanosphaera stadtmanae, to produce EVs. While the size (~ 130 nm) and morphology of these archaeal EVs were comparable to bacterial EVs, proteomic and metabolomic analyses revealed unique traits. The proteins found in both the EVs of M. smithii and Cand. M. intestini (n = 229) predominantly included adhesin and adhesin-like proteins, involved in archaea-surface and archaea-bacteria interaction. Additionally, the archaeal EVs contained glutamic acid and choline glycerophosphate, which could be involved in gut-brain signalling. Vesicles from all studied species were efficiently taken up by human monocytes and induced IL-8 production in epithelial HT-29 cells. The identification of archaeal EVs and their components provides novel insights into the complex interactions between the archaeal and bacterial microbiome and the host, potentially offering new paths to modulate vesicle-mediated impacts on host health.

Acute myeloid leukaemia (AML) and acute lymphoblastic leukaemia (ALL) remain significant challenges in haematological oncology. This review examines the pathophysiology, classification, and risk stratification of these aggressive malignancies, emphasising their impact on treatment strategies and prognosis. We discuss current standard-of-care treatments, including chemotherapy regimens and targeted therapies, while addressing the associated adverse effects and hypersensitivity reactions. Delving into the metabolic characteristics and vulnerabilities of leukaemia cells, the review highlights the key differences between lymphoid and myeloid leukaemia and how metabolic insights can be utilised for therapeutic purposes, with special focus on asparaginase therapy and its potential for improvement in both ALL and AML treatment. The review conveys the importance of personalised medicine approaches based on individual metabolic profiles and the challenges posed by metabolic heterogeneity and plasticity in leukaemia cells. Combining molecular and metabolic profiling can enhance and refine treatment strategies for acute leukaemia, potentially improving patient outcomes and quality of life. However, integrating these into routine clinical practice requires overcoming various practical, technical, and logistical issues.

One potential mechanism for microbiome-host, and microbiome constituents’ interaction and communication involves extracellular vesicles (EVs). Here, for the first time, we report the capability of two M. smithii strains (ALI and GRAZ-2), Candidatus M. intestini, and Methanosphaera stadtmanae, as underrepresented components of the gut microbiome, to produce EVs. Interesting, size, morphology, and composition of AEVs were comparable to bacterial EVs, as indicated by ultrastructure, composition, proteomic and metabolomic analyses; however, EVs were substantially less prevalent in the studied Archaea. When looking at the proteomics more precisely, although AEVs from M. smithii ALI and M. intestini were found to be carrying unique proteins (n=135 and n=30, respectively), the shared proteins in AEVs within this genus (n=229), were mostly adhesins(/like) proteins, or proteins with IG-like domains. One remarkable observation was the uptake of AEVs obtained from Methanosphaera stadtmanae and the studied Methanobrevibacter species by human monocytes and the subsequent IL-8 secretion. ### Competing Interest Statement The authors have declared no competing interest.

The death of dopamine-producing neurons in the substantia nigra in the base of the brain is a defining pathological feature in the development of Parkinson's disease (PD). PD is, however, a multi-systemic disease, also affecting the peripheral nervous system and gastrointestinal tract (GIT) that interact via the gut-brain axis (GBA). Our dual-flow GIT-brain microphysiological system (MPS) was modified to investigate the gut-to-brain translocation of the neurotoxin trigger of PD, 1-methyl-4-phenylpyridinium (MPP+), and its impact on key GIT and brain cells that contribute to the GBA. The modular GIT-brain MPS in combination with quantitative and morphometric image analysis methods reproduces cell specific neurotoxin-induced dopaminergic cytotoxicity and mitochondria-toxicity with the drug having no detrimental impact on the viability or integrity of cellular membranes of GIT-derived colonic epithelial cells. Our findings demonstrate the utility and capability of the GIT-brain MPS for measuring neuronal responses and its suitability for identifying compounds or molecules produced in the GIT that can exacerbate or protect against neuronal inflammation and cell death. (c) 2024 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International (CC BY-NC-ND) license (

Background: Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is an acquired disease with significant morbidity that affects both children and adults. Post-exertional malaise is a cardinal symptom of ME/CFS and impacts a patient’s functional capacity (FC). The absence of effective tools to assess FC has significant consequences for timely diagnosis, clinical follow-up, assessments for patient disability benefits, and research studies. In interventional studies, the inability to assess FC can result in an incomplete assessment of the potential benefit of the intervention, leading to beneficial treatment outcomes being missed. Methods: Using extensive, repeated patient feedback, we have developed a new questionnaire, FUNCAP, to accurately assess FC in ME/CFS patients. The questionnaire consists of eight domains divided by activity types: A. personal hygiene/basic functions, B. walking/movement, C. being upright, D. activities in the home, E. communication, F. activities outside the home, G. reactions to light and sound, and H. concentration. Results: Through five rounds of anonymous web-based surveys and a further test–retest validation round, two versions of the questionnaire were developed: a longer version comprising 55 questions (FUNCAP55), developed for improved diagnostic and disability benefit/insurance FC assessments; and a shorter version (FUNCAP27) for clinical patient follow-up and potential use in research. Good reliability and validity and negligible floor and ceiling effects were found, with comparable findings in all aspects in both a large Norwegian (n = 1263) and a separate English-language international sample (n = 1387) demonstrating the validity and reliability of FUNCAP. Conclusions: Our findings support the utility of FUNCAP as an effective, reliable and valid tool for assessing FC in ME/CFS patients.

The intestinal microbiota play a critical role in human health and disease, maintaining metabolic and immune/inflammatory health, synthesizing essential vitamins and amino acids and maintaining intestinal barrier integrity. The aim of this paper is to develop a mathematical model to describe the complex interactions between the microbiota, vitamin D/vitamin D receptor (VDR) pathway, epithelial barrier and immune response in order to understand better the effects of supplementation with probiotics and vitamin D. This is motivated by emerging data indicating the beneficial effects of vitamin D and probiotics individually and when combined. We propose a system of ordinary differential equations determining the time evolution of intestinal bacterial populations, concentration of the VDR:1,25(OH)$_{2}$D complex in epithelial and immune cells, the epithelial barrier and the immune response. The model shows that administration of probiotics and/or vitamin D upregulates the VDR complex, which enhances barrier function and protects against intestinal inflammation. The model also suggests co-supplementation to be superior to individual supplements. We explore the effects of inflammation on the populations of commensal and pathogenic bacteria and the vitamin D/VDR pathway and discuss the value of gathering additional experimental data motivated by the modelling insights.

Candida parapsilosis is a human fungal pathogen of increasing incidence and causes invasive candidiasis, notably in preterm or low-birthweight neonates. Here, we present the genome sequence of C. parapsilosis NCYC 4289, a fecal isolate from a preterm male infant.