Objective Gut microbiome alterations in Parkinson disease (PD) have been reported repeatedly, but their functional relevance remains unclear. Fecal metabolomics, which provide a functional readout of microbial activity, have scarcely been investigated. We investigated fecal microbiome and metabolome alterations in PD, and their clinical relevance. Methods Two hundred subjects (104 patients, 96 controls) underwent extensive clinical phenotyping. Stool samples were analyzed using 16S rRNA gene sequencing. Fecal metabolomics were performed using two platforms, nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography–mass spectrometry. Results Fecal microbiome and metabolome composition in PD was significantly different from controls, with the largest effect size seen in NMR‐based metabolome. Microbiome and NMR‐based metabolome compositional differences remained significant after comprehensive confounder analyses. Differentially abundant fecal metabolite features and predicted functional changes in PD versus controls included bioactive molecules with putative neuroprotective effects (eg, short chain fatty acids [SCFAs], ubiquinones, and salicylate) and other compounds increasingly implicated in neurodegeneration (eg, ceramides, sphingosine, and trimethylamine N‐oxide). In the PD group, cognitive impairment, low body mass index (BMI), frailty, constipation, and low physical activity were associated with fecal metabolome compositional differences. Notably, low SCFAs in PD were significantly associated with poorer cognition and low BMI. Lower butyrate levels correlated with worse postural instability–gait disorder scores. Interpretation Gut microbial function is altered in PD, characterized by differentially abundant metabolic features that provide important biological insights into gut–brain pathophysiology. Their clinical relevance further supports a role for microbial metabolites as potential targets for the development of new biomarkers and therapies in PD. ANN NEUROL 2021;89:546–559

Clinacanthus nutans has had a long history of use in folk medicine in Malaysia and Southeast Asia; mostly in the relief of inflammatory conditions. In this study, we investigated the effects of different extracts of C. nutans upon lipopolysaccharide (LPS) induced inflammation in order to identify its mechanism of action. Extracts of leaves and stem bark of C. nutans were prepared using polar and non-polar solvents to produce four extracts, namely polar leaf extract (LP), non-polar leaf extract (LN), polar stem extract (SP), and non-polar stem extracts (SN). The extracts were standardized by determining its total phenolic and total flavonoid contents. Its anti-inflammatory effects were assessed on LPS induced nitrite release in RAW264.7 macrophages and Toll-like receptor (TLR-4) activation in TLR-4 transfected human embryonic kidney cells (HEK-BlueTM-hTLR4 cells). The levels of inflammatory cytokines (TNF-α, IFN-γ, IL-1β, IL-6, IL-12p40, and IL-17) in treated RAW264.7 macrophages were quantified to verify its anti-inflammatory effects. Western blotting was used to investigate the effect of the most potent extract (LP) on TLR-4 related inflammatory proteins (p65, p38, ERK, JNK, IRF3) in RAW264.7 macrophages. All four extracts produced a significant, concentration-dependent reduction in LPS-stimulated nitric oxide, LPS-induced TLR-4 activation in HEK-BlueTM-hTLR4 cells and LPS-stimulated cytokines production in RAW264.7 macrophages. The most potent extract, LP, also inhibited all LPS-induced TLR-4 inflammatory proteins. These results provide a basis for understanding the mechanisms underlying the previously demonstrated anti-inflammatory activity of C. nutans extracts.

Understanding the basal gut bacterial community structure and the host metabolic composition is pivotal for the interpretation of laboratory treatments designed to answer questions pertinent to host-microbe interactions. In this study, we report for the first time the underlying gut microbiota and systemic metabolic composition in BALB/c mice during the acclimatisation period. Our results showed that stress levels were reduced in the first three days of the study when the animals were subjected to repetitive handling daily but the stress levels were increased when handling was carried out at lower frequencies (weekly). We also observed a strong influence of stress on the host metabolism and commensal compositional variability. In addition, temporal biological compartmental variations in the responses were observed. Based on these results, we suggest that consistency in the frequency and duration of laboratory handling is crucial in murine models to minimise the impact of stress levels on the commensal and host metabolism dynamics. Furthermore, caution is advised in consideration of the temporal delay effect when integrating metagenomics and metabonomics data across different biological matrices (i.e. faeces and urine).

Heat shock proteins (Hsps) 60 and 70 are postulated as a potential drug target for toxoplasmosis due to its importance in the developmental and survival of Toxoplasma gondii (T. gondii). As of today, there have been no reports on three-dimensional (3D) structure of Hsp60 and Hsp70 deposited in the Brookhaven Protein Data Bank. Hence, this study was conducted to predict 3D structures for Hsp60 and Hsp70 in T. gondii by homology modeling. Selection of the best predicted model was done based on multiple scoring functions. In addition, virtual screening was performed to short-list chemical compounds from the National Cancer Institute (NCI) Diversity Set III in search of potential inhibitor against Hsp60 and Hsp70 in T. gondii. Prior to virtual screening, binding sites of Hsp60 and Hsp70 were predicted using various servers and were used as the center in docking studies. The Hsps were docked against known natural ligands to validate the method used in estimating free energy of binding (FEB) and possible interactions between ligand and protein. Virtual screening was performed with a total of 1560 compounds from the NCI Diversity Set III. The compounds were ranked subsequently according to their FEB. Molecular basis of interactions of the top five ranked compounds was investigated using Ligplot. The major interactions exhibited were hydrogen bonding and hydrophobic interactions in binding to Hsp60 and Hsp70. The results obtained provided information and guidelines for the development of inhibitors for Hsp60 and Hsp70 in T. gondii.

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SUMMARYToxoplasmosis affects a third of the global population and is of particular concern for immunologically compromised individuals. Toxoplasmosis induces host physiological events ranging from immunological to metabolic responses across multiple biological compartments. To understand the sequence of host responses during acute and chronicToxoplasma gondiiinfection, eight male BALB/c mice were infected with 2000T. gondiiME49 tachyzoites with a further eight uninfected mice used as controls. Plasma cytokines status, urinary metabolic profiling and fecal microbial profiles were characterized to monitor temporal variation related toT. gondiiinfection. The results showed elevated serum interferon-γ(IFN-γ), interleukin-12p40 and  necrosis factor-αduring acute phase of infection with concomitant perturbation in host energy metabolism and host-gut microbiome co-metabolism of phenolics and a shift in microbial composition. However, the differences were less pronounced during the putative chronic phase of infection with elevated IFN-γ, differences in urinaryN-acetyls andO-acetyls of glycoproteins with no shift in gut microbial composition. Structural equation modelling on the current data showed host immune responses as the main driver for changes observed in urinary metabolites and gut microbial composition. Such an approach can be applied to other models of infectious diseases to aid understanding of host–pathogen interactions and potential biomarker discovery.