Biology subjects

Reynolds, K.

Publications and source records attributed to Reynolds, K..

6 recordsLinked to original sources

bioRxiv2026

Hunting for Helminths: short- and long-read shotgun metagenomics for parasite detection in faecal samples

Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infections were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author SummarySoil-transmitted helminths (STH) such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STH in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in stool samples from humans infected with STH, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read Illumina and long-read Oxford Nanopore Technologies, but short-read outperformed long-read shotgun metagenomics. Contamination of bacteria sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.

molecular biology↗

bioRxiv2025

Where is the melody? Spontaneous attention orchestrates melody formation during polyphonic music listening

Humans seamlessly process multi-voice music into a coherent perceptual whole. Yet the neural strategies supporting this experience remain unclear. One fundamental component of this process is the formation of melody, a core structural element of music. Previous work on monophonic listening has provided strong evidence for the neurophysiological basis of melody processing, for example indicating predictive processing as a foundational mechanism underlying melody encoding. However, considerable uncertainty remains about how melodies are formed during polyphonic music listening, as existing theories (e.g., divided attention, figure-ground model, stream integration) fail to unify the full range of empirical findings. Here, we combined behavioural measures with non-invasive electroencephalography (EEG) to probe spontaneous attentional bias and melodic expectation while participants listened to two-voice classical excerpts. Our uninstructed listening paradigm eliminated a major experimental constraint, creating a more ecologically valid setting. We found that attention bias was significantly influenced by both the high-voice superiority effect and intrinsic melodic statistics. We then employed transformer-based models to generate next-note expectation profiles and test competing theories of polyphonic perception. Drawing on our findings, we propose a weighted-integration framework in which attentional bias calibrates the overall degree of integration of the competing streams. In doing so, the proposed framework reconciles previous divergent accounts by showing that, even under free-listening conditions, melodies emerge through an attention-guided statistical integration mechanism. HighlightsO_LIEEG can be used to decode spontaneous attention during the uninstructed listening of polyphonic music. C_LIO_LIBehavioural and neural data indicate that spontaneous attention is influenced by both high-voice superiority and melodic contour. C_LIO_LIAttention bias impacts the neural encoding of the polyphonic streams, with strongest effects within 200 ms after note onset. C_LIO_LIStimuli that produced a stronger attention bias aligned with monophonic-model expectations, whereas stimuli with a weaker bias aligned with the Stream-Integration model. C_LIO_LIWe propose a bi-directional influence between attention and prediction mechanisms, with horizontal statistics impacting attention (i.e., salience), and attention impacting melody extraction. C_LI

neuroscience↗

bioRxiv2024

Glutamatergic regulation of miRNA-containing exosome precursor trafficking and secretion from cortical neurons

Neuronal exosomes are emerging secreted signals that play important roles in the CNS. Currently little is known about how glutamatergic signaling affects the subcellular localization of exosome precursor intraluminal vesicles (ILVs), microRNA (miR) packaging into ILVs, and in vivo neuronal exosome spreading. By selectively labeling ILVs and exosomes with GFP-tagged human CD63 (hCD63-GFP) in cortical neurons, we found that glutamate stimulation significantly redistributes subcellular localization of hCD63-GFP+ ILVs especially decreases its co-localization with multi-vesicular body (MVB) marker Rab7 while substantially promoting exosome secretion. Interestingly, glutamate stimulation only modestly alters exosomal miR profiles based on small RNA sequencing. Subsequent in vivo cortical neuronal DREADD activation leads to significantly more widespread hCD63-GFP+ area in hCD63-GFPf/+ mice, consistently supporting the stimulatory effect of glutamatergic activation on neuronal exosome secretion and spreading. Moreover, in situ localization of hCD63-GFP+ ILVs and secreted exosomes from specialized Hb9+ and DAT+ neurons were also illustrated in the CNS.

neuroscience↗

bioRxiv2021

Altered interactions between circulating and tissue-resident CD8 T cells with the colonic mucosa define colitis associated with immune checkpoint inhibitors

Therapeutic blockade of co-inhibitory immune receptors PD-1 and CTLA-4 has revolutionized oncology, but treatments are limited by immune-related adverse events (IRAEs). IRAE Colitis (irColitis) is the most common, severe IRAE affecting up to 25% of patients on dual PD-1 and CTLA-4 inhibition. Here, we present a systems biology approach to define the cell populations and transcriptional programs driving irColitis. We collected paired colon mucosal biopsy and blood specimens from 13 patients with irColitis, 8 healthy individuals, and 8 controls on immune checkpoint inhibitors (ICIs), and analyzed them with single-cell/nuclei RNA sequencing with paired TCR and BCR sequencing, multispectral fluorescence microscopy, and secreted factor analysis (Luminex). We profiled 299,407 cells from tissue and blood and identified 105 cell subsets that revealed significant tissue remodeling in active disease. Colon mucosal immune populations were dominated by tissue-resident memory (TRM) ITGAE-expressing CD8 T cells representing a phenotypic spectrum defined by gene programs associated with T cell activation, cytotoxicity, cycling, and exhaustion. CD8 TRM and effector CD4 T cells upregulated type 17 immune programs (IL17A, IL26) and Tfh-like programs (CXCL13, PDCD1). We also identified for the first time an increased abundance of two KLRG1 and ITGB2-expressing CD8 T cell populations with circulatory cell markers, including a GZMK TRM-like population and a CX3CR1 population that is predicted to be intravascular. These two populations were more abundant in irColitis patients treated with dual PD-1/CTLA-4 inhibition than those receiving anti-PD-1 monotherapy. They also had significant TCR sharing with PBMCs, suggesting a circulatory origin. In irColitis we observed significant epithelial turnover marked by fewer LGR5-expressing stem cells, more transit amplifying cells, and upregulation of apoptotic and DNA-sensing programs such as the cGAS-STING pathway. Mature epithelial cells with top crypt genes upregulated interferon-stimulated pathways, CD274 (PD-L1), anti-microbial genes, and MHC-class II genes, and downregulated aquaporin and solute-carrier gene families, likely contributing to epithelial cell damage and absorptive dysfunction. Mesenchymal remodeling was defined by increased endothelial cells, both in irColitis patients and specifically in patients on dual PD-1/CTLA-4 blockade. Cell-cell communication analysis identified putative receptor-ligand pairs that recruit CD8 T cells from blood to inflamed endothelium and positive feedback loops such as the CXCR3 chemokine system that retain cells in tissue. This study highlights the cellular and molecular drivers underlying irColitis and provides new insights into the role of CTLA-4 and PD-1 signaling in maintaining CD8 TRM homeostasis, regulating CD8 T recruitment from blood, and promoting epithelial-immune crosstalk critical to gastrointestinal immune tolerance and intestinal barrier function.

immunology↗

bioRxiv2021

Single cell transcriptomics and developmental trajectories of murine cranial neural crest cell fate determination and cell cycle progression

Cranial neural crest (NC) cells migrate long distances to populate the future craniofacial regions and give rise to various tissues, including facial cartilage, bones, connective tissues, and cranial nerves. However, the mechanism that drives the fate determination of cranial NC cells remains unclear. Using single-cell RNA sequencing combined genetic fate mapping, we reconstructed developmental trajectories of cranial NC cells, and traced their differentiation in mouse embryos. We identified four major cranial NC cell lineages at different status: pre-epithelial-mesenchymal transition, early migration, NC-derived mesenchymal cells, and neural lineage cells from embryonic days 9.5 to 12.5. During migration, the first cell fate determination separates cranial sensory ganglia, the second generates mesenchymal progenitors, and the third separates other neural lineage cells. We then focused on the early facial prominences that appear to be built by undifferentiated, fast-dividing NC cells that possess similar transcriptomic landscapes, which could be the drive for the facial developmental robustness. The post-migratory cranial NC cells exit the cell cycle around embryonic day 11.5 after facial shaping is completed and initiates further fate determination and differentiation processes. Our results demonstrate the transcriptomic landscapes during dynamic cell fate determination and cell cycle progression of cranial NC lineage cells and also suggest that the transcriptomic regulation of the balance between proliferation and differentiation of the post-migratory cranial NC cells can be a key for building up unique facial structures in vertebrates.

developmental biology↗

bioRxiv2018

High-order epistasis in catalytic power of dihydrofolate reductase gives rise to a rugged fitness landscape in the presence of trimethoprim selection

Evolutionary fitness landscapes of certain antibiotic target enzymes have been comprehensively mapped showing strong high order epistasis between mutations, but understanding these effects at the biochemical and molecular levels remained open. Here, we carried out an extensive experimental and computational study to quantitatively understand the evolutionary dynamics of Escherichia coli dihydrofolate reductase (DHFR) enzyme in the presence of trimethoprim induced selection. Biochemical and structural characterization of resistance-conferring mutations targeting a total of ten residues spanning the substrate binding pocket of DHFR revealed distinct resistance mechanisms. Next, we experimentally measured biochemical parameters (Km, Ki, and kcat) for a mutant library carrying all possible combinations of six resistance-conferring DHFR mutations and quantified epistatic interactions between them. We found that the epistasis between DHFR mutations is high-order for catalytic power of DHFR (kcat and Km), but less prevalent for trimethoprim affinity (Ki). Taken together our data provide a concrete illustration of how epistatic coupling at the level of biochemical parameters can give rise to complex fitness landscapes, and suggest new strategies for developing mutant specific inhibitors.

evolutionary biology↗