Biology subjects

Ryan, D.

Publications and source records attributed to Ryan, D..

14 recordsLinked to original sources

bioRxiv2026

Translating Innovation to Clinic: End-to-End Bioprocess Development and cGMP Manufacturing of N332-GT5 HIV Vaccine Candidate for First-in-Human Trials HVTN144

The successful translation of rationally designed HIV-1 immunogens into effective vaccines requires manufacturing platforms that maintain structural conformity while meeting clinical-grade quality standards. We developed and scaled a robust, cGMP-compliant process for N332-GT5 gp140, a germline-targeting envelope trimer designed to initiate broadly neutralizing antibody responses, which is now undergoing first-in-human evaluation in HVTN144. Starting with a stable CHO cell line developed using Leap-In(R) transposon technology, we established a production clone exhibiting high-titer expression (>200 mg/L) and genetic stability through 60 population doublings. The manufacturing process scaled efficiently from Ambr(R) 250 miniature bioreactors to 200-L single-use systems, delivering consistent product quality across multiple cGMP batches. A streamlined three-step purification strategy--affinity capture, multimodal polishing, and viral clearance- yielded >99% trimeric purity with preserved quaternary structure and native-like antigenicity. Orthogonal LC-MS analyses confirmed site-specific glycan occupancy matching design specifications, while robust viral clearance exceeded 18-log and 11-log reductions for model retroviruses. Clinical material manufactured through this platform has been successfully administered in HVTN144. This work establishes a scalable, reproducible manufacturing paradigm for structurally complex HIV-1 envelope immunogens, advancing the field toward rational vaccine design based on germline-targeting principles.

bioengineering↗

bioRxiv2026

Visual Cortical Response Variability in Infants at High Familial Likelihood for Autism

Visual processing undergoes rapid development in the first year of life, supporting the emergence of higher-order cognitive, language, and motor functions. Visual evoked potentials (VEPs) provide a non-invasive measure of visual system maturation that may shed light on heterogeneous developmental trajectories among infants at high familial likelihood for autism. Infants with an older sibling with autism spectrum disorder (N = 177 at 6 months; N = 132 at 12 months) participated in the Infant Brain Imaging Study-Early Prediction (IBIS-EP) study. Pattern-reversal VEPs were recorded at 6 and 12 months, and developmental skills were assessed at 24 months using the Bayley Scales of Infant and Toddler Development (Bayley-4). VEP components (P1 and N1) were characterized by their amplitude and latency, as well as trial-to-trial variability in these measures. Associations with 24-month cognitive, language, and motor scores were examined using general linear models controlling for age, site, sex, and trial count. Robust VEPs were observed at both time points, with age-appropriate morphology and expected developmental changes, including decreases in P1 latency and amplitude from 6 to 12 months. Greater trial-to-trial variability in P1 latency at both time points was associated with higher cognitive and language scores at 24 months. In contrast, conventional measures of mean P1 latency and amplitude were not associated with developmental outcomes. These findings suggest that temporal variability in early visual responses may index adaptive sensory-circuit flexibility during a period of rapid experience-dependent development. VEP response-timing variability may therefore provide an early mechanistic marker of sensory-circuit organization relevant to later developmental trajectories. Research HighlightsO_LIGreater trial-to-trial variability in visual cortical response timing was associated with higher cognitive and language scores in infants at high familial likelihood for autism. C_LIO_LIConventional average VEP measures were unrelated to outcomes, suggesting temporal variability may more sensitively capture relevant neural-circuit differences in this population. C_LIO_LIGreater variability in early visual responses may reflect adaptive sensory-circuit flexibility during a critical period of neurodevelopment. C_LIO_LIVEP response-timing measures provide an early mechanistic window into sensory-circuit organization and its relation to later developmental trajectories. C_LI

neuroscience↗

bioRxiv2026

Co-Designing Research Priorities in Developmental Neuroscience: A Community-Based Participatory Approach

Children and adolescents, the primary participants in developmental neuroscience research, are rarely consulted or included in the research design process. By neglecting their perspectives and needs, we risk limiting the relevance and effectiveness of translational research that may emerge from this work. Here, we report on a process of co-design with adolescents to inform the generation of a new population neuroimaging research programme. We employed a two-study community-based participatory approach and used mixed methods to identify the research priorities of this community. In the first study, 79 secondary school students from four schools were introduced to fundamental neuroscience concepts during five one-hour workshops. In groups, they then designed research studies, centred around topics they deemed important, and were asked to present their ideas through poster presentations, role playing as neuroscientists. Reflexive thematic analysis was conducted on these posters, extracting ten key themes. In the second study, 376 students from four different schools, ranked these ten themes in order of importance. Mental health and stress consistently emerged as the highest priorities. Further exploratory analysis revealed that the priority for exploring everyday routines and understanding anti-social behaviours varied across schools and age groups, while the importance of mental health varied significantly between genders. This work provides an illustration of how to implement place-based participatory approaches at the early stages of a developmental neuroscience research project to maximise inclusivity and potential downstream translation and impact.

neuroscience↗

bioRxiv2024

Capacity building needed to reap the benefits of access to biodiversity collections

SummaryO_LIThis research examines biodiversity specimens from two areas of the Caribbean to understand patterns of collection and the roles of the people involved. Using open data from the Global Biodiversity Information Facility (GBIF) and Wikidata, we aimed to uncover geographic and historical trends in specimen use. This study aims to provide concrete evidence to guide collaboration between collection-holding institutions and the communities that need their resources most. C_LIO_LIWe analysed biodiversity specimens from Montserrat and the Cayman Islands in three steps. First, we extracted specimen data from GBIF, disambiguated collector names, and linked them to unique biographical entries. Next, we connected collectors to their publications and specimens. Finally, we analysed the modern use of these specimens through citation data, mapping author affiliations and research themes. C_LIO_LISpecimens are predominantly housed in the Global North and were initially used by their collectors, whose focus was largely on taxonomy and biogeography. With digitisation, use of these collections remains concentrated in the Global North and covers a broader range of subjects, although Brazil and China stand out as significant users of digital collection data compared to other similar countries. C_LIO_LIThe availability of open digital data from collections in the Global North has led to a substantial increase in the reuse of these data across biodiversity science. Nonetheless, most research using these data is still conducted in the Global North. For the non-monetary benefits of digitisation to extend to the countries of origin, capacity building in the Global South is crucial, Open Data alone are insufficient. C_LI Societal Impact StatementDigital biodiversity data from herbaria and museums hold significant potential for nature conservation in the Global South, yet many regions, like Montserrat and the Cayman Islands in the Caribbean, are, for multiple reasons, unable to fully leverage this information. This lack of skills and resources limits local conservation efforts, showing the need for more investment in training, facilities, and expertise. Although past funding has helped improve coordination and build skills, our findings show that more work is needed to make sure conservation in these biodiverse areas can continue in the long term.

scientific communication and education↗

bioRxiv2023

The RNA-binding protein RbpB is a central regulator of polysaccharide utilization in gut Bacteroides

Paramount to human health, symbiotic bacteria in the gastrointestinal tract rely on the breakdown of complex polysaccharides to thrive in this sugar-deprived environment. Gut Bacteroides are metabolic generalists and deploy dozens of polysaccharide utilization loci (PULs) to forage diverse dietary and host-derived glycans. The expression of the multi-protein PUL complexes is tightly regulated at the transcriptional level. However, how PULs are orchestrated at translational level in response to the fluctuating levels of their cognate substrates is unknown. Here, we identify the RNA-binding protein RbpB and a family of noncoding RNAs as key players in post-transcriptional PUL regulation. Ablation of RbpB in Bacteroides thetaiotaomicron displays compromised colonization in the mouse gut in a host diet-dependent manner. Current dogma holds that individual PULs are regulated by dedicated transcriptional regulators. We demonstrate that RbpB acts as a global RNA binder that directly interacts with several hundred cellular transcripts. This includes a paralogous noncoding RNA family comprised of 14 members, the FopS (family of paralogous sRNAs) cluster. Through a series of in-vitro and in-vivo assays, we reveal that FopS sRNAs repress the translation of a SusC-like glycan transporter when substrates are limited--an effect antagonized by RbpB. Together, this study implicates RNA-coordinated metabolic control as an important, yet previously overlooked, factor contributing to the in-vivo fitness of predominant microbiota species in dynamic nutrient landscapes.

microbiology↗

bioRxiv2023

Global analysis of the RNA-RNA interactome in Acinetobacter baumannii AB5075 uncovers a small regulatory RNA repressing the virulence-related outer membrane protein CarO

Acinetobacter baumannii is an opportunistic Gram-negative pathogen that infects critically ill patients. The emergence of antimicrobial resistant A. baumannii has exacerbated the need to functionally characterise environmental adaptation, antibiotic resistance and pathogenicity of this organism and their genetic regulators to inform intervention strategies. Critical to rapid adaptation to changing environments in bacteria are small regulatory RNAs (sRNAs), however, the role that sRNAs play in the biology of A. baumannii is poorly understood. To assess the regulatory function of sRNAs and to uncover their RNA interaction partners in A. baumannii, we employed an RNA proximity ligation and sequencing method (Hi-GRIL-seq) in three different environmental conditions. We found that 40 sRNA candidates were ligated to sRNA-RNA chimeric sequencing reads, suggesting that sRNA-mediated gene regulation is pervasive in A. baumannii and that sRNAs act as direct regulators of mRNA molecules through antisense base-pairing. In-depth characterisation uncovered the sRNA Aar to be a post-transcriptional regulator of four mRNA targets including that of the outer membrane protein CarO and the siderophore receptor BfnH. We show that Aar initiates base-pairing with these mRNA molecules using a conserved seed region of nine nucleotides, sequestering the ribosome binding sites and inhibiting translation. Aar is differentially expressed in response to multiple stress stimuli suggesting a role in fine-tuning translation of the Aar-target molecules in A. baumannii under hostile conditions. Together, our study provides mechanistic insights into sRNA-mediated gene expression control in A. baumannii and represents a valuable resource for future RNA-centric research endeavours in this ESKAPE pathogen.

microbiology↗

bioRxiv2023

The Drosophila cardiogenic transcription factors Myocyte enhancer factor-2 and Tinman contribute to heart lumen enlargement through direct activation of the collagen gene Multiplexin.

During embryogenesis, the Drosophila heart forms a lumen, the posterior region of which is increased in diameter and corresponds to the heart proper. To identify the transcriptional control of this morphogenetic process, we analyzed the formation and enlargement of the heart lumen in mutants for the myogenic transcription factor gene Myocyte enhancer factor-2 (Mef2). We found that Mef2 contributes to both lumen formation and lumen expansion, the latter through a requirement for both Mef2 and the cardiogenic gene tinman (tin) to activate the collagen gene Multiplexin (Mp). To determine if Tin and MEF2 act directly upon the Mp gene, we identified an enhancer whose activity recapitulates the cardiac expression of Mp. This enhancer contains binding sites for both Tin and MEF2 and is activated in tissue culture by MEF2 but not Tin. We did not observe synergistic activation of the enhancer when the factors were in combination, despite documenting a direct physical interaction between Tin and MEF2 in vitro. In vivo, the Tin sites are required for normal enhancer activity, whereas mutation of the MEF2 sites results in expanded expression of an enhancer-lacZ reporter, suggesting that transcriptional repression may also contribute to regulation of Mp. Our studies underline how transcription factors must utilize combinatorial interactions to achieve organ-specific and region-specific patterns of gene expression and cell morphogenesis.

developmental biology↗

bioRxiv2023

An integrated transcriptomics-functional genomics approach reveals a small RNA that modulates Bacteroides thetaiotaomicron sensitivity to tetracyclines

Gene expression plasticity allows bacteria to adapt to diverse environments, tie their metabolism to available nutrients, and cope with stress. This is particularly relevant in a niche as dynamic and hostile as the human intestinal tract, yet transcriptional networks remain largely unknown in gut Bacteroides spp. Here, we map transcriptional units and profile their expression levels in Bacteroides thetaiotaomicron over a suite of 15 defined experimental conditions that are relevant in vivo, such as variation of temperature, pH, and oxygen tension, exposure to antibiotic stress, and growth on simple carbohydrates or on host mucin-derived glycans. Thereby, we infer stress- and carbon source-specific transcriptional regulons, including conditional expression of capsular polysaccharides and polysaccharide utilization loci, and expand the annotation of small regulatory RNAs (sRNAs) in this organism. Integrating this comprehensive expression atlas with transposon mutant fitness data, we identify conditionally important sRNAs. One example is MasB, whose inactivation led to increased bacterial tolerance of tetracyclines. Using MS2 affinity purification coupled with RNA sequencing, we predict targets of this sRNA and discuss their potential role in the context of the MasB-associated phenotype. Together, this transcriptomic compendium in combination with functional sRNA genomics--publicly available through a new iteration of the Theta-Base web browser (www.helmholtz-hiri.de/en/datasets/bacteroides-v2)--constitutes a valuable resource for the microbiome and sRNA research communities alike.

molecular biology↗

bioRxiv2021

Foxd3 controls heterochromatin-mediated silencing of repeat elements in mouse embryonic stem cells and represses the 2-cell transcription program

Repeat element transcription plays a vital role in early embryonic development. Expression of repeats such as MERVL characterises mouse embryos at the 2-cell stage, and defines a 2-cell-like cell (2CLC) population in a mouse embryonic stem cell culture. Repeat element sequences contain binding sites for numerous transcription factors. We identify the forkhead domain transcription factor FOXD3 as a regulator of repeat element transcription in mouse embryonic stem cells. FOXD3 binds to and recruits the histone methyltransferase SUV39H1 to MERVL and major satellite repeats, consequentially repressing the transcription of these repeats by the establishment of the H3K9me3 heterochromatin modification. Notably, depletion of FOXD3 leads to the de-repression of MERVL and major satellite repeats as well as a subset of genes expressed in the 2-cell state, shifting the balance between the stem cell and 2-cell like population in culture. Thus, FOXD3 acts as a negative regulator of repeat transcription, ascribing a novel function to this transcription factor.

developmental biology↗

bioRxiv2019

CHD4 slides nucleosomes by decoupling entry- and exit-side DNA translocation

Chromatin remodellers hydrolyse ATP to move nucleosomal DNA against histone octamers. The mechanism, however, is only partially resolved, and unclear if it is conserved among the four remodeller families. Here we use single-molecule assays to examine the mechanism of action of CHD4, which is part of the least well understood family of remodellers. We demonstrate that the binding energy for CHD4-nucleosome complex formation - even in the absence of nucleotide - triggers significant conformational changes in DNA at the entry side, effectively priming the system for remodelling. During remodelling, flanking DNA enters the nucleosome in a continuous, gradual manner but exits in concerted 4-6 base-pair steps. This decoupling of entry- and exit-side translocation suggests that ATP-driven movement of entry-side DNA builds up strain inside the nucleosome that is subsequently released at the exit side by DNA expulsion. We propose a mechanism for nucleosome sliding based on these and published data.

biochemistry↗

bioRxiv2017

Community-driven data analysis training for biology

The primary problem with the explosion of biomedical datasets is not the data itself, not computational resources, and not the required storage space, but the general lack of trained and skilled researchers to manipulate and analyze these data. Eliminating this problem requires development of comprehensive educational resources. Here we present a community-driven framework that enables modern, interactive teaching of data analytics in life sciences and facilitates the development of training materials. The key feature of our system is that it is not a static but a continuously improved collection of tutorials. By coupling tutorials with a web-based analysis framework, biomedical researchers can learn by performing computation themselves through a web-browser without the need to install software or search for example datasets. Our ultimate goal is to expand the breadth of training materials to include fundamental statistical and data science topics and to precipitate a complete re-engineering of undergraduate and graduate curricula in life sciences.

bioinformatics↗

bioRxiv2017

Pair Matcher (PaM): Fast Model-Based Optimisation Of Treatment/Case-Control Matches Using Demographic And Genetic Data

In clinical trials, individuals are matched for demographic criteria, paired, and then randomly assigned to treatment and control groups to determine a drugs efficacy. The successful completion of pilot trials is a prerequisite to larger and more expensive Phase III trials. One of the chief causes for the irreproducibility of results across pilot to Phase III trials is population stratification bias caused by the uneven distribution of ancestries in the treatment and control groups. Pair Matcher (PaM) addresses stratification bias by optimising pairing assignments a priori- and\\or posteriori to the trial using both genetic and demographic criteria. Using simulated and real datasets, we show that PaM identifies ideal and near-ideal pairs that are more genetically homogeneous than those identified based on racial criteria or Principal Component Analysis (PCA) alone. Homogenising the treatment (or case) and control groups can be expected to improve the accuracy and reproducibility of the study. PaMs ability to infer the ancestry of the participants further allows identifying subgroup of responders and developing a precision medicine approach to treatment. PaM is simple to execute, fast, and can be used for clinical trials and association studies. PaM is freely available via R scripts and a web interface.

bioinformatics↗

bioRxiv2017

Establishment In Culture Of Expanded Potential Stem Cells

Mouse embryonic stem cells are derived from in vitro explantation of blastocyst epiblasts1,2 and contribute to both the somatic lineage and germline when returned to the blastocyst3 but are normally excluded from the trophoblast lineage and primitive endoderm4-6. Here, we report that cultures of expanded potential stem cells (EPSCs) can be established from individual blastomeres, by direct conversion of mouse embryonic stem cells (ESCs) and by genetically reprogramming somatic cells. Remarkably, a single EPSC contributes to the embryo proper and placenta trophoblasts in chimeras. Critically, culturing EPSCs in a trophoblast stem cell (TSC) culture condition permits direct establishment of TSC lines without genetic modification. Molecular analyses including single cell RNA-seq reveal that EPSCs share cardinal pluripotency features with ESCs but have an enriched blastomere transcriptomic signature and a dynamic DNA methylome. These proof-of-concept results open up the possibility of establishing cultures of similar stem cells in other mammalian species.

developmental biology↗

bioRxiv2017

Targeted gene correction of FKRP by CRISPR/Cas9 restores functional glycosylation of α-dystroglycan in cortical neurons derived from human induced pluripotent stem cells

Mutations in genes required for functional glycosylation of -dystroglycan cause a group of congenital muscular dystrophies associated with brain malformations, referred to as dystroglycanopathies. The lack of isogenic, physiology-relevant human cellular models has limited our understanding of the cortical abnormalities in dystroglycanopathies. Here we generate induced pluripotent stem cells (iPSCs) from a severe dystroglycanopathy patient with homozygous mutations in the ribitol-5-phosphate transferase gene, FKRP. We carry out targeted gene correction in FKRP-iPSCs using CRISPR/Cas9-mediated genome editing. We characterise the directed differentiation of FKRP- and corrected-iPSCs to neural stem cells, cortical progenitors and cortical neurons. Importantly, we show that targeted gene correction of FKRP restores functional glycosylation of -dystroglycan in iPSC-derived cortical neurons. We independently validate this result by showing targeted gene mutation of FKRP disrupts functional glycosylation of -dystroglycan. This work demonstrates the feasibility of using CRISPR/Cas9-engineered human iPSCs for modelling dystroglycanopathies and provides a foundation for therapeutic development.\n\nHighlightsO_LIGeneration of FKRP-iPSCs for modelling cortical abnormalities in dystroglycanopathies\nC_LIO_LIPrecise gene correction by CRISPR/Cas9-mediated genome editing\nC_LIO_LIDirected differentiation of isogenic control and FKRP-iPSC to cortical neurons\nC_LIO_LIFunctional glycosylation of -dystroglycan is restored in cortical neurons derived from CRISPR/Cas9-corrected iPSCs\nC_LIO_LITargeted gene mutation of FKRP disrupts functional glycosylation of -dystroglycan in cortical neurons\nC_LI

molecular biology↗