Biology subjects

Richardson, C.

Publications and source records attributed to Richardson, C..

16 recordsLinked to original sources

bioRxiv2026

Heterogeneous causes of acute respiratory distress syndrome correlate with distinct peripheral polyunsaturated fatty acid metabolites

Acute Respiratory Distress Syndrome (ARDS), a heterogeneous syndrome of hypoxic respiratory failure secondary to dysregulated pulmonary inflammation, is caused by diverse insults. Because of this heterogeneity, mechanisms and treatments are difficult to study. As a treatment, n-3 polyunsaturated fatty acid (PUFA) supplementation has had mixed results. PUFAs and downstream oxylipins are important to pulmonary inflammation but are not well defined in ARDS. We hypothesized that differences in fatty acid metabolism, as measured by levels of n-3 and n-6 PUFAs and oxylipins, are associated with differences in ARDS outcomes, ARDS causes, and inflammation. To test this, PUFAs/oxylipins were measured by LC MS/MS in plasma samples from 90 patients with ARDS. Inflammatory cytokines (IL-6, IL-8) were measured by ELISA. Multivariate linear regressions modeled the relationship between PUFAs/oxylipins, inflammation, and ARDS mortality, severity and cause. Multiple n-3 and n-6 PUFA derived oxylipins were decreased in severe ARDS. We did not detect differences in PUFAs/oxylipins by mortality. PUFAs/oxylipins varied by cause of ARDS, especially between patients with sepsis and those with trauma. Furthermore, specific oxylipins were associated with IL-6 and IL-8. Based on our identification of oxylipins that vary by disease severity and injury, these metabolites should be considered as potential biomarkers and mechanisms of lung repair biomarkers. Furthermore, differences in PUFAs did not directly correlate with changes in oxylipins, suggesting differences in lipid metabolism by etiology of injury. Further consideration of differences in lipid metabolism in ARDS could identify potential subgroups that could benefit from n-3 PUFA supplementation or other therapies.

molecular biology↗

bioRxiv2025

SFPQ Promotes Homologous Recombination via mRNA Stabilization of RAD51 and Its Paralogs

Double-strand break (DSB) repair occurs through non-homologous end joining (NHEJ) or homologous recombination (HR). To identify non-canonical factors that influence DSB repair outcomes, we parsed data from pooled genetic screens. Through this approach, we identified the splicing factor SFPQ, which has been previously reported to associate with DSBs and promote repair. Here, we show that SFPQ depletion alters DSB repair via HR. However, in contrast to other published work, we find that SFPQ does not localize to DSBs but instead stabilizes the expression of RAD51 and its paralogs independently of p53 activation or DNA damage. Our findings suggest that SFPQ contributes to constitutive DSB repair by maintaining RAD51 paralog mRNA stability rather than through direct interaction with DSBs or RAD51 protein and highlight indirect mechanisms by which RNA-binding proteins can influence genome stability.

molecular biology↗

bioRxiv2025

Massively parallel assessment of designed protein solution properties using mass spectrometry and peptide barcoding

Library screening and selection methods can determine the binding activities of individual members of large protein libraries given a physical link between protein and nucleotide sequence, which enables identification of functional molecules by DNA sequencing. However, the solution properties of individual protein molecules cannot be probed using such approaches because they are completely altered by DNA attachment. Mass spectrometry enables parallel evaluation of protein properties amenable to physical fractionation such as solubility and oligomeric state, but current approaches are limited to libraries of 1,000 or fewer proteins. Here, we improved mass spectrometry barcoding by co-synthesizing proteins with barcodes optimized to be highly multiplexable and minimally perturbative, scaling to libraries of >5,000 proteins. We use these barcodes together with mass spectrometry to assay the solution behavior of libraries of de novo-designed monomeric scaffolds, oligomers, binding proteins and nanocages, rapidly identifying design failure modes and successes.

biochemistry↗

bioRxiv2024

Wisteria floribunda agglutinin enhances Zaire ebolavirus entry through interactions at specific N-linked glycosylation sites on the virus glycoprotein complex

Entry of Zaire ebolavirus (EBOV) into a host cell is a complex process requiring interactions between the viral glycoproteins (GP) and cellular factors. These entry factors are cell-specific and can include cell surface lectins and phosphatidylserine receptors. NPC1 is critical to the late stage of the entry process. Entry has been demonstrated to be enhanced by interactions between the virion and surface-expressed lectins, which interact with carbohydrate moieties attached to the GP. In addition, soluble lectins, including mannose binding lectin (MBL), can enhance entry in vitro. However, the mechanism of lectin-mediated enhancement remains to be defined. This study investigated the potential of three plant lectins, Wisteria floribunda agglutinin (WFA), soybean agglutinin (SBA) and Galanthus nivalis agglutinin (GNA), which possess different carbohydrate binding specificities, to enhance EBOV entry by binding to the GP. WFA was observed to potently enhance entry of lentiviral pseudotype viruses (PVs) expressing the GP of three Ebolavirus species (Zaire, Sudan [SUDV] and Reston [RESTV]), with the greatest impact on EBOV. SBA had a modest enhancing effect on entry that was specific to EBOV, while GNA had no impact on entry of any of the Ebolavirus species. None of the lectins enhanced entry of control PVs expressing the surface proteins of other RNA viruses tested. WFA was demonstrated to bind directly with the EBOV-GP via the glycans, and mutational analysis implicated N238 as contributing to the interaction. Furthermore, enhancement was observed in both human and bat cell lines indicating a highly conserved mechanism of action. We conclude that binding of WFA to EBOV GP through interactions including the glycan at N238 results in GP alterations that enhance entry, providing evidence of a mechanism for lectin-mediated virus entry enhancement. Targeting lectin-ligand interactions presents a potential strategy for restricting Ebolavirus entry.

microbiology↗

bioRxiv2024

Flight-Scope: microscopy with microfluidics in microgravity

With the European Space Agency (ESA) and NASA working to return humans to the moon and onwards to Mars, it has never been more important to study the impact of altered gravity conditions on biological organisms. These include astronauts but also useful micro-organisms they may bring with them to produce food, medicine, and other useful compounds by synthetic biology. Parabolic flights are one of the most accessible microgravity research platforms but present their own challenges: relatively short periods of altered gravity ([~]20s) and aircraft vibration. Live-imaging is necessary in these altered-gravity conditions to readout any real-time phenotypes. Here we present Flight-Scope, a new microscopy and microfluidics platform to study dynamic cellular processes during the short, altered gravity periods on parabolic flights. We demonstrated Flight-Scopes capability by performing live and dynamic imaging of fluorescent glucose uptake by yeast, S. cerevisiae, on board an ESA parabolic flight. Flight-Scope operated well in this challenging environment, opening the way for future microgravity experiments on biological organisms.

biophysics↗

bioRxiv2024

Phased ERK-responsiveness and developmental robustness regulate teleost skin morphogenesis

Elongation of the vertebrate embryonic axis necessitates rapid expansion of the epidermis to accommodate the growth of underlying tissues. Here, we generated a toolkit to visualize and quantify signaling in entire cell populations of periderm, the outermost layer of the epidermis, in live developing zebrafish. We find that oriented cell divisions facilitate growth of the early periderm during axial elongation rather than cell addition from the basal layer. Activity levels of ERK, a downstream effector of MAPK pathway, gauged by a live biosensor, predicts cell cycle entry, and optogenetic ERK activation controls proliferation dynamics. As development proceeds, rates of peridermal cell proliferation decrease, ERK activity becomes more pulsatile and functionally transitions to promote hypertrophic cell growth. Targeted genetic blockade of cell division generates animals with oversized periderm cells, yet, unexpectedly, development to adulthood is not impaired. Our findings reveal stage-dependent differential responsiveness to ERK signaling and marked developmental robustness in growing teleost skin.

developmental biology↗

bioRxiv2023

Engineering water exchange is a safe and effective method for magnetic resonance imaging in diverse cell types

Aquaporin-1 (Aqp1), a water channel, has garnered significant interest for cell-based medicine and in vivo synthetic biology due to its ability to be genetically encoded to produce magnetic resonance signals by increasing the rate of water diffusion in cells. However, concerns regarding the effects of Aqp1 overexpression and increased membrane diffusivity on cell physiology have limited its widespread use as a deep-tissue reporter. In this study, we present evidence that Aqp1 generates strong diffusion-based magnetic resonance signals without adversely affecting cell viability or morphology in diverse cell lines derived from mice and humans. Our findings indicate that Aqp1 overexpression does not induce ER stress, which is frequently associated with heterologous expression of membrane proteins. Furthermore, we observed that Aqp1 expression had no detrimental effects on native biological activities, such as phagocytosis, immune response, insulin secretion, and tumor cell migration in the analyzed cell lines. These findings should serve to alleviate any lingering safety concerns regarding the utilization of Aqp1 as a genetic reporter and should foster its broader application as a noninvasive reporter for in vivo studies.

bioengineering↗

bioRxiv2023

Effects of aluminum-salt, CpG and emulsion adjuvants on the stability and immunogenicity of a virus-like particle displaying the SARS-CoV-2 receptor binding domain (RBD)

Second-generation COVID-19 vaccines with improved immunogenicity (e.g., breadth, duration) and availability (e.g., lower costs, refrigerator stable) are needed to enhance global coverage. In this work, we formulated a clinical-stage SARS-CoV-2 receptor binding domain (RBD) virus-like particle (VLP) vaccine candidate (IVX-411) with widely available adjuvants. Specifically, we assessed the in vitro storage stability and in vivo mouse immunogenicity of IVX-411 formulated with aluminum-salt adjuvants (Alhydrogel, AH and Adjuphos, AP), without or with the TLR-9 agonist CpG-1018 (CpG), and compared these profiles to IVX-411 adjuvanted with an oil-in-water nano-emulsion (AddaVax, AV). Although IVX-411 bound both AH and AP, lower binding strength of antigen to AP was observed by Langmuir binding isotherms. Interestingly, AH- and AP-adsorbed IVX-411 had similar storage stability profiles as measured by antigen binding assays (competitive ELISAs), but the latter displayed higher pseudovirus neutralizing titers (pNT) in mice, at levels comparable to titers elicited by AV-adjuvanted IVX-411. CpG addition to alum (AP or AH) resulted in a marginal trend of improved pNTs in stressed samples only, yet did not impact the storage stability profiles of IVX-411. In contrast, previous work with AH-formulations of a monomeric RBD antigen showed greatly improved immunogenicity and decreased stability upon CpG addition to alum. At elevated temperatures (25, 37{degrees}C), IVX-411 formulated with AH or AP displayed decreased in vitro stability compared to AV-formulated IVX-411and this rank-ordering correlated with in vivo performance (mouse pNT values). This case study highlights the importance of optimizing antigen-adjuvant interactions to develop low cost, aluminum-salt adjuvanted recombinant subunit vaccine candidates.

immunology↗

bioRxiv2023

Synthetic STING agonists elicit powerful vaccine adjuvancy providing robust central memory and anti-tumour effects

Drugs that target the innate immune sensor STING are known to be effective in modulating the immune infiltrate of the tumour microenvironment. STING agonists have potential to enhance responses to checkpoint inhibitor therapy, however, their ability to influence and shape adaptive immune responses is poorly understood. Here, we investigated the impact of a range of synthetic STING agonists on antigen specific CD8+ T-cell responses to soluble antigen using the murine OT-1 adoptive transfer model with Ovalbumin as the antigen to monitor T cell responses. Our data demonstrate that synthetic STING agonists are able to stimulate antigen specific T-cell expansion in response to challenge in mice. This effect required expression of STING, an intact myeloid compartment and Type-I IFN and TNF signalling. Expanded T-cells post treatment differed from those induced by the established immune adjuvant, anti-CD40 antibody through lower induction of the immune checkpoint receptor PD-1. Furthermore, our data revealed a marked increase in the induction and persistence of CD8+ central memory cells after STING agonist and antigen challenge. Finally, we demonstrate that following rechallenge, STING agonism produced larger secondary responses that could be translated into enhanced tumour protection and survival. Therefore, synthetic STING agonists are capable of acting as potent immune adjuvants and can induce robust memory formation leading to better recall and tumour control. Critically, these benefits along with the lower expression of PD-1, have implications for their use as adjuvants for multiple immunotherapy and vaccine applications.

immunology↗

bioRxiv2021

A NET4-RabG3 couple mediate the link between actin and the tonoplast and is essential for normal actin cytoskeletal remodelling in stomatal closure to flg22

Members of the NETWORKED (NET) family are involved in actin-membrane interactions. They tether the cells plasma membrane (PM) to the actin network. Moreover, in a similar manner, they are also involved in the tethering of membrane bound organelles to the actin cytoskeleton; the endoplasmic reticulum (ER) and the ER to the PM. This raises the question as to whether NET proteins are involved in actin cytoskeletal remodelling. Here we show that two members of the NET family, NET4A and NET4B, are essential for normal guard cell actin reorganization, which is a process critical for stomatal closure in plant immunity. NET4 proteins interact with F-actin and with members of the Rab7 GTPase RABG3 family through two distinct domains, allowing for simultaneous localization to actin filaments and the tonoplast. NET4 proteins interact with GTP-bound, active RABG3 members, suggesting their function as downstream effectors. We also show that RABG3b is critical for stomatal closure induced by microbial patterns. Taken together, we conclude that the actin cytoskeletal remodelling during stomatal closure depends on a molecular link between actin filaments and the tonoplast, which is mediated by the NET4-RABG3b interaction. We propose that stomatal closure to microbial patterns involves the coordinated action of immune signalling events and proper actin cytoskeletal remodelling.

plant biology↗

bioRxiv2021

Cascading indirect genetic effects in a clonal vertebrate

Understanding how individual differences among organisms arise and how their effects propagate through groups of interacting individuals are fundamental questions in biology.Individual differences can arise from genetically-based variation in the conspecifics with which an individual interacts, and these effects might then be propagated to other individuals. Using a clonal species, the Amazon molly (Poecilia formosa), we test the hypothesis that such indirect genetic effects (IGE) propagate beyond individuals that experience them firsthand. We tested this hypothesis by exposing genetically identical Amazon mollies to social partners of different genotypes, and then moving these individuals to new social groups in which they were the only member to have experienced the IGE. We found that genetically different social environments induced different levels of aggression experienced by the focal animals, and that these genetically-based social effects carried over into new social groups to influence the behavior of individuals that did not directly experience the previous social environments. Our data reveal that IGE can cascade beyond the individuals that directly experience them to influence phenotypes even when there is no genetically-based variation present within interacting groups. Theoretical and empirical expansion of the quantitative genetic framework developed for IGE to include cascading and other types of carry-over effects will improve understanding of social behavior and its evolution.

animal behavior and cognition↗

bioRxiv2021

Male sex and age biases viral burden, viral shedding, and type 1 and 2 interferon responses during SARS-CoV-2 infection in ferrets

SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) hospitalizations and deaths disportionally affect males and the elderly. Here we investigated the impact of male sex and age by infecting adult male, aged male, and adult female ferrets with SARS-CoV-2. Aged male ferrets had a decrease in temperature which was accompanied by prolonged viral replication with increased pathology in the upper respiratory tract after infection. Transcriptome analysis of the nasal turbinates and lungs indicated that female ferrets had significant increases in interferon response genes (OASL, MX1, ISG15, etc.) on day 2 post infection which was delayed in aged males. In addition, genes associated with taste and smell such as RTP1, CHGA, and CHGA1 at later time points were upregulated in males but not in females. These results provide insight into COVID-19 and suggests that older males may play a role in viral transmission due to decreased antiviral responses.

microbiology↗

bioRxiv2020

STING Nuclear Partners Contribute to Innate Immune Signalling Responses

STING and cGAS initiate innate immune responses (IIR) by recognizing cytoplasmic pathogen dsDNA and activating signaling cascades from the ER; however, another less investigated pool of STING resides in the nuclear envelope. We find that STING in the inner nuclear membrane increases mobility and changes localization upon IIR activation both from dsDNA and poly(I:C) stimuli. We next identified nuclear partners of STING from isolated nuclear envelopes. These include several known nuclear membrane proteins, bromodomain and epigenetic enzymes, and RNA- or DNA-binding proteins. Strikingly, 17 of these DNA and RNA-binding STING partners are known to bind direct partners of the IRF3/7 transcription factors that are central drivers of IIR. We find that several of these STING partners --SYNCRIP, Men1, Ddx5, snRNP70, RPS27a, Aatf-- can contribute to IIR activation and SYNCRIP can moreover protect against influenza A virus infection. These data suggest that the many roles identified for STING likely reflect its interactions with multiple RNA and DNA-binding proteins that also function in IIR.

immunology↗

bioRxiv2018

Resolving Cytosolic Diffusive States in Bacteria by Single-Molecule Tracking

The trajectory of a single protein in the cytosol of a living cell contains information about its molecular interactions in its native environment. However, it has remained challenging to accurately resolve and characterize the diffusive states that can manifest in the cytosol using analytical approaches based on simplifying assumptions. Here, we show that multiple intracellular diffusive states can be successfully resolved if sufficient single-molecule trajectory information is available to generate well-sampled distributions of experimental measurements and if experimental biases are taken into account during data analysis. To address the inherent experimental biases in camera-based and MINFLUX-based single-molecule tracking, we use an empirical data analysis framework based on Monte Carlo simulations of confined Brownian motion. This framework is general and adaptable to arbitrary cell geometries and data acquisition parameters employed in 2D or 3D single-molecule tracking. We show that, in addition to determining the diffusion coefficients and populations of prevalent diffusive states, the timescales of diffusive state switching can be determined by stepwise increasing the time window of averaging over subsequent single-molecule displacements. Time-averaged diffusion (TAD) analysis of single-molecule tracking data may thus provide quantitative insights into binding and unbinding reactions among rapidly diffusing molecules that are integral for cellular functions.

biophysics↗

bioRxiv2017

C11orf70 mutations causing primary ciliary dyskinesia disrupt a conserved step in the intraflagellar transport-dependent assembly of multiple axonemal dyneins

Primary ciliary dyskinesia (PCD) is a genetically and phenotypically heterogeneous disorder characterized by destructive respiratory disease and laterality abnormalities due to randomised left-right body asymmetry. PCD is mostly caused by mutations affecting components of the core axoneme structure of motile cilia that are essential for cilia movement. In addition, there is a growing group of PCD genes that encode proteins essential for the assembly of the ciliary dynein motors and the active transport process that delivers them from their cytoplasmic assembly site into the axoneme. We screened a cohort of affected individuals for disease-causing mutations using a targeted next generation sequencing panel and identified 2 unrelated families (3 affected children) with mutations in the uncharacterized C11orf70 gene. The affected children share a consistent PCD phenotype from early life with laterality defects and immotile respiratory cilia displaying combined loss of inner and outer dynein arms (IDA+ODA). Phylogenetic analysis shows C11orf70 is highly conserved, distributed across species similarly to proteins involved in the intraflagellar transport (IFT)-dependant assembly of axonemal dyneins. Paramecium C11orf70 RNAi knockdown led to combined loss of ciliary IDA+ODA with reduced cilia beating and swim velocity. Fluorescently tagged C11orf70 in Paramecium and Chlamydomonas localises mainly in the cytoplasm with a small amount in the ciliary component, its abundance in the axoneme being IFT-dependant. During ciliogenesis, C11orf70 accumulates at the ciliary tips in a similar distribution to the IFT-B protein IFT46. In summary, C11orf70 is essential for IFT-dependant assembly of dynein arms and C11orf70 mutations cause defective cilia motility and PCD.

genetics↗

bioRxiv2017

Single-molecule tracking in live Yersinia enterocolitica reveals distinct cytosolic complexes of injectisome subunits

In bacterial type 3 secretion, substrate proteins are actively transported from the bacterial cytoplasm into the host cell cytoplasm by a large membrane-embedded machinery called the injectisome. Injectisomes transport secretion substrates in response to specific environmental signals, but the molecular details by which the cytosolic secretion substrates are selected and transported through the type 3 secretion pathway remain unclear. Secretion activity and substrate selectivity are thought to be controlled by a sorting platform consisting of the proteins SctK, SctQ, SctL, and SctN, which together localize to the cytoplasmic side of membrane-embedded injectisomes. However, recent work revealed that sorting platform proteins additionally exhibit substantial cytosolic populations and that SctQ reversibly binds to and dissociates from the cytoplasmic side of membrane-embedded injectisomes. Based on these observations, we hypothesized that dynamic molecular turnover at the injectisome and cytosolic assembly among sorting platform proteins is a critical regulatory component of type 3 secretion. To determine whether sorting platform complexes exist in the cytosol, we measured the diffusive properties of the two central sorting platform proteins, SctQ and SctL, using live cell high-throughput 3D single-molecule tracking microscopy. Single-molecule trajectories, measured in wild-type and mutant Yersinia enterocolitica cells, reveal that both SctQ and SctL exist in several distinct diffusive states in the cytosol, indicating that these proteins form stable homo- and hetero-oligomeric complexes in their native environment. Our findings provide the first diffusive state-resolved insights into the dynamic regulatory network that interfaces stationary membrane-embedded injectisomes with the soluble cytosolic components of the type 3 secretion system.

biophysics↗