Biology subjects

Wolf, C.

Publications and source records attributed to Wolf, C..

16 recordsLinked to original sources

bioRxiv2026

Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

Chemical modifications such as 2'-O-methyl (2'-OMe) are central to the efficacy and tolerability of RNA therapeutics. We recently identified that 2'-OMe RNA fragments as short as three nucleotides can exert opposing effects on Toll-like receptor 8 (TLR8) sensing in a motif-dependent manner. This discovery raises important considerations for degradation products of chemically modified RNA therapeutics, which may generate such immunologically active fragments. Here, leveraging their short length, we systematically map how base and sugar modifications within 3-mer oligonucleotides regulate TLR7 and TLR8 responses, and we resolve the structural basis for both TLR8 potentiation and TLR7/8 antagonism by RNA fragments. Building on these insights, we report the development of a dual TLR7/8 inhibitory oligonucleotide with therapeutic potential in autoimmune disease. Together, these findings provide unprecedented resolution of the immunomodulatory properties of oligonucleotide modifications on TLR7/8 and establish 3-mer oligonucleotides as the shortest functional class of RNA therapeutics described to date.

immunology↗

bioRxiv2025

Truncating RELA variants drive autoinflammation and autoimmunity by impairing the negative feedback control of NF-kB

The NF-{kappa}B signaling pathway coordinates inflammation, cell survival, and proliferation, while restraining excessive cell death to maintain immune homeostasis. Truncating mutations in RELA, encoding the NF-{kappa}B subunit p65, have been linked to autoinflammation and autoimmunity, but the underlying mechanisms remain incompletely defined. We investigated six patients from five unrelated families carrying novel heterozygous truncating RELA variants. Despite reduced p65 expression, patients exhibited a broad spectrum of inflammatory manifestations alongside elevated baseline and stimulus-induced pro-inflammatory cytokines. Functional analyses in patient-derived cells and mutant RELA knock-in models showed that upstream NF-{kappa}B signaling was intact, but induction of inhibitory regulators such as I{kappa}B and A20 was impaired. This defective feedback control shifted immune homeostasis toward amplified inflammatory responses that depended on the residual activity of the remaining functional RELA allele. Single-cell transcriptomics revealed distinct cell type-specific consequences: monocytes displayed constitutive type I interferon and NF-{kappa}B activation, B cells retained partial compensatory signaling, whereas T and NK cells exhibited transcriptional signatures of cell death pathways. Patient fibroblasts and mutant RELA knock-in cells further confirmed enhanced TNF-induced inflammatory gene expression and hypersensitivity to apoptosis and necroptosis. These findings establish RELA haploinsufficiency as a cause of systemic immune dysregulation, and link defective NF-{kappa}B feedback control to unchecked inflammation and inflammatory cell death. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=152 SRC="FIGDIR/small/687461v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1f0bfe6org.highwire.dtl.DTLVardef@c6cb60org.highwire.dtl.DTLVardef@1522accorg.highwire.dtl.DTLVardef@177c724_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2025

Sample-multiplexed FACS-preprocessing of PBMCs enables scalable scRNA-seq without compromising transcriptomic or cellular integrity

Efficient preprocessing of peripheral blood mononuclear cells (PBMCs) for single-cell RNA-Sequencing (scRNA-seq) is crucial to ensure high sample throughput while maintaining sample integrity. In particular, when enrichment of rare immune cell populations is necessary to enable their representative profiling among more common PBMCs, sample preprocessing may become a detrimental bottleneck. Here, we present an optimized fluorescence-activated cell sorting (FACS)-based preprocessing workflow designed to enrich rare immune cells while conserving overall PBMC composition. The protocol integrates dead cell removal, targeted rare cell enrichment, channel splitting, and hash-based sample multiplexing together with a new powerful yet lightweight demultiplexing tool (YAHD), improving throughput and cell yield, reducing batch effects, and preserving biological context. Validation across cryopreserved human PBMCs obtained from different scientifically relevant sources (clinical routine and laboratory setting) demonstrated improved sample viability and representation of rare subsets in the final scRNA-seq data. Thorough transcriptomic assessment confirmed non-concerning levels of stress induction and T cell activation as well as low technical variability, removing concerns around FACS-processing, cross-donor multiplexing and channel splitting. The presented approach enables scalable and biologically faithful PBMC preprocessing for scRNA-seq, advancing the study of immune heterogeneity in health and disease.

genomics↗

bioRxiv2025

The role of feedback for sensorimotor decisions under risk

For goal-directed movements like throwing darts or shooting a soccer penalty, the optimal location to aim depends on the endpoint variability of an individual. Currently, there is no consensus whether people can optimize their movement planning based on information about their motor variability. Here, we tested the role of different types of feedback for movement planning under risk. We measured saccades towards a bar that consisted of a reward and a penalty region. Participants either received error-based feedback about their endpoint or reinforcement feedback about the resulting reward. We additionally manipulated the feedback schedule to assess the role of feedback frequency and whether feedback focusses on individual trials or a group of trials. Participants with trial-by-trial reinforcement feedback performed best and had the least endpoint deviation from optimality. Our results are consistent with a slow gradual drift in the participants internal aiming location under summary feedback. Feedback focusing on individual trials reduces this drift, thereby enabling consistent movement planning from one trial to the next. Our results therefore suggest that reinforcement feedback about a single movement is most effective to optimize movement planning under risk.

neuroscience↗

bioRxiv2025

Wnt11 Positively Regulates Neonatal Cardiomyocyte Maturation at the Interphase of Life via Frizzled 4 Receptor

Congenital heart defects (CHDs) affect 1% of live births and remain the leading cause of infant morbidity and early mortality. While most studies focus on the genetic basis of CHDs, relatively little is known about the interplay between intrinsic signaling and external environmental factors in the progression of CHDs after birth during the perinatal circulatory transition window when environmental stress factors are prevalent. We recently explored such interplay through a newly identified gene-environment regulatory circuit involving Wnt11 signaling and systemic hypoxia. Specifically, we demonstrated that activation of the Wnt11/Rb1 axis is critical for normal chamber-specific development after birth. This regulatory switch is disrupted by systemic hypoxia more robustly in the right ventricle (RV) than the left ventricle (LV), leading to enhanced neonatal cardiomyocyte cell cycle activity in an RV-specific manner, resulting in delayed maturation and attenuation of ventricular patterning in response to systemic hypoxia stress in the neonatal heart. Furthermore, we found that the Wnt11/Rb1 axis is also inactivated in infantile hearts with cyanotic CHDs, such as tetralogy of Fallot (TOF), potentially contributing to hypoxia-associated RV abnormalities in this context. However, the molecular players of this signaling cascade in neonatal cardiomyocyte remain largely unknown. Herein, we report that Frizzled 4 (Fzd4) acts as a specific upstream receptor for Wnt11 in neonatal cardiomyocytes. Specifically, Fzd4 exhibited an expression pattern like Wnt11 in neonatal heart perinatal circulatory transition under normal and hypoxemic environments. Furthermore, Fzd4 loss in neonatal cardiomyocytes stimulated cardiomyocyte cell cycle activity and disrupted the Wnt11-Rb1 signaling axis mirroring the impact of the Wnt11-deficient cardiomyocyte phenotype. Finally, co-immunoprecipitation analysis confirmed the Wnt11-Fzd4 binding in isolated neonatal cardiomyocytes and intact hearts. These results demonstrate that Fzd4 is a specific and required upstream receptor for the Wnt11-Rb1 signaling activity in the neonatal heart and provides mechanistic insights into the essential role of Wnt11 as a key positive regulator of neonatal cardiomyocyte transition from proliferative to mature phenotype at the interphase of life.

cell biology↗

bioRxiv2025

Needle in a haystack: A droplet digital polymerase chain reaction assay to detect rare helminth parasites infecting natural host populations

Helminths infect humans, livestock, and wildlife, yet remain understudied despite their significant impact on public health and agriculture. Because many of the most prevalent helminth-borne diseases are zoonotic, the health of diverse host species are closely interconnected. Therefore, understanding helminth transmission among wildlife could improve predictions and management of infection risks across species. A key challenge to understanding helminth transmission dynamics in wildlife is accurately and quantitatively tracking infection levels across hosts and environments. Traditional methods, such as visual parasite identification from environmental samples or infected hosts, are time-consuming, while standard molecular techniques (e.g., PCR and qPCR) often lack the sensitivity to reliably detect lower parasit burdens. These limitations often underestimate the prevalence and severity of infection, hindering efforts to manage infectious diseases. Here, we developed a multiplexed droplet digital PCR (ddPCR) assay to quantify helminth levels in aquatic habitats using 18S rRNA target genes. Using Schistocephalus solidus and their copepod hosts as a case study, we demonstrate ddPCRs sensitivity and precision. By establishing a 1:1 infection standard in the lab, we contextualize ddPCR gene concentration data to quantify both host and parasite numbers in field samples. The assay is highly reproducible, reliably detecting target genes at concentrations as low as 1 picogram of DNA in lab standards and field samples (multi-species and eDNA). Thus, we provide a toolkit for quantifying infection loads in intermediate hosts and monitoring infection dynamics across spatio-temporal scales in multiple helminth systems of concern for public health, agriculture, and conservation biology. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/634533v1_ufig1.gif" ALT="Figure 1"> View larger version (66K): org.highwire.dtl.DTLVardef@1bc2e40org.highwire.dtl.DTLVardef@180822eorg.highwire.dtl.DTLVardef@1e2c2c8org.highwire.dtl.DTLVardef@621909_HPS_FORMAT_FIGEXP M_FIG Applications of ddPCR probe-primer design to parallel systems. Cyclopoid copepods serve as initial hosts for diverse helminthic diseases distributed globally. The primers designed in this assay are suitable for other systems, with minimal work required for probe design specific to each helminth species. C_FIG

molecular biology↗

bioRxiv2024

Effort in Oculomotor Control: Role of Instructions and Reward on Spatio-temporal Eye Movement Dynamics

Effort is an important construct in several psychological disciplines, yet there is little consensus on how it manifests in behavior. Here, we focus on effort in terms of performance improvements beyond speed-accuracy tradeoffs and argue that oculomotor kinematics are conducive to a more fine-grained understanding of effortful behavior. Specifically, we investigated the efficiency and persistence of mere task instructions to induce transient effort. In a saccadic selection task, participants were instructed to look at targets as quickly and accurately as possible (standard instructions) or to mobilize all resources and respond even faster and more accurately ("to give 110%", effort instructions). We compared results to standard speeded performance (baseline block) and to a potential upper performance limit linking effort to performance-contingent rewards (reward block). Performance improved beyond speed-accuracy tradeoffs when reward was present. Effort instructions reduced saccade latencies, increased amplitudes and changed the saccade main sequence relationship. Yet, these effects were more strongly pronounced and more persistent over time when effort was additionally rewarded. Altogether, the present findings underscore the possibility to intentionally activate cognitive resources for regulating oculomotor performance. Yet, its effectiveness and maintenance over time are more successful when behavior is rendered purposeful by the presence of reward. Significance StatementThis study shows that people can exert voluntary control over the cognitive resources they devote to a task and that better task performance also goes along with changes in eye movement behavior. These findings thus suggest that eye movements are a particularly valuable tool when studying effort and cognitive control. Furthermore, eye movements can help to discriminate between phases in which people engage in a task and when they do not.

neuroscience↗

bioRxiv2024

Destabilized host-parasite dynamics in newly founded populations

When species disperse into previously unoccupied habitats, new populations encounter unfamiliar species interactions such as altered parasite loads. Theory predicts that newly founded populations should exhibit destabilized eco-evolutionary fluctuations in infection rates and immune traits. However, to understand founder effects biologists typically rely on retrospective studies of range expansions, missing early-generation infection dynamics. To remedy this, we experimentally founded whole-lake populations of threespine stickleback. Infection rates were temporally stable in native source lakes. In contrast, newly founded populations exhibit destabilized host-parasite dynamics: high starting infection rates led to increases in a heritable immune trait (peritoneal fibrosis), suppressing infection rates. The resulting temporal auto-correlation between infection and immunity suggest that newly founded populations can exhibit rapid host-parasite eco-evolutionary dynamics.

evolutionary biology↗

bioRxiv2023

Mapping Chromatin Occupancy of Ppp1r1b-lncRNA Genome-Wide Using Chromatin Isolation by RNA Purification (ChIRP)-seq

Long non-coding RNA (lncRNA) mediated transcriptional regulation is increasingly recognized as an important gene regulatory mechanism during development and disease. LncRNAs are emerging as critical regulators of chromatin state; yet the nature and the extent of their interactions with chromatin remain to be fully revealed. We have previously identified Ppp1r1b-lncRNA as an essential epigenetic regulator of myogenic differentiation in cardiac and skeletal myocytes in mice and humans. We further demonstrated that Ppp1r1b-lncRNA function is mediated by the interaction with the chromatin-modifying complex polycomb repressive complex 2 (PRC2) at the promoter of myogenic differentiation transcription factors, TBX5 and MyoD1. Herein, we employed an unbiased chromatin isolation by RNA purification (ChIRP) and high throughput sequencing to map the repertoire of Ppp1r1b-lncRNA chromatin occupancy genome-wide in the mouse muscle myoblast cell line. We uncovered a total of 99732 true peaks corresponding to Ppp1r1b-lncRNA binding sites at high confidence (P-value < 1e-5 and enrichment score [≥] 10). The Ppp1r1b-lncRNA-binding sites averaged 558 bp in length and were distributed widely within the coding and non-coding regions of the genome. Approximately 46% of these true peaks were mapped to gene elements, of which 1180 were mapped to experimentally validated promoter sequences. Importantly, the promoter-mapped binding sites were enriched in myogenic transcription factors and heart development while exhibiting focal interactions with known motifs of proximal promoters and transcription initiation by RNA polII, including TATA, transcription initiator, CCAAT-box, and GC-box, supporting Ppp1r1b-lncRNA role in transcription initiation of myogenic regulators. Remarkably, nearly 40% of Ppp1r1b-lncRNA-binding sites mapped to gene introns, were enriched with the Homeobox family of transcription factors, and exhibited TA-rich motif sequences, suggesting potential motif specific Ppp1r1b-lncRNA-bound introns. Lastly, more than 136521enhancer sequences were detected in Ppp1r1b-lncRNA-occupancy sites at high confidence. Among these enhancers,12% exhibited cell type/tissue-specific enrichment in fetal heart and muscles. Together, our findings provide further insights into the genome-wide Ppp1r1b-lncRNA: Chromatin interactome that may potentially dictate its function in myogenic differentiation and potentially other cellular and biological processes.

genomics↗

bioRxiv2023

Endosome dysfunction leads to gain-of-function TLR7 and human lupus

Hyperactive Toll-like receptor (TLR) 7 signaling has long been appreciated as a driver of autoimmune disease by breaking tolerance to self-nucleic acids in mouse models1-5. Recently, mutations in TLR7 or its associated regulator UNC93B16, 7, were identified as monogenic causes of human lupus; the unifying feature of these mutations being TLR7 gain-of-function. TLR7 is an intracellular transmembrane receptor, sensing RNA breakdown products within late endosomes8, 9. Hence, its function depends on intricate transport mechanisms and membrane interactions within the endomembrane network. Whether perturbations of any of these endosome-related processes can give rise to TLR7 gain-of-function and facilitate self-reactivity has not been investigated. Here, we show that a dysregulated endosomal compartment leads to unrestricted TLR7 signaling and human lupus. The late endosomal BLOC-1-related protein complex (BORC) together with the small Arf1-like GTPase Arl8b controls TLR7 protein levels, and a direct interaction between Arl8b and Unc93b1 is required to regulate TLR7 turnover. We identified an amino acid insertion in UNC93B1 in a patient with childhood-onset lupus, which reduces the interaction with the BORC-Arl8b complex and leads to endosomal TLR7 accumulation. Therefore, a failure to control the proper progression of TLR7 through its endocytic life cycle is sufficient to break immunological tolerance to nucleic acids in humans. Our results highlight the importance of an intact endomembrane system to prevent autoimmune disease. As the cellular mechanisms restricting TLR7 signaling can be manifold, identifying and stratifying lupus patients based on a TLR7-driven pathogenesis could be a viable strategy towards a targeted therapy.

immunology↗

bioRxiv2022

Virus-like particle displaying SARS-CoV-2 receptor binding domain elicits neutralizing antibodies and is protective in a challenge model

While the effort to vaccinate people against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has largely been successful, particularly in the developed world, the rise of new variants as well as waning immunity illustrate the need for a new generation of vaccines that provide broader and/or more durable protection against infection and severe disease. Here we describe the generation and characterization of IVX-411, a computationally designed, two-component virus-like particle (VLP) displaying the ancestral SARS-CoV-2 receptor binding domain (RBD) on its surface. Immunization of mice with IVX-411 generates neutralizing antibodies against the ancestral strain as well as three variants of concern. Neutralizing antibody titers elicited by IVX-411 are durable and significantly higher than those elicited by immunization with soluble RBD and spike antigens. Furthermore, immunization with IVX-411 is shown to be protective in a Syrian Golden hamster challenge model using two different strains of SARS-CoV-2. Overall, these studies demonstrate that IVX-411 is highly immunogenic and capable of eliciting broad, protective immunity.

immunology↗

bioRxiv2021

Comprehensive characterization of the antibody responses to SARS-CoV-2 Spike protein after infection and/or vaccination

BackgroundControl of the COVID-19 pandemic will rely on SARS-CoV-2 vaccine-elicited antibodies to protect against emerging and future variants; an understanding of the unique features of the humoral responses to infection and vaccination, including different vaccine platforms, is needed to achieve this goal. MethodsThe epitopes and pathways of escape for Spike-specific antibodies in individuals with diverse infection and vaccination history were profiled using Phage-DMS. Principal component analysis was performed to identify regions of antibody binding along the Spike protein that differentiate the samples from one another. Within these epitope regions we determined potential escape mutations by comparing antibody binding of peptides containing wildtype residues versus peptides containing a mutant residue. ResultsIndividuals with mild infection had antibodies that bound to epitopes in the S2 subunit within the fusion peptide and heptad-repeat regions, whereas vaccinated individuals had antibodies that additionally bound to epitopes in the N- and C-terminal domains of the S1 subunit, a pattern that was also observed in individuals with severe disease due to infection. Epitope binding appeared to change over time after vaccination, but other covariates such as mRNA vaccine dose, mRNA vaccine type, and age did not affect antibody binding to these epitopes. Vaccination induced a relatively uniform escape profile across individuals for some epitopes, whereas there was much more variation in escape pathways in in mildly infected individuals. In the case of antibodies targeting the fusion peptide region, which was a common response to both infection and vaccination, the escape profile after infection was not altered by subsequent vaccination. ConclusionsThe finding that SARS-CoV-2 mRNA vaccination resulted in binding to additional epitopes beyond what was seen after infection suggests protection could vary depending on the route of exposure to Spike antigen. The relatively conserved escape pathways to vaccine-induced antibodies relative to infection-induced antibodies suggests that if escape variants emerge, they may be readily selected for across vaccinated individuals. Given that the majority of people will be first exposed to Spike via vaccination and not infection, this work has implications for predicting the selection of immune escape variants at a population level. FundingThis work was supported by NIH grants AI138709 (PI Overbaugh) and AI146028 (PI Matsen). Julie Overbaugh received support as the Endowed Chair for Graduate Education (FHCRC). The research of Frederick Matsen was supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute and the Simons Foundation. Scientific Computing Infrastructure at Fred Hutch was funded by ORIP grant S10OD028685.

immunology↗

bioRxiv2021

GDAP1 loss of function inhibits the mitochondrial pyruvate dehydrogenase complex by altering the actin cytoskeleton

Charcot-Marie-Tooth (CMT) disease 4A is an autosomal-recessive polyneuropathy caused by mutations of ganglioside-induced differentiation-associated protein 1 (GDAP1), a putative glutathione transferase, which affects mitochondrial shape and alters cellular Ca2+ homeostasis. Here, we identify the underlying mechanism. We found that patient-derived motoneurons and GDAP1 knockdown SH-SY5Y cells display two phenotypes: more tubular mitochondria and a metabolism characterized by glutamine dependence and fewer cytosolic lipid droplets. GDAP1 interacts with the actin-depolymerizing protein Cofilin-1 in a redoxdependent manner, suggesting a role for actin signaling. Consistently, GDAP1 loss causes less F-actin close to mitochondria, which restricts mitochondrial localization of the fission factor dynamin-related protein 1, instigating tubularity. Changes in the actin cytoskeleton also disrupt mitochondria-ER contact sites. This results in lower mitochondrial Ca2+ levels and inhibition of the pyruvate dehydrogenase complex, explaining the metabolic changes upon GDAP1 loss of function. Together, these findings reconcile GDAP1-associated phenotypes and implicate disrupted actin signaling in CMT4A pathophysiology.

neuroscience↗

bioRxiv2021

NECAB2 orchestrates an endosomal pathway of mitochondrial quality control at striatal synapses

Synaptic signaling depends on ATP generated by mitochondria. Due to extensive connectivity, the striatum is especially vulnerable to mitochondrial dysfunction and thus requires efficient mitochondrial quality control and repair. We found that global knockout of the neuronal calcium-binding protein 2 (NECAB2) in the mouse causes loss of striatal synapses and behavioral phenotypes related to striatal dysfunction such as reduced motivation and altered sensory gating. Striatal mitochondria from Necab2 knockout mice are more abundant and smaller. They are characterized by increased respiration and superoxide production resulting in oxidative stress. This accumulation of dysfunctional mitochondria is caused by a defective assembly of mitochondria with early endosomes in a pathway that involves the small GTPase Rab5 and its guanine nucleotide exchange factor Alsin/ALS2. NECAB2 therefore participates in an endosomal pathway of mitochondrial stress response and repair important for striatal function.

neuroscience↗

bioRxiv2019

Resilience trinity: safeguarding ecosystem services across three different time horizons and decision contexts

Ensuring ecosystem resilience is an intuitive approach to safeguard future provisioning of ecosystem services (ES). However, resilience is an ambiguous concept and difficult to operationalize. Focusing on resilience mechanisms, such as diversity, network architectures or adaptive capacity, has recently been suggested as means to operationalize resilience. Still, the focus on mechanisms is not specific enough because the usefulness of a mechanism is context-dependent. We suggest a conceptual framework, resilience trinity, to facilitate management of resilience mechanisms in three distinctive decision contexts and time-horizons. i) reactive, when there is an imminent threat to ES resilience and a high pressure to act, ii) adjustive, when the threat is known in general but there is still time to adapt management, and iii) provident when time horizons are very long and the nature of the threats is uncertain, leading to a low willingness to act. This emphasizes that resilience has different interpretations and implications at different time horizons which however need to be reconciled. The inclusion of time into resilience thinking ensures that longer-term management actions are not missed while urgent threats to ES are given priority.

ecology↗

bioRxiv2016

Quantifying predator dependence in the functional response of generalist predators.

A longstanding debate concerns whether functional responses are best described by prey-dependent versus ratio-dependent models. Theory suggests that ratio dependence can explain many food web patterns left unexplained by simple prey-dependent models. However, for logistical reasons, ratio dependence and predator dependence more generally have seen infrequent empirical evaluation and then only so in specialist predators, which are rare in nature. Here we develop an approach to simultaneously estimate the prey-specific attack rates and predator-specific interference rates of predators interacting with arbitrary numbers of prey and predator species. We apply the approach to field surveys and two field experiments involving two intertidal whelks and their full suite of potential prey. Our study provides strong evidence for the presence of weak predator dependence that is closer to being prey dependent than ratio dependent over manipulated and natural ranges of species abundances. It also indicates how, for generalist predators, even the qualitative nature of predator dependence can be prey-specific.\n\nAuthor contributionsCW contributed to method development, KC and IS performed the caging experiment, and MN conceived of the study, carried out the fieldwork and analyses, and wrote the manuscript.

ecology↗